WO2018112226A1 - Sharpin-based polypeptides and their uses - Google Patents

Sharpin-based polypeptides and their uses Download PDF

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Publication number
WO2018112226A1
WO2018112226A1 PCT/US2017/066463 US2017066463W WO2018112226A1 WO 2018112226 A1 WO2018112226 A1 WO 2018112226A1 US 2017066463 W US2017066463 W US 2017066463W WO 2018112226 A1 WO2018112226 A1 WO 2018112226A1
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polypeptide
amino acid
amino acids
sequence
alanine
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PCT/US2017/066463
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French (fr)
Inventor
Federico Bernal
Amanda Lee WHITING
Kazuhiro Iwai
Hiroaki Fujita
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The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Kyoto University
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Publication of WO2018112226A1 publication Critical patent/WO2018112226A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/19—Omega peptidases (3.4.19)
    • C12Y304/19012—Ubiquitinyl hydrolase 1 (3.4.19.12)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • LUBAC ubiquitin chain assembly complex
  • the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, the polypeptide comprising at least two non-natural ammo acids that form a covalent cross-link with each other, wherein the non-natural amino acids are the same or are different, and wherein the cross-link is internal to the polypeptide, and (a) wherein the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine; or the polypeptide has from the N- to C-terminus a glutamine one amino acid position before a first valine, and optionally (i) wherein the first valine is three amino acid positions before a second valine, and (ii) wherein the second valine is one amino acid position before a leucine; and (b) wherein the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
  • LUBAC linear ubiquitin chain assembly complex
  • the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, comprising a modified amino acid sequence of EELAGSLARAI (SEQ ID NO: 1), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO: 4); wherein the modification of the sequence is of at least two of the amino acids within the sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein the non-natural amino acids form a covalent cross-link with each other, wherein the covalent cross-link is internal to the sequence; and wherein the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
  • LUBAC linear ubiquitin chain assembly complex
  • the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, the polypeptide comprising at least two non-natural amino acids capable of forming an internal cross-link, wherein the non-natural amino acids are the same or are different, wherem each of the non-natural amino acids includes a moiety, wherein the moieties are capable of undergoing a reaction to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the polypeptide; and (a) wherein the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine; or the polypeptide has from the N- to C-terminus (1 ) a glutamine one amino acid position before a first valine, and (2) optionally (i) wherein the first valine is three amino acid positions before a second valine, and (ii) wherein the second valine is one amino acid position before
  • the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, comprising a modified amino acid sequence of EELAGSLARAI (SEQ ID NO: 1 ), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO: 4); wherein the modification of the sequence is of at least two of the amino acids within the sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein each of the non- natural amino acids include a moiety, wherein each moiety is capable of undergoing a reaction with a moiety of one other of the non-natural amino acids to form a covalent crosslink with each other, wherein the covalent cross-link is internal to the sequence; and wherein if the non-natural amino acids form an internal cross-link with each other, the
  • the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence modified according to the formula (I):
  • Ri and R? are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
  • R3 of formula (I) is alkylene, alkenylene, alkynylene, or [R4-K'-R 4 ']n, each of which is substituted with 0-6 R5;
  • R4 and R ⁇ are independently alkylene, alkenylene, or alkynylene;
  • R5 is independently halo, alkyl, OR6, N(R 6 )2, SR6, SOR6, SO2R6, CO2R6, R&, a fluorescent moiety, or a radioisotope;
  • K' is independently O, S, SO, S0 2 , CO, CO2, CONR&, or R-6 is independently H, alkyl, or a therapeutic agent;
  • n is independently an integer from 1-4;
  • [Xaa]x has 2 to
  • the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence modified according to the formula (II):
  • Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
  • R3 and R3' of formula (II) are independently alkenyl, alkynyl, azido, amino, carboxylic acid, or sulfide;
  • [Xaa] has 2 to 6 amino acids;
  • [Xaa] w , [Xaa] x , and [Xaa] y taken together have the amino acid sequence of EELAGSLARAI (SEQ ID NO: 1 ), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the ammo acids within the sequence are replaced with the residues A and B; and wherein if A and B form an internal cross-link, the polypeptid
  • the invention also provides a method of inhibiting the linear ubiquitin chain assembly complex (LUBAC) in a subject, which method comprises administering an effective amount of an inventive polypeptide described herein or a pharmaceutical composition of an inventive polypeptide described herein to the subject.
  • LUBAC linear ubiquitin chain assembly complex
  • the invention also provides a method of treating activated B- cell like diffuse large B cell lymphoma (ABC DLBCL) in a subject, which method comprises administering an effective amount of an inventive polypeptide described herein or a pharmaceutical composition of an inventive polypeptide described herein to the subject.
  • ABSC DLBCL activated B- cell like diffuse large B cell lymphoma
  • the invention also provides a method of treating rheumatoid arthritis in a subject, which method comprises administering an effective amount of an inventive polypeptide described herein or a pharmaceutical composition of an inventive polypeptide described herein to the subject.
  • the invention also provides a method of treating cancer that is resistant to cytotoxic chemotherapy, radiation therapy, vaccine therapy, or cytokine therapy in a subject, which method comprises administering an effective amount of an inventive polypeptide described herein or a pharmaceutical composition of an inventive polypeptide described herein to the subject.
  • the invention also provides a method of treating chronic autoinflammation, systemic lupus erythematosus, Crohn's inflammatory bowel disease, or psoriasis in a subject, which method comprises administering an effective amount of an inventive polypeptide described herein or a pharmaceutical composition of an inventive polypeptide described herein to the subject.
  • Figure 1 A is a schematic representation of domains of HOIL-1 L, HOIP and SHARP IN.
  • Figure I B shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Cell lysates and anti-FLAG immunoprecipitates were immunoblotted with the indicated antibody.
  • Figure 1C presents a surface plasmon resonance line graph. GST-mHOIP UBA (466-630aa) was immobilized on the sensor chip of surface plasmon resonance via GST antibody. Binding between mHOIP UBA and MBP-mSHARPIN UBL (163-301 aa) was measured.
  • Figure ID shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Cell lysates and anti-FLAG immunoprecipitates were immunoblotted with the indicated antibody.
  • Figure IE shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Cell lysates and anti-FLAG immunoprecipitates were immunoblotted with the indicated antibody.
  • Figure 2D presents a surface plasmon resonance line graph.
  • GST-tagged mHOIP UBA (466-630 aa) was immobilized on sensor chip of SPR via GST antibody.
  • mSHARPIN (163-301 aa) alone (8.5 ⁇ ) or both (8.5 ⁇ each) were used as analytes. See also Figure 8A.
  • Figure 2E shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Cell lysates were immunoblotted with the indicated antibody.
  • Figure 2F shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Anti-Myc immunoprecipitates were immunoblotted with the indicated antibody.
  • Figure 2G shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Anti-FLAG immunoprecipitates were immunoblotted with the indicated antibody.
  • Figure 2H shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Cell lysates were analyzed by western blotting.
  • Figure 21 shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Anti-FLAG immunoprecipitates were analyzed by western blotting.
  • Figure 3A shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T HOIP KO cells. Cell lysates were analyzed by immunoblotting.
  • Figure 3B shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T HOIP KO cells. Anti-Myc immunoprecipitates were analyzed by immunoblotting.
  • Figure 3C presents a surface plasmon resonance line graph.
  • GST-mHOIP UBA WT (466-630 aa) was immobilized on sensor chip of SPR via GST antibody. Binding ability to the UBLs containing or lacking Na region was analyzed as described in Figure 2D.
  • Figure 3D shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T HOIP KO cells. Cell lysates were analyzed by immunoblotting.
  • Figure 3E shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T HOIP KO cells. Anti-FLAG immunoprecipitates were analyzed by immunoblotting.
  • Figure 3F shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Cell lysates were analyzed by immunoblotting.
  • Figure 3G shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Anti-FLAG immunoprecipitates were analyzed by immunoblotting.
  • Figure 4A presents immunoblot analysis of lysates from LUBAC TKO cells reconstituted with the indicated proteins.
  • FIG. 4B presents a surface plasmon resonance line graph.
  • TKO cells reconstituted with the indicated proteins were stimulated with TNF-a ( l Ong/ml) and cell viability was measured continuously by using impedance-based real-time cell analyzer (RTCA).
  • RTCA real-time cell analyzer
  • Figure 4C presents immunoblot analysis of lysates from LUBAC TKO cells reconstituted with the indicated proteins.
  • Figure 4D presents a surface plasmon resonance line graph. TKO cells reconstituted with the indicated proteins were stimulated with TNF-a (lOng/ml) and analyzed as described in Figure 4B.
  • Figure 4E presents sequences of ct-helical SHARPIN polypeptides. The asterisks show the location of the hydrocarbon cross-linker.
  • Figure 4F presents a surface plasmon resonance line graph.
  • GST-mHOIP UBA (466-630 aa) was immobilized on the sensor chip of SPR via GST antibody.
  • Figure 4G shows immunoblot gel analysis. Trimeric LUBAC (0.2 ⁇ ) was incubated with stapled polypeptides (80 ⁇ ) on ice for 3 hours. Then, a mixture of El, E2 and ubiquitin was added and incubated at 37°C for 30 min followed by immunoblotting.
  • Figure 4H shows immunoblot gel analysis. 10 ⁇ g of SI 00 lysate of HOIP KO Jurkat cells and trimeric LUBAC (0.1 ⁇ ) was incubated with stapled polypeptides (80 ⁇ ) on ice for 3 hours. Then, a mixture of El , E2 and ubiquitin was added and incubated at 37°C for 30 min followed by immunoblotting.
  • Figure 41 shows immunoblot gel analysis. HBL-1 cells were treated with the indicated polypeptides for 24 h. Cell lysates and anti-SHARPIN immunoprecipitates were analyzed by immunoblotting.
  • Figure 4J presents a gel. HBL-1 cells were treated with the indicated polypeptides for 24 h. Cell viability was measured by CellTiter-Glo Luminescent Cell Viability assay and NF-KB activity was measured by EMSA assay.
  • Figure 5A presents immunoblot analysis of MEF cells from indicated genotypes of mice.
  • Figure 5B presents quantification of genotypes of animals obtained after crossing HOIL-l L +/nu " mice.
  • Figure 5C presents the numbers of embryos obtained at each embryonic stage (E9.5, 10.5, 1 1.5, and 12.5) after crossing HOIL-l L +/nu " mice.
  • Figure 6A shows immunoblot gel analysis. Cell lysates of TKO cells expressing the indicated proteins were analyzed by immunoblotting.
  • Figure 6B presents a schematic of sequences of polypeptides used in Figure 6C.
  • Figure 6C shows immunoblot gel analysis. Cell lysates of TKO cells
  • FIG. 6D shows immunoblot gel analysis.
  • TKO cells reconstituted with indicated proteins were stimulated with TNF-a (5 ng/ml) for indicated periods and cell lysates were analyzed by immunoblotting.
  • Figure 6E shows immunoblot gel analysis. TKO cells reconstituted with indicated proteins were stimulated with TNF-a (1 ng/ml) plus CHX (20 ⁇ g/ml) for indicated periods and cell lysates were analyzed by immunoblotting.
  • Figure 6F shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T HOIP KO cells. Cell lysates were analyzed by immunoblotting.
  • Figure 6G shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T HOIP KO cells. Anti-FLAG immunoprecipitates were analyzed by immunoblotting.
  • Figure 7A shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Cell lysates were analyzed by immunoblotting.
  • Figure 7B shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Anti-FLAG immunoprecipitates were analyzed by immunoblotting.
  • Figure 7C presents a surface plasmon resonance line graph.
  • GST-HOIP UBA (466-630 aa) was immobilized on the sensor chip for SPR via a GST antibody and binding affinity between MBP-HOIL- 1 L UBL (1 -140 aa) was determined.
  • Figure 7D shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Cell lysates were analyzed by immunoblotting.
  • Figure 7E shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Anti-FLAG immunoprecipitates were analyzed by immunoblotting.
  • Figure 7F presents sequences, showing the conserved residues of HOIL-1 L and SHARPIN. Asterisks show the amino acids which are thought to be involved in interactions with HOIP UBA1 or UBA2, respectively.
  • Figure 8A shows the line graphs of Figure 2D, with responses normalized at the time of stopped applying the UBL.
  • Figure 8B presents a surface plasmon resonance line graph.
  • mHOIP UBA UBA2 mut (466-630 aa) was immobilized and binding ability to UBLs was analyzed as shown in Figure 2D.
  • Figure 9A is a schematic representation of guide R A sequence against hHOIP. 293T HOIP KO cells were homozygous for a 1 bp insertion (depicted as underlined). The protospacer-adjacent motif (P AM) sequence is depicted (GGG at end).
  • P AM protospacer-adjacent motif
  • Figure 9B presents immunoblotting analysis of 293T HOIP KO cells.
  • Cell lysates of parent and HOIP KO 293T cells were immunoblotted with the indicated antibody.
  • Figure 9C presents sequences showing conserved residues in Na regions of HOIL-lL and SHARPIN.
  • Figure 9D presents a surface plasmon resonance line graph.
  • GST-mHOIP UBA WT (466-630 aa) was immobilized on a sensor chip for SPR via a GST antibody. Binding ability between UBLs containing or lacking Na regions and HOIP UBA were analyzed.
  • Figure 9E presents a surface plasmon resonance line graph.
  • GST-HOIP UBA (466-630 aa) was immobilized on sensor chip of SPR via GST antibody and HOIL-1L UBL (1 -189 aa) alone (500 ⁇ g/ml) or SHARPIN UBL (500 ⁇ ) (163-340 aa) or both (500 g/ml each) were used as analyte.
  • Figure 9F shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Cell lysates were analyzed by immunoblotting.
  • Figure 9G shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Anti-FLAG immunoprecipitates were analyzed by immunoblotting.
  • Figure 9H presents sequences showing conserved residues of Na regions of HOIL-1L in various species.
  • Figure 91 presents sequences showing conserved residues of Na regions of SHARPIN in various species.
  • Figure 1 OA is a schematic representation of guide RNA sequences against mHOIP or mHOIL-l L.
  • TKO cells carry one allele with 1 bp deletion and one allele with 1 1 bp deletion in HOIL- 1 L loci, and one allele with 2 bp deletion and one allele with 1 bp deletion in HOIP loci.
  • Figure 1 OB is a schematic representation of guide RNA sequences against mHOIP or mHOIL-lL.
  • TKO cells carry one allele with 1 bp deletion and one allele with 1 1 bp deletion in HOIL-I L loci, and one allele with 2 bp deletion and one allele with 1 bp deletion in HOIP loci.
  • Figure 10 C presents immunoblot analysis of lysates from TKO cells.
  • Figure 1 1A shows immunoblot gel analysis. Cell lysates of TKO cells reconstituted with the indicated proteins were immunoblotted with the indicated antibody.
  • Figure 1 IB shows immunoblot gel analysis. Cell lysates from TKO cells reconstituted with indicated proteins and anti-FLAG immunoprecipitates were
  • Figure 1 1C presents a line graph showing RTCA results.
  • Figure 1 I D shows immunoblot gel analysis.
  • TKO cells reconstituted with the indicated proteins were stimulated with TNF-a (1 ng/ml) plus CHX (20 ⁇ g/ml) for the indicated periods.
  • Cell lysates were immunoblotted with the indicated antibody.
  • Figure 1 I E presents RT-PCR analysis of expression levels of N-terminal part or
  • Figure 1 I F shows immunoblot gel analysis. Lysates from TKO cells reconstituted with HOIP, HOIL- I L WT or UBL (HOIL-I L 1 -140 aa) in the presence or absence of
  • SHARP IN were immunoblotted with indicated antibody.
  • Figure 1 1 G shows immunoblot gel analysis.
  • TKO cells reconstituted with indicated proteins were stimulated with TNF-a (3 ng/ml) plus CHX (20 ⁇ g/ml) for the indicated periods. Lysates were analyzed by immunoblotting.
  • Figure 1 1 H is a schematic representation of guide RNA sequences against mHOIL- l L.
  • Figure 1 I presents sequences showing mutated alleles. Mutated allele #1 was obtained using guideRNA# l and mutated allele W2. was obtained using guideRNA#2.
  • Figure 1 1 J shows quantification of genotypes of animals obtained after crossing HOIL- l L +/null#2 mice.
  • Figure 1 IK presents numbers of embryos obtained at each embryonic stage (E9.5, 10.5, 1 1.5, and 12.5) after crossing HOIL-lL +/null#2 mice.
  • Figure 1 1L shows immunoblot gel analysis. Immunoblot analysis of HOIL-1L null MEFs reconstituted HOIL-1L or SHARPIN. Lysates were immunoblotted with the indicated antibody.
  • Figure 12A presents sequences showing conserved residues of HOIP UBA domain between human and mouse.
  • Figure 12B shows immunoblot gel analysis. Indicated plasmids were transfected HEK293T HOIP KO cells. Cell lysates and anti-FLAG immunoprecipitates were analyzed by immunoblotting.
  • Figure 12C shows immunoblot gel analysis. Indicated plasmids were transfected HEK293T cells. Cell lysates and anti-HA immunoprecipitates were analyzed by western blotting.
  • Figure 12D shows immunoblot gel analysis. Indicated plasmids were transfected HEK293T cells. Cell lysates and anti-HA immunoprecipitates were analyzed by western blotting.
  • Figure 13 presents helical wheel diagrams of some of the inventive polypeptides as described herein, in accordance with embodiments of the invention.
  • Figures 14A-14F are as described in Example 6.
  • Figures 15A-15I are as described in Example 6.
  • the invention is based on the discovery of SHARPIN polypeptides that can inhibit the linear ubiquitin chain assembly complex (LUBAC), which includes three protein subunits: HOIP (also known as RNF31), HOIL-1L (also known as RBCK1 ), and SHARPIN (also known as SHANK Associated RH domain INteractor).
  • LUBAC linear ubiquitin chain assembly complex
  • the polypeptides of the invention can be used to disrupt LUBAC activity in vitro or in vivo.
  • the polypeptides of the invention can be used to inhibit LUBAC-mediated cell signaling, e.g., NF- B signaling. Since LUBAC-mediated cell signaling is specific to certain cell types, the polypeptide inhibitors of the invention can be used to selectively inhibit LUBAC-mediated signaling, e.g., NF- ⁇ signaling in specific cells.
  • Polypeptide refers to a polypeptide chain of amino acids having two or more amino acids.
  • the polypeptides of the invention are based on the sequence of wild-type human SHARPIN at position 171-201 :
  • EREELAGSLARAIAGGDEKGAAQVAAVLAQH (SEQ ID NO: 5) and wild-type mouse SHARPIN at position 168-198: KKEELATRLS Q AI AGGDEKA AAQ V A A VLAQH (SEQ ID NO: 6).
  • UBL ubiquitin-like domain
  • the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, the polypeptide comprising at least two non-natural amino acids that form a covalent cross-link with each other, wherein the non-natural amino acids are the same or are different, and wherein the cross-link is internal to the polypeptide, and (a) wherein the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine; or the polypeptide has from the N- to C-terminus a glutamine one amino acid position before a first valine, and optionally (i) wherein the first valine is three amino acid positions before a second valine, and (ii) wherein the second valine is one amino acid position before a leucine; and (b) wherein the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
  • LUBAC linear ubiquitin chain assembly complex
  • the polypeptide of the invention is a hydrocarbon-stapled - helical polypeptide.
  • the polypeptide is not stapled but contains amino acids that can be cross-linked to form a stapled polypeptide, e.g., when the polypeptide is contacted with a cross-linking catalyst and/or placed into an environment in which cross- linking can occur.
  • a cross-linking catalyst that could be used is Grubbs' ruthenium metathesis catalyst or similar catalysts.
  • other cross-linking catalyst examples include copper for cross-linking alkynes and azides, and oxidizing agents for cross-linking disulfides.
  • a stapled polypeptide refers to a polypeptide that includes at least one pair of non-natural amino acids that are covalently cross-linked to each other and thereby form an internal hydrocarbon "staple" within the polypeptide.
  • Such internal cross-links can function as "braces' * or "locks” that stabilize the alpha-helical conformation of a polypeptide and/or improves cell penetration, target affinity, proteolytic resistance, or serum half-life of the polypeptide.
  • Non-natural amino acid as used herein means any amino acid that may form a polypeptide, excluding the “natural amino acids” that form polypeptides in mammals.
  • the "natural amino acids” are alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • the polypeptide can be modified to include one, two, three, or four hydrocarbon staples.
  • the stapled polypeptides of the invention are cell-permeable or cell penetrating and therefore, useful for inhibiting LUBAC in vitro (e.g., in cells cultures, tissue cultures, or explants) or in vivo (e.g. in a "subject," as defined herein).
  • the terms “modified,” “is modified,” “modification,” “replaced,” “is replaced,” etc. are used herein as a way to describe the end resulting polypeptide sequence as if the modification had occurred to a starting polypeptide sequence, without limiting the process by which the modification had or can occur.
  • the phrases "wherein the modification of the sequence is of at least two of the amino acids within the sequence” and “an amino acid sequence modified according to the formula” do not limit the invention such that the modification(s) had to actually occur on the sequence.
  • the resulting sequence may be, e.g., synthesized such that the modifications are incorporated during the synthesis to produce the end resulting polypeptide such that the unmodified sequence may itself not have been actually obtained/produced and subsequently modified, and/or had amino acids replaced.
  • a polypeptide according to the invention can include a first non-natural amino acid substitution.
  • the first non-natural amino acid can be cross- linked to a second non-natural amino acid that is substituted or inserted at a position in the polypeptide which is four residues away.
  • the relative positions of the first and second non- natural amino acids in this stapled polypeptide are designated as (z, i + 4).
  • the first non-natural amino acid can be cross-linked to a second non-natural amino acid located seven residues away (i, i + 7) in the polypeptide.
  • the first non-natural amino acid can be cross-linked to a second non-natural amino acid located three residues away (/, + 3) in the polypeptide.
  • the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine, and wherein the cross-link is formed from a non-natural amino acid at position z within the polypeptide and another non-natural amino acid at position i + 3, i + 4, or i + 7 within the polypeptide; and wherein the amino acid at position i + 6 within the polypeptide is the leucine, the amino acid at position i + 9 within the polypeptide is the alanine, and the amino acid at position i + 10 within the polypeptide is the isoleucine.
  • the polypeptide has from the N- to C-terminus (1) a glutamine one amino acid position before a first valine, and (2) optionally (i) wherein the first valine is three amino acid positions before a second valine, and (ii) wherein the second valine is one amino acid position before a leucine, and wherein the cross-link is formed from a non- natural amino acid at position i within the polypeptide and another non-natural amino acid at position i + 3, i + 4, or i + 7 within the polypeptide; and wherein the amino acid at position i + 1 within the polypeptide is the glutamine, and the amino acid at position i + 2 within the polypeptide is the first valine.
  • the cross-link of the polypeptide is formed from the amino acid at position i within the polypeptide and another amino acid at position i + 4 within the polypeptide, and the amino acid at position i is (S)-2-(4'-pentenyl)alanine (S5) and the amino acid at position i + 4 is S5; or formed from the amino acid at position i within the polypeptide and another amino acid at position i + 3 within the polypeptide, and the amino acid at position i is (i?)-2-(4'-pentenyl)alanine (R5) or (R)-2-(2'-propenyl)alanine (R3) and the amino acid at position i + 3 is S5; or formed from the amino acid at position i within the polypeptide and another amino acid at position i + 7 within the polypeptide, and the amino acid at position i is (R)-2-(7'-octenyl)alanine (R8) and the amino acid at position i
  • the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine, wherein the polypeptide is a first polypeptide, and wherein the polypeptide further comprises a second polypeptide having non-natural amino acids that form a covalent cross-link internally within the second polypeptide, wherein the second polypeptide has from the N- to C-terminus (1) a glutamine one amino acid position before a first valine, and (2) optionally (i) wherein the first valine is three amino acid positions before a second valine, and (ii) wherein the second valine is one amino acid position before a leucine.
  • the cross-link in the first polypeptide is formed from a non-natural amino acid at position i within the first polypeptide and another non-natural amino acid at position i + 3, + 4, or i + 7 within the first polypeptide; and wherein the amino acid at position i + 6 within the first polypeptide is the leucine, the amino acid at position i + 9 within the first polypeptide is the alanine, and the amino acid at position i + 10 within the first polypeptide is the isoleucine; and (b) the cross-link in the second polypeptide is formed from a non-natural amino acid at position i within the second polypeptide and another non- natural amino acid at position i + 3, i + 4, or i + 7 within the second polypeptide; and wherein the amino acid at position i + 1 within the second polypeptide is the glutamine, and the amino acid at position i + 2 within the second polypeptide is the first valine.
  • the cross-link of the first polypeptide is formed from the amino acid at position z within the first polypeptide and another amino acid at position i + 4 within the first polypeptide, and the amino acid at position i is (S)-2-(4'-pentenyl)alanine (S5) and the amino acid at position z + 4 is S5; or formed from the amino acid at position z " within the first polypeptide and another amino acid at position i + 3 within the first polypeptide, and the amino acid at position z is (R)-2-(4'-pentenyl)alanine (R5) or (R)-2-(2'- propenyl)alanine (R3) and the amino acid at position i + 4 is S5; or formed from the amino acid at position z " within the first polypeptide and another amino acid at position z + 7 within the first polypeptide, and the amino acid at position i is (R)-2-(7'-octenyl)a
  • the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, comprising a modified amino acid sequence of EELAGSLARAI (SEQ ID NO: 1), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO: 4); wherein the modification of the sequence is of at least two of the amino acids within the sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein the non-natural amino acids form a covalent cross-link with each other, wherein the covalent cross-link is internal to the sequence; and wherein the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
  • LUBAC linear ubiquitin chain assembly complex
  • exactly two of the amino acids are non-natural amino acids.
  • two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5.
  • the modified amino acid sequence is of EELAGSLARAI (SEQ ID NO: 1), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second modified amino acid sequence of
  • EKGAAQVAAVLAQ (SEQ ID NO: 2); wherein the modification of the second sequence is of at least two of the amino acids within the second sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein the non- natural amino acids form a covalent cross-link with each other, wherein the covalent crosslink is internal to the second sequence.
  • the modified amino acid sequence is of EELATRLSQAI (SEQ ID NO: 3), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second modified amino acid sequence of
  • EKAAAQVAAVLAQ (SEQ ID NO: 4); wherein the modification of the second sequence is of at least two of the amino acids within the second sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein the non- natural amino acids form a covalent cross-link with each other, wherein the covalent crosslink is internal to the second sequence.
  • exactly four of the amino acids are non-natural amino acids.
  • two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5, and another two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5.
  • the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, the polypeptide comprising at least two non-natural amino acids capable of forming an internal cross-link, wherein the non-natural amino acids are the same or are different, wherein each of the non-natural amino acids includes a moiety, wherein the moieties are capable of undergoing a reaction to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the polypeptide; and (a) wherein the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine; or the polypeptide has from the N- to C-terminus (1) a glutamine one amino acid position before a first valine, and (2) optionally (i) wherein the first valine is three amino acid positions before a second valine, and (ii) wherein the second valine is one amino acid position before a
  • An amino acid capable of being cross-linked or having a moiety capable of undergoing a reaction to form a covalent cross-link is a non-natural amino acid that includes a moiety capable of undergoing a reaction with a second moiety on another non-natural amino acid in the polypeptide to form a covalent cross-link formed from the at least two non- natural amino acids.
  • Such amino acids may be the same non-natural amino acids or different non-natural amino acids within the polypeptide, provided they are suitable for cross-linking to each other.
  • amino acids include, e.g., ⁇ , ⁇ -disubstituted cross-linking amino acids; a-methyl, a-alkenyl cross-linking amino acids; and a-hydro, a-alkenyl cross-linking amino acids.
  • Such non-natural cross-linking amino acids are commercially available, e.g., from Sigma-Aldrich, St. Louis, MO, USA or EMD Chemicals.
  • Suitable non-natural cross-linking amino acids suitable for use in a stapled polypeptide of the invention as well as methods for cross-linking them are described in U.S. Patent Application Publication 201 1/0144306 Al, which is incorporated by reference herein in its entirety.
  • the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine, and wherein the non-natural amino acids capable of being cross-linked are at position i within the polypeptide and at position i + 3, / + 4, or i + 7 within the polypeptide; and wherein the amino acid at position i + 6 within the polypeptide is the leucine, the amino acid at position i + 9 within the polypeptide is the alanine, and the amino acid at position i + 10 within the polypeptide is the isoleucine.
  • the polypeptide has from the N- to C-terminus (1 ) a glutamine one amino acid position before a first valine, and (2) optionally (i) wherein the first valine is three amino acid positions before a second valine, and (ii) wherein the second valine is one amino acid position before a leucine; and wherein the non-natural amino acids capable of being cross-linked are at position i within the polypeptide and at position i + 3, + 4, or + 7 within the polypeptide; and wherein the amino acid at position i + 1 within the polypeptide is the glutamine, and the amino acid at position i + 2 within the polypeptide is the first valine.
  • the amino acids capable of being cross-linked are at position within the polypeptide and at position i + 4 within the polypeptide, and the amino acid at position i is (S)-2-(4'-pentenyl)alanine (S5) and the amino acid at position i + 4 is S5; or at position i within the polypeptide and another amino acid at position i + 3 within the polypeptide, and the amino acid at position i is (R)-2-(4'-pentenyl)alanine (R5) or (R)-2-(2' ⁇ propenyl)alanine (R3) and the amino acid at position i + 3 is S5; or the amino acids capable of being cross-linked are at position i within the polypeptide and at position i + 7 within the polypeptide, and the amino acid at position i is (R)-2-(7'-octenyl)alanine (R8) and the amino acid at position i + 7 is S5.
  • the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine, wherein the polypeptide is a first polypeptide, and wherein the polypeptide further comprises a second polypeptide comprising at least two non-natural amino acids capable of forming an internal cross-link, wherein the non-natural amino acids are the same or are different, wherein each of the non-natural amino acids includes a moiety, wherein the moieties are capable of undergoing a reaction to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the second polypeptide, wherein the second polypeptide has from the N- to C-terminus (1 ) a glutamine one amino acid position before a first valine, and (2) optionally (i) wherein the first valine is three amino acid positions before a second valine, and (ii) wherein the second valine is
  • the non-natural amino acids capable of being cross- linked are at position i within the first polypeptide and at position i + 3, i + 4, or i + 7 within the first polypeptide; and wherein the amino acid at position i + 6 within the first polypeptide is the leucine, the amino acid at position i + 9 within the first polypeptide is the alanine, and the amino acid at position / + 10 within the first polypeptide is the isoleucine; and (b) non- natural the amino acids capable of being cross-linked are at position i within the second polypeptide and at position i + 3, i + 4, or i + 1 within the second polypeptide; and wherein the amino acid at position i + 1 within the second polypeptide is the glutamine, and the amino acid at position i + 2 within the second polypeptide is the first valine.
  • the non-natural amino acids capable of being cross- linked in the first polypeptide are at position i within the first polypeptide and at position i + 4 within the first polypeptide, and the amino acid at position i is (S)-2-(4'-pentenyl)alanine (S5) and the amino acid at position i + 4 is S5; or at position i within the first polypeptide and another amino acid at position i + 3 within the first polypeptide, and the amino acid at position i is (i?)-2-(4'-pentenyl)alanine (R5) or (R)-2-(2'-propenyl)alanine (R3) and the amino acid at position i + 3 is S5; or at position i within the first polypeptide and at position i + 7 within the first polypeptide, and the amino acid at position i is (R)-2-(7'-octenyl)alanine (R8) and the amino acid at position i + 7
  • the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, comprising a modified amino acid sequence of EELAGSLARAI (SEQ ID NO: 1), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO: 4); wherein the modification of the sequence is of at least two of the amino acids within the sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein each of the non- natural amino acids include a moiety, wherein each moiety is capable of undergoing a reaction with a moiety of one other of the non-natural amino acids to form a covalent crosslink with each other, wherein the covalent cross-link is internal to the sequence; and wherein if the non-natural amino acids form an internal cross-link with each other, the poly
  • the modified amino acid sequence is of EELAGSLARAI (SEQ ID NO: 1), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second modified amino acid sequence of
  • EKGAAQVAAVLAQ (SEQ ID NO: 2); wherein the modification of the second sequence is of at least two of the amino acids within the second sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein each of the non-natural amino acids include a moiety, wherein each moiety is capable of undergoing a reaction with a moiety of one other of the non-natural amino acids to form a covalent crosslink with each other, wherein the covalent cross-link is internal to the second sequence.
  • the modified amino acid sequence is of EELATRLSQAI (SEQ ID NO: 3), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second modified amino acid sequence of
  • EKAAAQVAAVLAQ (SEQ ID NO: 4); wherein the modification of the second sequence is of at least two of the amino acids within the second sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein each of the non-natural amino acids include a moiety, wherein each moiety is capable of undergoing a reaction with a moiety of one other of the non-natural amino acids to form a covalent crosslink with each other, wherein the covalent cross-link is internal to the second sequence.
  • amino acids wherein exactly four of the amino acids are non-natural amino acids.
  • two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5, and another two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5.
  • R3 of formula (I) is independently selected of R3 of formula (la).
  • the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence modified according to the formula (I):
  • Ri and R 2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
  • R3 of formula (I) is alkylene, alkenylene, alkynylene, or [R 4 -K'-R 4 , ]n, each of which is substituted with 0-6 R5;
  • R4 and Rr are independently alkylene, alkenylene, or alkynylene;
  • R5 is independently halo, alkyl, OR6, N(R 6 ) 2 , SR6, SOR6, SO2R6, CO2R6, 6, a fluorescent moiety, or a radioisotope;
  • K' is independently O, S, SO, SO2, CO, C0 2 , CONR 6 , or
  • Re is independently H, alkyl, or a therapeutic agent; n is independently an integer from 1-4; [Xaa] x has 2 to 6 amino acids; [Xaa] w , [Xaa] x , and [Xaa] y taken together have the amino acid sequence of EELAGSLARAI (SEQ ID NO: 1), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the amino acids within the sequence are replaced with the residues A and B; and wherein the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex
  • the modified amino acid sequence is of EELAGSLARAI (SEQ ID NO: 1), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second amino acid sequence modified according to the formula (la):
  • Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
  • R3 is alkylene, alkenylene, alkynylene, or [R4-K'-R4']n, each of which is substituted with 0-6 R5;
  • R4 and ⁇ are independently alkylene, alkenylene, or alkynylene;
  • R5 is independently halo, alkyl, OR6, N(R6) 2 , SR6, SOR6, SO2R6, CO2R6, R6, a fluorescent moiety, or a radioisotope;
  • K' is independently O, S, SO, SO2, CO, CO2, CONR6, or
  • R6 is independently H, alkyl, or a therapeutic agent; n is independently an integer from 1 -4; [Xaa]x has 2 to 6 amino acids; [Xaa] w , [Xaa] x , and [Xaa] y taken together have the amino acid sequence of EKGAAQVAAVLAQ (SEQ ID NO: 2), wherein two of the amino acids within the sequence are replaced with the residues A and B.
  • the modified amino acid sequence is of EELATRLSQAI (SEQ ID NO: 3), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second amino acid sequence modified according to the formula (la):
  • Ri and R 2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
  • R3 is alkylene, alkenylene, alkynylene, or [R4-K'-R4']n, each of which is substituted with 0-6 R 5 ;
  • R4 and R4' are independently alkylene, alkenylene, or alkynylene;
  • R5 is independently halo, alkyl, OR6, N(R 6 ) 2 , SR6, SOR6, SO2R6, CO2R6, 6, a fluorescent moiety, or a radioisotope;
  • K' is independently O, S, SO, SO2, CO, CO2, CONR6, or
  • R6 is independently H, alkyl, or a therapeutic agent; n is independently an integer from 1-4; [Xaa]x has 2 to 6 amino acids; [Xaa] w , [Xaa] x , and [Xaa] y taken together have the amino acid sequence of EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the amino acids within the sequence are replaced with the residues A and B.
  • the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence modified according to the formula (II):
  • Ri and R 2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
  • R3 and R of formula (II) are independently alkenyl, alkynyl, azido, amino, carboxylic acid, or sulfide;
  • [Xaa] x has 2 to 6 amino acids;
  • [Xaa] w , [Xaa]x, and [Xaa] y taken together have the amino acid sequence of EELAGSLARAI (SEQ ID NO: 1), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the amino acids within the sequence are replaced with the residues A and B; and wherein if A and B form an internal cross-link, the polypeptide is capable of inhibit
  • both R3 and R3' are alkenyl.
  • the modified amino acid sequence is of EELAGSLARAI (SEQ ID NO: 1), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second amino acid sequence modified according to the formula (Ila):
  • Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
  • R3 and R3' are independently alkenyl, alkynyl, azido, amino, carboxylic acid, or sulfide;
  • [Xaa] x has 2 to 6 amino acids;
  • [Xaa] w , [Xaa] x , and [Xaa] y taken together have the amino acid sequence of EKGAAQVAAVLAQ (SEQ ID NO: 2), wherein two of the amino acids within the sequence are replaced with the residues A and B.
  • both R3 and R3* are alkenyl.
  • the modified amino acid sequence is of EELATRLSQAI (SEQ ID NO: 3), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second amino acid sequence modified according to the formula (Ila):
  • Ri and R 2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl; R3 and R3' are independently alkenyl, alkynyl, azido, amino, carboxylic acid, or sulfide; [Xaa] x has 2 to 6 amino acids; [Xaa] w , [Xaa] x , and [Xaa] y taken together have the amino acid sequence of EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the amino acids within the sequence are replaced with the residues A and B. In certain embodiments, both R3 and R3' are alkenyl.
  • [Xaa] x has 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, or 6 amino acids.
  • Halo includes any halogen, e.g., F, CI, Br, I.
  • alkyl means a saturated straight chain or branched non-cyclic hydrocarbon having an indicated number of carbon atoms (e.g., C1 -C20, Ci -Ci o, C1 -C4, C1 -C6, etc.).
  • An alky group may have 1 , 2, 3, 4, 5, 6, 7, 8, or more carbons.
  • saturated straight chain alkyls include -methyl, -ethyl, -n- propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n-nonyl and -n-decyl; while representative saturated branched alkyls include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, - isopentyl, 2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2- methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylbutyl, 2,3- dimethylpentyl, 2,4-dimethylpentyl, 2,3 -dimethylhexyl, 2,4-dimethylhe
  • Alkenyl means an unsaturated straight chain or branched non-cyclic hydrocarbon having an indicated number of carbon atoms (e.g., C1 -C20, C1 -C10, C1-C4, C1 -C6, etc.), where at least one carbon-carbon bond is a double bond.
  • An alkenyl group may have 1, 2, 3, 4, 5, 6, 7, 8, or more carbons.
  • Alkynyl means an unsaturated straight chain or branched non-cyclic hydrocarbon having an indicated number of carbon atoms (e.g., C1 -C20, C1 -C10, C1-C4, C1-C6, etc.), where at least one carbon-carbon bond is a triple bond.
  • An alkynyl group may have 1 , 2, 3, 4, 5, 6, 7, 8, or more carbons.
  • alkylene alkenylene
  • alkynylene are the bivalent radical forms of alkyl, alkenyl, and alkynyl, respectively.
  • cycloalkyl means a cyclic alkyl moiety containing from, for example, 3 to 6 carbon atoms, preferably from 5 to 6 carbon atoms. Examples of such moieties include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
  • Cycloalkylalkyl is a cycloalkyl as defined above substituted with an alkyl as defined above.
  • heterocyclyl means a cycloalkyl moiety having one or more heteroatoms selected from nitrogen, sulfur, and/or oxygen.
  • a heterocyclyl is a 5 or 6-membered monocyclic ring and contains one, two, or three heteroatoms selected from nitrogen, oxygen, and/or sulfur.
  • the heterocyclyl can be attached to the parent structure through a carbon atom or through any heteroatom of the heterocyclyl that results in a stable structure. Examples of such heterocyclic rings are pyrrolinyl, pyranyl, piperidyl,
  • Heterocyclylalkyl is a heterocyclyl as defined above substituted with an alkyl as defined above.
  • alkylamino means
  • cycloalkylamino means -NH(cycloalkyl) or -N(alkyl)(alkyl), wherein alkyl is defined above.
  • cycloalkylamino means -NH(cycloalkyl) or
  • aryl refers to an unsubstituted or substituted aromatic carbocyclic moiety, as commonly understood in the art, and includes monocyclic and polycyclic aromatics such as, for example, phenyl, biphenyl, naphthyl, anthracenyl, pyrenyl, and the like.
  • An aryl moiety generally contains from, for example, 6 to 30 carbon atoms, preferably from 6 to 18 carbon atoms, more preferably from 6 to 14 carbon atoms and most preferably from 6 to 10 carbon atoms.
  • Arylalkyl means an aryl as defined above substituted with an alkyl as defined above.
  • heteroaryl refers to aromatic 4, 5, or 6 membered monocyclic groups, 9 or 10 membered bicyclic groups, and 11 to 14 membered tricyclic aryl groups having one or more heteroatoms (O, S, or N).
  • Each ring of the heteroaryl group containing a heteroatom can contain one or two oxygen or sulfur atoms and/or from one to four nitrogen atoms provided that the total number of heteroatoms in each ring is four or less and each ring has at least one carbon atom.
  • the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen atoms may optionally be quatemized.
  • heteroaryl groups are pyridinyl, pyridazinyl, pyrimidyl, pyrazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1 ,2,3,)- and (l ,2,4)-triazolyl, pyrazinyl, pyrimidinyl, tetrazolyl, furyl, thiophenyl, isothiazolyl, thiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrrolo[2,3-c]pyridinyl, pyrrolo[3,2- c]pyridinyl, pyrrolo[2,3-b]pyridinyl, pyrrolo[3,2-b]pyridinyl, pyrrolo[3,2-d]pyrimidinyl, and pyrrolo[2,3-d]pyrimidinyl.
  • a range of the number of atoms in a structure is indicated (e.g., a Ci -Cs, Ci-C 6 , C1 -C4, or C1 -C3 alkyl, haloalkyl, alkylamino, alkenyl, etc.), it is specifically contemplated that any sub-range or individual number of carbon atoms falling within the indicated range also can be used.
  • the recitation of a range of 1-8 carbon atoms e.g., Ci -Cs
  • 1-6 carbon atoms e.g., C1 -C6
  • 1-4 carbon atoms e.g., C1-C4
  • 1-3 carbon atoms e.g., C1 -C3
  • 2-8 carbon atoms e.g., C2-Cg
  • any chemical group e.g., alkyl, haloalkyl, alkylamino, alkenyl, etc.
  • any sub-range thereof e.g., 1-2 carbon atoms, 1 -3 carbon atoms, 1 -4 carbon atoms,
  • R3 and R3 described in the formulas herein are capable of forming a cross-link.
  • the following provides exemplary reactions for forming a cross-link between R3 and R3 .
  • Alkene-alkene, alkene-alkyne, and alkyne-alkyne pairs can react by olefin metathesis.
  • a sulfide-sulfide pair can react under oxidizing conditions.
  • Azide-alkyne pairs can react to form triazoles.
  • Carboxylic acid-amine pairs can react to form lactams.
  • Carboxylic acid- sulfide pairs could react to form thioesters.
  • any of the above embodiments of the inventive polypeptides may have a sequence such that a polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine.
  • Any of the above embodiments of the inventive polypeptides may have a sequence such that a polypeptide has from the N- to C-terminus a glutamine one amino acid position before a first valine, optionally wherein the first valine is three amino acid positions before a second valine, wherein the second valine is one amino acid position before a leucine.
  • These amino acids may be positioned within the polypeptide such that the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
  • LUBAC linear ubiquitin chain assembly complex
  • Tables 1 and 2 below present certain polypeptide sequences of the invention compared to wild-type SHARPIN sequences, showing the amino acids that are conserved among the inventive polypeptides, and including possible locations for cross-linkages.
  • Non- natural amino acids are shown.
  • R5/R3 indicates that either R5 or R3 may be used at that amino acid position.
  • the polypeptides with non-natural amino acids may include any amino acids shown to be N-terminal to the amino acids of the polypeptide with non-natural amino acids and in the natural sequence.
  • any polypeptide with non-natural amino acids can include the arginine and/or glutamic acid- arginine directly N-terminal to the amino acids shown in the SEQ ID NOs.
  • the C-terminus of any of the polypeptides of Table 1A or IB may be linked to the N-terminus of any of the polypeptides of Tables 1C-1E, directly or with the use of one or more spacer amino acids.
  • Such a polypeptide may have 0, 1 , or 2 cross-links.
  • the C-terniinus of any of the polypeptides of Table 2A or 2B may be linked to the N-tenninus of any of the polypeptides of Tables 2C-2E, directly or with the use of one or more spacer amino acids.
  • Such a polypeptide may have 0, 1 , or 2 cross-links.
  • any of the inventive polypeptides described herein can have a tryptophan added directly to the N- or C- terminus or through the use of one or more spacer amino acids.
  • the polypeptides may be synthesized using any suitable method.
  • the process may include synthesis, ring closing metathesis (RCM) and capping for a single helix and partial synthesis, RCM, hydrogenation, synthesis, RCM, and capping for two helices.
  • RCM ring closing metathesis
  • the side chains of non-natural amino acids wherein each non-natural ammo acid includes a moiety, wherein each moiety is capable of undergoing a reaction with a moiety of one other of the non-natural amino acids to form a covalent cross-link
  • can be covalently linked e.g., R3 to S5, S5 to S5, R5 to S5, or R8 to S5 in the presence of a catalyst to produce the "staple" of the polypeptide.
  • the polypeptide includes a capping group, a linker group, or both a capping group and linker group.
  • Capping groups include fluorescein thiourea (FITC) and biotin (Bt).
  • FITC fluorescein thiourea
  • Bt biotin
  • An example of an N-terminal capping group is acetyl.
  • An example of a C-terminal capping group is the amino group, such that the C-terminus is amidated.
  • the linker group can be one or more naturally occurring a-amino acids (e.g., A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V), non-natural a-amino acids, natural ⁇ -amino acids (e.g., ⁇ -alanine or in the form of N-P-Fmoc-P-alanine), and/or non-natural ⁇ -amino acids.
  • the polypeptide includes a linker group, wherein the linker group is ⁇ - alanine or N ⁇ -Fmoc ⁇ -alanine.
  • the polypeptide includes an N- terminal amino protecting group, a C-terminal carboxyl protecting group, or both an N- terminal amino protecting group and a C-terminal carboxyl protecting group.
  • Protecting groups include fluorenylmethoxy-carbonyl (Fmoc) and any suitable amino protecting group disclosed in Greene et al., Protecting Groups in Organic Synthesis, 3 rd ed., (John Wiley & Sons, 1999), the entirety of which is incorporated herein by reference.
  • Carboxylic acid protecting groups include groups that form an amino-, silyl-, alkyl-, alkenyl-, aryl-, or arylalkyl-protected carboxylic acid.
  • the polypeptide includes a linker group, wherein the linker group is ⁇ -alanine or N-P-Fmoc ⁇ -alanine located between an N- terminus protective group or capping group and the first amino acid position corresponding to SHARPIN-derived sequence.
  • the polypeptide includes a linker group, wherein the linker group is ⁇ -alanine or located between a C- terminus protective group or capping group and the last amino acid position corresponding to SHARPIN-derived sequence.
  • one or more peptide bonds may be replaced by a different bond that may increase the stability of the polypeptide in the body.
  • Peptide bonds can be replaced by: a retro-inverso bond (C(O)-NFI); a reduced amide bond (NH-CHb); a thiomethylene bond (S-CH? or CFb-S); an oxomethylene bond (O-CH?
  • the inventive polypeptides can be modified by: acetylation, amidation, biotinylation, cinnamoylation, farnesylation, fluoresceination, formylation, myristoylation, palmitoylation, phosphorylation (Ser, Tyr or Thr), stearoylation, succinylation and sulfurylation.
  • the polypeptides of the invention may also be conjugated to, for example, polyethylene glycol (PEG); alkyl groups (e.g., C1-C20 straight or branched alkyl groups); fatty acid radicals; and combinations thereof.
  • amino acids of the inventive polypeptides that are not identified specifically, any non-natural amino acid capable of forming a cross-link that is recited is specifically recited, may be substituted using amino acid substitutions, so long as the polypeptides are capable of inhibiting LUBAC. Such substitutions are preferably not amoung the conserved amino acids of the inventive polypeptides as shown herein. Such substitutions may be conservative substitutions. Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same or similar chemical or physical properties.
  • the conservative amino acid substitution can be an acidic/negatively charged polar amino acid substituted for another acidic/negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic/positively charged polar amino acid substituted for another basic/positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain substituted for another uncharged amino acid with a polar side chain (e.g., Gly, Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid with a beta-branched side-chain substituted for another amino acid with a beta-branched side-chain (e.g., He, Thr, and Val), an amino acid with an aromatic side-chain substituted for another amino acid
  • the invention also provides a substance, the substance comprising a polypeptide, the polypeptide having a sequence consisting essentially of or consisting of ELAGSLARAIA (SEQ ID NO: 78), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAIA (SEQ ID NO: 79), or EKAAAQVAAVLAQ (SEQ ID NO: 4).
  • the invention also provides a polypeptide, the polypeptide consisting essentially of or consisting of the sequence ELAGSLARAIA (SEQ ID NO: 78), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAIA (SEQ ID NO: 79), or EKAAAQVAAVLAQ (SEQ ID NO: 4). Any of these may be isolated, purified, modified, or any combination thereof, as described elsewhere herein.
  • Table 3 below presents certain polypeptide sequences of the invention compared to wild-type SHARPIN sequences.
  • inventive polypeptides can be any suitable length of amino acids.
  • any of the inventive sequences can have an additional 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids on either the N-terminus or C-terminus or both, as long as when cross- linked the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex.
  • any of the inventive polypeptides may be isolated. Any of the inventive polypeptides may be purified.
  • isolated is meant the removal of a substance (e.g., a polypeptide) from its natural environment.
  • purified is meant that a given substance (e.g., a polypeptide), whether one that has been removed from nature (e.g., a protein enzymatically cleaved into polypeptides) or synthesized (e.g., by polypeptide synthesis), has been increased in purity, wherein “purity” is a relative term, not “absolute purity.” It is to be understood, however, that polypeptides may be formulated with diluents or adjuvants and still for practical purposes be isolated.
  • polypeptides can be mixed with an acceptable earner or diluent when used for introduction into cells.
  • inventive polypeptides described herein may be provided in the form of a salt, e.g., a pharmaceutically acceptable salt.
  • Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example, -toluenesulphonic acid.
  • the invention also provides a pharmaceutical composition comprising an effective amount of an inventive polypeptide described herein.
  • a composition e.g., formulated in a pharmaceutically acceptable composition.
  • a composition comprises a carrier (e.g., a pharmaceutically acceptable carrier), such as those known in the art.
  • a pharmaceutically acceptable carrier preferably is chemically inert to the inventive polypeptide and has few or no detrimental side effects or toxicity under the conditions of use. The choice of earner is determined, in part, by the particular method used to administer the composition.
  • Carrier formulations suitable for parenteral, oral, nasal (and otherwise inhaled), topical, and other administrations can be found in Remington 's Pharmaceutical Scie?ices 17 th ed., Mack Publishing Co., Easton, PA (2000), which is incorporated herein in its entirety by reference thereto.
  • Requirements for effective pharmaceutical carriers in parenteral and injectable compositions are well known to those of ordinary skill in the art. See, e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 ( 1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 ( 1986). Accordingly, there is a wide variety of suitable formulations of the composition.
  • the composition can contain suitable buffering agents, including, for example, acetate buffer, citrate buffer, borate buffer, or a phosphate buffer.
  • suitable buffering agents including, for example, acetate buffer, citrate buffer, borate buffer, or a phosphate buffer.
  • suitable preservatives such as benzalkonium chloride, chlorobutanol, parabens, and thimerosal.
  • composition can be presented in unit dosage form and can be prepared by any suitable method, many of which are well known in the art of pharmacy. Such methods include the step of bringing the inventive polypeptide into association with a carrier that constitutes one or more accessory ingredients. In general, the composition is prepared by uniformly and intimately bringing the inventive polypeptide into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • composition can be administered using any suitable method including, but not limited to parenteral, oral, nasal (or otherwise inhaled), and topical administration.
  • Delivery systems useful in the context of the invention include time-released, delayed-release, and sustained-release delivery systems.
  • a composition suitable for parenteral administration conveniently comprises a sterile aqueous preparation of the inventive polypeptide, which may be isotonic with the blood of the recipient.
  • This aqueous preparation can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
  • Sterile powders for sterile injectable solutions can be prepared by vacuum drying and/or freeze-drying to yield a powder of the inventive polypeptide, optionally, in association with a filler or diluent.
  • a composition suitable for oral administration can be formulated in discrete units such as capsules, cachets, tablets, or lozenges, each containing a predetermined amount of the inventive polypeptide as a powder or granules.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine, with the inventive polypeptide being in a free-flowing form, such as a powder or granules, which optionally is mixed with a binder, disintegrant, lubricant, inert diluent, surface inventive polypeptide, or discharging agent.
  • Molded tablets comprised of a mixture of the inventive polypeptide with a suitable carrier may be made by molding in a suitable machine.
  • Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example,
  • proteins, polypeptides, and polypeptides of the invention are mixed with solubilizing agents such a Cremophor, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, or any combination thereof.
  • solubilizing agents such as Cremophor, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, or any combination thereof.
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution, and 1 ,3-butanediol.
  • sterile, fixed oils can be employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • Topical formulations comprise at least one inventive polypeptide dissolved or suspended in one or more media, such as mineral oil, petroleum, polyhydroxy alcohols, or other bases used for topical pharmaceutical formulations.
  • Transdermal formulations may be prepared by incorporating the inventive polypeptide in a thixotropic or gelatinous carrier such as a cellulosic medium, e.g., methyl cellulose or hydroxyethyl cellulose, with the resulting formulation then being packed in a transdermal device adapted to be secured in dermal contact with the skin of a wearer.
  • inventive polypeptide suitable for administration depends on the specific inventive polypeptide used and the particular route of administration.
  • inventive polypeptide can be administered in a dose of about 0.5 ng to about 900 ng (e.g., about 1 ng, 25 ng, 50 ng, 100, ng, 200 ng, 300 ng, 400 ng, 500, ng, 600 ng, 700 ng, 800 ng, or any range bounded by any two of the aforementioned values), in a dose of about 1 ⁇ g to about 900 ⁇ g (e.g., about 1 ⁇ g, 2 g, 5 ⁇ g, 10 ⁇ g, 15 ⁇ g, 20 ⁇ g, 25 ⁇ g, 30 ⁇ g, 40 ⁇ g, 50 ⁇ g, 60 ⁇ g, 70 ⁇ g, 80 ⁇ g, 90 ⁇ g, 100 ⁇ g, 200 ⁇ g, 300 ⁇ g, 400 ⁇ g, 500
  • the invention also provides a method of inhibiting the linear ubiquitin chain assembly complex (LUBAC) in a subject, which method comprises administering an effective amount of an inventive polypeptide described herein or a pharmaceutical composition of an inventive polypeptide described herein to the subject.
  • LUBAC linear ubiquitin chain assembly complex
  • the invention also provides a method of treating activated B- cell like diffuse large B cell lymphoma (ABC DLBCL) in a subject, which method comprises administering an effective amount of an inventive polypeptide described herein or a pharmaceutical composition of an inventive polypeptide described herein to the subject.
  • ABSC DLBCL activated B- cell like diffuse large B cell lymphoma
  • the invention provides a method of killing ABC DLBCL that comprises administering a therapeutically effective amount of an inventive polypeptide described herein to ABC DLBCL and thereby killing (or inhibiting proliferation) of ABC DLBCL.
  • the method can include administering a therapeutically effective amount of the inventive polypeptide to ABC DLBCL in vitro or in a subject (i.e., in vivo).
  • the inventive polypeptide can be administered to ABC DLBCL that is in, for example, a primary cell culture or an animal model of ABC DLBCL.
  • the invention provides a method of treating a subject that is suffering from ABC DLBCL or at risk for ABC DLBCL.
  • the method can include administering a pharmaceutical composition comprising a therapeutically effective amount of one or more inventive polypeptides described herein to the subject.
  • a subject that is suffering from or at risk for ABC DLBCL can be a subject diagnosed with ABC DLBCL, a subject undergoing treatment for ABC DLBCL, a subject suspected to have ABC DLBCL, or a subject at risk for having ABC DLBCL (for example, a subject at risk for recurrence of ABC DLBCL).
  • the methods of using the inventive polypeptides as described herein can each further include the co-administration a second therapeutic agent.
  • the method of killing ABC DLBCL or the method of treating ABC DLBCL by administration of an inventive polypeptide can further include the co-administration of a cytotoxic, cystostatic, or antiangiogenic agent suitable for use against DLBCL.
  • Such a method can include, for example, the co-administration of SAH-RNF31 -N, polypeptides described in US
  • the invention provides, for example, methods that include co-administration of an inventive polypeptide described herein and one or more cytotoxic agents used in CHOP, EPOCH, R-CHOP, therapeutic regimens.
  • cytotoxic agents include cyclophosphamide, doxorubicin, vincristine, prednisone, etoposide, and derivatives thereof.
  • the invention also provides a method of treating rheumatoid arthritis in a subject, which method comprises administering an effective amount of an inventive polypeptide described herein or a pharmaceutical composition of an inventive polypeptide described herein to the subject.
  • the invention also provides a method of treating cancer that is resistant to cytotoxic chemotherapy, radiation therapy, vaccine therapy, or cytokine therapy in a subject, which method comprises administering an effective amount of an inventive polypeptide described herein or a pharmaceutical composition of an inventive polypeptide described herein to the subject.
  • the invention also provides a method of sensitizing ABC DLBCL to cytotoxic agents.
  • the method includes administering an inventive polypeptide described herein to ABC DLBCL and thereby reducing or inactivating resistance to cytotoxic therapy in the ABC DLBCL.
  • the invention provides a method of treating a disease or pathological condition mediated by LUBAC activity.
  • the invention provides a method of treating a subject with cancer that is resistant to cytotoxic
  • This method of the invention comprises administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an inventive polypeptide described herein.
  • the method further includes co-administering a second therapeutic agent which is an agent for cytotoxic chemotherapy, radiation therapy, cytokine therapy, or vaccine therapy.
  • the invention also provides a method for screening whether a subject suffering from cancer that is resistant to cytotoxic chemotherapy, radiation therapy, or cytokine therapy is a candidate for treatment with a LUBAC inhibitor.
  • the method includes obtaining a sample (biopsy) from the subject that includes cancer cells, then administering to the sample an inventive polypeptide as described herein, and co-administering to the sample one or more second therapeutic agents for cytotoxic chemotherapy, radiation therapy, or cytokine therapy.
  • the cancer cells in the sample are assayed for viability before the inventive polypeptide and the second therapeutic agent(s) can exert any cytotoxic effect (e.g., just before administration of the inventive polypeptide and second therapeutic agent(s)).
  • the cells are subsequently assayed for viability at one or more times after the co-administration of the inventive polypeptide and the second therapeutic agent(s) to determine whether there is a significant decrease in the number of viable cancer cells. Such a decrease indicates that the subject is a candidate for treatment with a LUBAC inhibitor.
  • the method of screening can further include treating a second control sample from the subject.
  • An equivalent dose or amount of the second therapeutic agent(s), without inventive polypeptide, is administered to the control sample. Cancer cells in the sample are assayed for viability before the second therapeutic agent(s) can exert any effect and after administration of the second therapeutic agent(s). If the number of viable cells remaining in the control sample is greater than the number of viable cells in the sample treated by co-administration with an inventive polypeptide and the second therapeutic agent, then the inventive polypeptide has sensitized the cancer cells to the cytotoxic chemotherapy, radiation therapy, or cytokine therapy, and the subject is a candidate for treatment with a LUBAC inhibitor.
  • the invention also provides a method of treating chronic autoinflammation, systemic lupus erythematosus, Crohn's inflammatory bowel disease, or psoriasis in a subject, which method comprises administering an effective amount of an inventive polypeptide described herein or a pharmaceutical composition of an inventive polypeptide described herein to the subject.
  • the invention provides a method of treating a condition associated with a dysregulated inflammatory response, such as rheumatoid arthritis, chronic autoinflammation, systemic lupus erythematosus, Crohn's inflammatory bowel disease, and psoriasis.
  • the method can include administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an inventive polypeptide described herein.
  • the inventive method can provide any amount of any level of treatment.
  • the treatment provided by the inventive method can include the treatment of one or more conditions or symptoms of the disease being treated.
  • the terms “treat,” “treating,” “treatment,” “therapeutically effective,” “inhibit,” etc. used herein do not necessarily imply 100% or complete treatment/inhibition/reduction. Rather, there are varying degrees, which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect.
  • the inventive polypeptides and methods can provide any amount of any level of treatment/inhibition/reduction.
  • the treatment provided by the inventive method can include the treatment of one or more conditions or symptoms of the disease being treated.
  • Inhibiting the linear ubiquitin chain assembly complex (LUBAC) means at least a 20% reduction in LUBAC activity as assayed using the in vitro ubiquitin assay as described in the Example.
  • co-administering refers to the administration of an inventive polypeptide described herein and one or more additional therapeutic agents sufficiently close in time to (i) enhance the effectiveness of the inventive polypeptide or the one or more additional therapeutic agents and/or (ii) reduce an undesirable side effect of the inventive polypeptide or the one or more additional therapeutic agents.
  • inventive polypeptide can be administered first, and the one or more additional therapeutic agents can be administered second, or vice versa.
  • inventive polypeptide and the one or more additional therapeutic agents can be co-administered simultaneously.
  • subject is used herein, for example, in connection with therapeutic and screening methods, to refer to human or animal subjects (e.g., mammals).
  • Animal subjects include, but are not limited to, animal models, such as, mammalian models of conditions or disorders associated dysregulated LUBAC-signaling.
  • the subject can be an animal model of ABC DLBCL, or a cancer that is resistant to cytotoxic chemotherapy, radiation therapy, or cytokine therapy.
  • a subject can also be an animal model of an autoimmune disorder associated with dysregulated innate immune response.
  • a subject can be a human patient suffering from or at risk for (i) ABC DLBCL, (ii) a cancer that is resistant to cytotoxic chemotherapy, radiation therapy, or cytokine therapy, or (iii) an autoimmune disorder associated with dysregulated innate immune response.
  • kits suitable for carrying out the methods of the invention typically comprises two or more components required for performing a therapeutic or screening method of the invention.
  • Kit components include, but are not limited to, one or more inventive polypeptides of the invention, appropriate reagents, and/or equipment.
  • a kit can comprise one or more inventive polypeptides of the invention and a second therapeutic agent, e.g., a cytotoxic, cystostatic, or antiangiogenic agent.
  • the kit includes inventive polypeptide of the invention suitably packaged, e.g., in a vial, pouch, ampoule, and/or any container appropriate for a therapeutic or screening method.
  • Kit components can be provided as concentrates (including lyophilized compositions), which may be further diluted prior to use, or the kit components can be provided at the
  • concentration intended for use When a polypeptide of the invention is intended to be used in vivo, single dosages may be provided in sterilized containers having the desired amount and concentration of agents.
  • the invention provides a method for identifying and optimizing LUBAC inhibitors.
  • the method includes binding an inventive polypeptide described herein to the HOIL- 1 L UBL domain and evaluating the ability of a test compound to disrupt or inhibit the binding reaction.
  • the UBL domain can be provided in context of the entire HOIL-1L protein or as a fragment thereof that includes the portion of the UBL that binds to the LUBAC inhibitor of the invention.
  • the method can include tagging an inventive polypeptide described herein (or tagging the UBL domain) to a fluorescent moiety.
  • inventive polypeptide or the UBL domain can be fixed (e.g., immobilized or covalently bound) to a substrate and contacted to its fluorescently tagged partner to thereby create a substrate -bound dimer.
  • the amount of fluorescently tagged polypeptide or UBL domain that binds to substrate can be determined, e.g., by detecting the strength of signal from the fluorescent moiety that is bound to the substrate. A fluorescence binding curve can be generated.
  • a test compound can be added to the reaction and the ability of the test compound to disrupt or inhibit the fluorescently tagged polypeptide or UBL domain from forming a substrate-bound dimer can be measured, for example, by detecting a reduction in the signal produced by the fluorescent moiety that is bound to the substrate.
  • a test compound that significantly reduces the fluorescence signal is a candidate inhibitor or a candidate optimized inhibitor of LUBAC.
  • a fluorescent moiety can be attached to either the polypeptide of the invention or the UBL domain, and a quencher moiety can be attached to the other.
  • a baseline signal of the fluorescent moiety is measured.
  • a test compound can be added and the ability of the test compound to disrupt or inhibit the binding reaction of the polypeptide inhibitor and the UBL domain can be measured, for example, by detecting an increase in the signal produced by the fluorescent moiety.
  • a test compound that significantly increases the fluorescence signal in this assay is a candidate inhibitor or a candidate optimized inhibitor of LUBAC.
  • Other techniques for detecting the ability of test compounds to disrupt protein binding can be used. These include, for example, surface plasmon resonance (SPR) binding assays, co-immunoprecipitation, affinity chromatography, and the like.
  • SPR surface plasmon resonance
  • test compound can be a small molecule compound.
  • test compound can be any polypeptide of the invention which has been modified to include one, two, three, or four substitutions, deletions, or insertions.
  • non-natural amino acids that form a covalent cross-link with each other, wherein the non-natural amino acids are the same or are different, and wherein the cross-link is internal to the polypeptide, and
  • the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine; or the polypeptide has from the N- to C-terminus a glutamine one amino acid position before a first valine, and optionally
  • polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
  • LUBAC linear ubiquitin chain assembly complex
  • polypeptide of aspect 1 wherein the polypeptide has from the N- to C- terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine, and
  • cross-link is formed from a non-natural amino acid at position i within the polypeptide and another non-natural amino acid at position z + 3, + 4, or i + 7 within the polypeptide; and wherein the amino acid at position i + 6 within the polypeptide is the leucine, the amino acid at position i + 9 within the polypeptide is the alanine, and the amino acid at position i + 10 within the polypeptide is the isoleucine.
  • polypeptide of aspect 1 wherein the polypeptide has from the N- to C- terminus (1) a glutamine one amino acid position before a first valine, and (2) optionally
  • cross-link is formed from a non-natural amino acid at position i within the polypeptide and another non-natural amino acid at position i + 3, i + 4, or i + 7 within the polypeptide;
  • amino acid at position / ' + 1 within the polypeptide is the glutamine, and the amino acid at position i + 2 within the polypeptide is the first valine.
  • polypeptide of aspect 2 or 3, wherein the cross-link of the polypeptide is: formed from the amino acid at position i within the polypeptide and another amino acid at position i + 4 within the polypeptide, and the amino acid at position i is (5)-2-(4'- pentenyl)alanine (S5) and the amino acid at position i + 4 is S5; or
  • amino acid at position i is (R)-2- (4'-pentenyl)alanine (R5) or (i?)-2-(2'-propenyl)alanine (R3) and the amino acid at position i + 3 is S5; or
  • polypeptide of aspect 1 wherein the polypeptide has from the N- to C- terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine, wherein the polypeptide is a first polypeptide, and wherein the polypeptide further comprises a second polypeptide having non-natural amino acids that form a covalent cross-link internally within the second polypeptide, wherein the second polypeptide has from the N- to C-terminus (1 ) a glutamine one amino acid position before a first valine, and (2) optionally
  • the cross-link in the first polypeptide is formed from a non-natural amino acid at position i within the first polypeptide and another non-natural amino acid at position i + 3, i + 4, or i + 7 within the first polypeptide;
  • amino acid at position i + 6 within the first polypeptide is the leucine
  • amino acid at position i + 9 within the first polypeptide is the alanine
  • amino acid at position / + 10 within the first polypeptide is the isoleucine
  • the cross-link in the second polypeptide is formed from a non-natural amino acid at position i within the second polypeptide and another non-natural amino acid at position i + 3, i + 4, or i + 7 within the second polypeptide;
  • amino acid at position i + 1 within the second polypeptide is the glutamine, and the amino acid at position i + 2 within the second polypeptide is the first valine.
  • amino acid at position i is (S)-2-(4 ? -pentenyl)alanine (S5) and the amino acid at position + 4 is S5; or
  • amino acid at position i within the first polypeptide formed from the amino acid at position i within the first polypeptide and another ammo acid at position i + 3 within the first polypeptide, and the amino acid at position is (7?)-2-(4'-pentenyl)alanine (R5) or (i?)-2-(2'-propenyl)alanine (R3) and the amino acid at position + 4 is S5; or
  • amino acid at position i within the second polypeptide formed from the amino acid at position i within the second polypeptide and another amino acid at position i + 4 within the second polypeptide, and the amino acid at position is (S)-2-(4'-pentenyl)alanine (S5) and the amino acid at position / ' + 4 is S5; or
  • amino acid at position i within the second polypeptide and another ammo acid at position / ' + 3 within the second polypeptide, and the amino acid at position is (R)-2-(4'-pentenyl)alanine (R5) or (R)-2-(2'-propenyl)alanine (R3) and the amino acid at position ; ' + 4 is S5; or
  • amino acid at position i is (R)-2-(7'-octenyl)alanine (R8) and the amino acid at position + 7 is S5.
  • the modification of the sequence is of at least two of the amino acids within the sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids;
  • non-natural amino acids form a covalent cross-link with each other, wherein the covalent cross-link is internal to the sequence
  • polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
  • LUBAC linear ubiquitin chain assembly complex
  • polypeptide of aspect 8 or 9, wherein two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5.
  • polypeptide further comprises a second modified amino acid sequence of EKGAAQVAAVLAQ (SEQ ID NO: 2);
  • the modification of the second sequence is of at least two of the amino acids within the second sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids;
  • polypeptide further comprises a second modified amino acid sequence of EKAAAQVAAVLAQ (SEQ ID NO: 4);
  • the modification of the second sequence is of at least two of the amino acids within the second sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids;
  • non-natural amino acids form a covalent cross-link with each other, wherein the covalent cross-link is internal to the second sequence.
  • non-natural amino acids capable of forming an internal cross-link, wherein the non-natural amino acids are the same or are different
  • each of the non-natural amino acids includes a moiety, wherein the moieties are capable of undergoing a reaction to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the polypeptide;
  • the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine; or the polypeptide has from the N- to C-terminus (1) a glutamine one amino acid position before a first valine, and (2) optionally
  • polypeptide (b) wherein if the non-natural amino acids form an internal cross-link, the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
  • LUBAC linear ubiquitin chain assembly complex
  • non-natural amino acids capable of being cross-linked are at position i within the polypeptide and at position i + 3, i + 4, or i + 7 within the polypeptide;
  • amino acid at position i + 6 within the polypeptide is the leucine
  • amino acid at position i + 9 within the polypeptide is the alanine
  • amino acid at position i + 10 within the polypeptide is the isoleucine
  • polypeptide of aspect 15 wherein the polypeptide has from the N- to C- terminus (1) a glutamine one amino acid position before a first valine, and (2) optionally
  • amino acid at position i + 1 within the polypeptide is the glutamine, and the amino acid at position i + 2 within the polypeptide is the first valine.
  • polypeptide of aspect 16 or 17, wherein the amino acids capable of being cross-linked are:
  • amino acid at position i is (S)-2-(4'-pentenyl)alanine (S5) and the amino acid at position i + 4 is S5; or
  • amino acid at position i within the polypeptide and another amino acid at position i + 3 within the polypeptide is (R)-2-(4'-pentenyl)alanine (R5) or ( ?)-2-(2'- propenyl)alanine (R3) and the amino acid at position i + 3 is S5; or
  • amino acids capable of being cross-linked are at position i within the polypeptide and at position i + 7 within the polypeptide, and the amino acid at position i is (K)-2-(T- octenyl)alanine (R8) and the amino acid at position i + 7 is S5.
  • polypeptide of aspect 15 wherein the polypeptide has from the N- to C- terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine, wherein the polypeptide is a first polypeptide, and wherein the polypeptide further comprises a second polypeptide comprising at least two non-natural amino acids capable of forming an internal cross-link, wherein the non- natural amino acids are the same or are different, wherein each of the non-natural amino acids includes a moiety, wherein the moieties are capable of undergoing a reaction to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the second polypeptide,
  • the second polypeptide has from the N- to C-terminus (1) a glutamine one amino acid position before a first valine, and (2) optionally
  • the non-natural amino acids capable of being cross-linked are at position i within the first polypeptide and at position + 3, i + 4, or / ' + 7 within the first polypeptide;
  • amino acid at position i + 6 within the first polypeptide is the leucine
  • amino acid at position i + 9 within the first polypeptide is the alanine
  • amino acid at position i + 10 within the first polypeptide is the isoleucine
  • the non-natural amino acids capable of being cross-linked are at position i within the second polypeptide and at position i + 3, i + 4, or i + 7 within the second polypeptide;
  • amino acid at position i + 1 within the second polypeptide is the glutamine, and the amino acid at position + 2 within the second polypeptide is the first valine.
  • amino acid at position i is (5)-2-(4'-pentenyl)alanine (S5) and the amino acid at position i + 4 is S5; or
  • amino acid at position i is (S)-2-(4'-pentenyl)alanine (S5) and the amino acid at position i + 4 is S5; or
  • amino acid at position i within the second polypeptide and another amino acid at position i + 3 within the second polypeptide, and the amino acid at position i is (R)-2-(4'-pentenyl)alanine (R5) or (R)-2-(2'-propenyl)alanine (R3) and the amino acid at position i + 3 is S5; or
  • the modification of the sequence is of at least two of the amino acids within the sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids;
  • each of the non-natural amino acids include a moiety, wherein each moiety is capable of undergoing a reaction with a moiety of one other of the non-natural amino acids to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the sequence;
  • the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
  • LUBAC linear ubiquitin chain assembly complex
  • polypeptide of aspect 22 wherein the modified amino acid sequence is of EELAGSLARAI (SEQ ID NO: 1 ), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second modified amino acid sequence of EKGAAQVAAVLAQ (SEQ ID NO: 2);
  • the modification of the second sequence is of at least two of the amino acids within the second sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids;
  • each of the non-natural amino acids include a moiety, wherein each moiety is capable of undergoing a reaction with a moiety of one other of the non-natural amino acids to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the second sequence.
  • polypeptide further comprises a second modified amino acid sequence of EKAAAQVAAVLAQ (SEQ ID NO: 4);
  • the modification of the second sequence is of at least two of the amino acids within the second sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids;
  • each of the non-natural amino acids include a moiety, wherein each moiety is capable of undergoing a reaction with a moiety of one other of the non-natural amino acids to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the second sequence.
  • Ri and R 2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
  • R3 is alkylene, alkenylene, alkynylene, or each of which is substituted with 0-6 R 5 ;
  • R 4 and R4' are independently alkylene, alkenylene, or alkynylene;
  • R 5 is independently halo, alkyl, OR 6 , N(R 6 ) 2 , SR 6 , SOR 6 , S0 2 R 6 , CO?R 6 , Re, a fluorescent moiety, or a radioisotope;
  • K' is independently O, S, SO, S0 2 , CO, C0 2 , CONR 6 , or
  • R6 is independently H, alkyl, or a therapeutic agent
  • n is independently an integer from 1 -4;
  • [Xaa]x has 2 to 6 amino acids
  • EELAGSLARAI SEQ ID NO: 1
  • EKGAAQVAAVLAQ SEQ ID NO: 2
  • EELATRLSQAl SEQ ID NO: 3
  • EKAAAQVAAVLAQ SEQ ID NO: 4
  • polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
  • LUBAC linear ubiquitin chain assembly complex
  • Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
  • R3 is alkylene, alkenylene, alkynylene, or [R4-K'-R 4' ] n , each of which is substituted with 0-6 R 5 ;
  • R4 and R 4 ' are independently alkylene, alkenylene, or alkynylene;
  • R 5 is independently halo, alkyl, OR 6 , N(R 6 ) 2 , SR 6 , SOR 6 , SO2R6, C0 2 R 6 , 3 ⁇ 4 > , a fluorescent moiety, or a radioisotope;
  • K' is independently O, S, SO, SO2, CO, CO2, CONR 6 , or
  • R6 is independently H, alkyl, or a therapeutic agent
  • n is independently an integer from 1-4;
  • [Xaa]x has 2 to 6 amino acids
  • EKGAAQVAAVLAQ (SEQ ID NO: 2), wherein two of the amino acids within the sequence are replaced with the residues A and B.
  • polypeptide further comprises a second amino acid sequence modified according to the formula (la): A B
  • Ri and R 2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
  • R3 is alkylene, alkenylene, alkynylene, or [R4-K'-R4 ⁇ ] n , each of which is substituted with 0-6 R 5 ;
  • R4 and R4' are independently alkylene, alkenylene, or alkynylene;
  • R 5 is independently halo, alkyl, OR 6 , N(R 6 ) 2 , SR 6 , SOR 6 , S0 2 R 6 , C0 2 R 6 , R & , a fluorescent moiety, or a radioisotope;
  • K' is independently O, S, SO, S0 2 , CO, CO2, CONR 6 , or
  • R6 is independently H, alkyl, or a therapeutic agent
  • n is independently an integer from 1 -4;
  • [Xaa]x has 2 to 6 amino acids
  • EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the amino acids within the sequence are replaced with the residues A and B.
  • Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
  • R3 and R 3 ' are independently alkenyl, alkynyl, azido, amino, carboxylic acid, or sulfide;
  • [Xaa] x has 2 to 6 amino acids
  • EELAGSLARAI SEQ ID NO: 1
  • EKGAAQVAAVLAQ SEQ ID NO: 2
  • EELATRLSQAI SEQ ID NO: 3
  • EKAAAQVAAVLAQ SEQ ID NO: 4
  • polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
  • LUBAC linear ubiquitin chain assembly complex
  • polypeptide further comprises a second amino acid sequence modified according to the formula (Ila):
  • Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
  • R3 and R3' are independently alkenyl, alkynyl, azido, amino, carboxylic acid, or sulfide;
  • [Xaa]x has 2 to 6 amino acids
  • EKGAAQVAAVLAQ (SEQ ID NO: 2), wherein two of the amino acids within the sequence are replaced with the residues A and B.
  • polypeptide further comprises a second amino acid sequence modified according to the formula (Ila): A B
  • Ri and R 2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
  • R3 and R3' are independently alkenyl, alkynyl, azido, amino, carboxylic acid, or sulfide;
  • [Xaa] x has 2 to 6 amino acids
  • EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the amino acids within the sequence are replaced with the residues A and B.
  • a polypeptide comprising the amino acid sequence of any one of SEQ ID NOS: 8-21 , 23-41 , 43-57, or 59-77.
  • polypeptide of aspect 41 wherein the polypeptide includes a linker group, wherein the linker group is beta-alanine.
  • a pharmaceutical composition comprising an effective amount of a polypeptide of any one of aspects 1-45.
  • a method of inhibiting the linear ubiquitin chain assembly complex (LUBAC) in a subject comprises administering an effective amount of a polypeptide of any one of aspects 1-45 or a pharmaceutical composition of aspect 46 to the subject.
  • LUBAC linear ubiquitin chain assembly complex
  • a method of treating activated B-cell like diffuse large B cell lymphoma (ABC DLBCL) in a subject comprises administering an effective amount of a polypeptide of any one of aspects 1-45 or a pharmaceutical composition of aspect 46 to the subject.
  • a method of treating rheumatoid arthritis in a subject which method comprises administering an effective amount of a polypeptide of any one of aspects 1-45 or a pharmaceutical composition of aspect 46 to the subject.
  • a method of treating cancer that is resistant to cytotoxic chemotherapy, radiation therapy, vaccine therapy, or cytokine therapy in a subject comprises administering an effective amount of a polypeptide of any one of aspects 1-45 or a pharmaceutical composition of aspect 46 to the subject.
  • erythematosus erythematosus, Crohn's inflammatory bowel disease, or psoriasis in a subject
  • method comprises administering an effective amount of a polypeptide of any one of aspects 1 -45 or a pharmaceutical composition of aspect 46 to the subject.
  • the LUBAC ubiquitin ligase which is composed of three subunits, the catalytic HOIP and two accessory HOIL-IL and SHARPIN subunits, is important in various biological phenomena such as NF- ⁇ activation and cell death protection (Iwai et al., Nat Rev Mol Cell Biol, 15: 503-508 (2014)).
  • NF- ⁇ activation and cell death protection Iwai et al., Nat Rev Mol Cell Biol, 15: 503-508 (2014).
  • structural modeling based on the reported structures of homologous proteins and mutational analyses, it is shown there are important roles for the three interactions between each subunit of LUBAC in stabilization of the complex. It was also found that mHOIP and mSHARPIN cannot form the stable complex, which indicated the important role of mHOIL-lL in formation of the mouse LUBAC complex.
  • HOIL-IL""" 7 "” mice were established, which has the loss of function mutation in the exon encoding the UBL, and it was found that HOIL-IL”"" "”” mice are embryonic lethal and exhibit phenotypes almost identical to mice lacking LUBAC ligase activity although HOIL-I L "7” mice established previously do not exhibit any overt phenotypes.
  • Small N-terminal truncated alternative splicing product of HOIL-IL (RBCK2-like) that contains intact Not and UBL exist in the previously described HOIL-I L 7" , but not HOIL-I L"""”” mice.
  • HOIL- 1 L deficiency has been separately reported in humans: one report showed that patients exhibit immunodeficiency and autoinfiammation and polyglucosan body myopathy in skeletal muscle and heart (Boisson et al., Nat Immunol, 13: 1 178-1 186 (2012)), whereas patients in other reports showed polyglucosan body myopathy without having immunological symptoms (Nilsson et al., Ann Neurol, 74: 2246-2257 (2013)). Most of the genomic mutations found in patients who exhibit only polyglucosan body myopathy seem not to affect expression of RBCK2-like product (Nilsson et al., Ann Neurol, 74: 2246-2257 (2013)).
  • Presence of the RBCK2-like product, corresponding mRNA of which is reported to exist in human, may determine the phenotypical difference of patients with HOIL-I L mutations.
  • the A 1 8P mutation in the Na-region of HOIL-I L impaired HOIL-1 L-SHARP1N interaction likely because the A18P mutation may disrupt a-helix of the Na region as it is known that proline breaks a-helix.
  • disruption of SHARPIN-HOIL-1L interaction profoundly destabilized LUBAC, tiny amount of trimeric LUBAC may still exist because HOIL-I L A18P could bind to HOIP.
  • SHARPIN can stabilize HOIP in human, but not in mouse.
  • trace amounts of LUBAC composed of SHARPIN and HOIP might exist in immunodeficiency and autoinflamrnation patients.
  • SHARPIN could barely be detected in MEFs of HOIL-IL""'"""” but not HOIL-IL 7" mice.
  • HOIL-IL can dimerize with SHARPIN via the Na region in the absence of HOIP
  • a LUBAC-independent function of SHARPIN as indicated by several previous reports (Pouwels et al., Cell Rep, 5: 619-628 (2013); Rantala et al., Nat Cell Biol, 13 : 13 15-1324 (201 1)) might be exerted by the HOIL- 1 L-SHARPIN complex.
  • HOIL-1 which is a shorter alternative product of HOIL-I L, lacks part of Na and the spacer region between Na and UBL (Yamanaka et al., Nat Cell Biol, 5: 336-340 (2003)).
  • HOIL-1 is resistant to incorporation into the LUBAC complex (Kirisako et al., EMBO J, 25: 4877-4887 (2006)), possibly because HOIL-1 cannot bind to SHARPIN on account of the lack of these regions. It might be plausible that HOIL-1 exert LUBAC-independent functions (Donley et al, Oncogene, 33 : 3441 -3450 (2014);
  • Loss of LUBAC ligase activity is embryonic lethal (Shimizu et al, Mol Cell Biol, 36: 1569-1583 (2016)), however, the LUBAC ligase activity cannot be completely lost by inhibition of SHARPIN-HOIL-IL interaction because the inhibition does not affect the other interactions between the LUBAC components and LUBAC composed of HOIL-1L-HOIP or SHARPIN-HOIP can exist in human cells.
  • cDNA used in this study were described previously (Fujita et al., 2014; Tokunaga et al., Nature, 471 : 633-636 (201 1 ); Tokunaga et al, Nat Cell Biol, 1 1 : 123-132 (2009)).
  • the following full-length proteins, deletion mutants, and fragments were generated from the amplified ORF of mouse HOIP; wild type (WT) (amino acids 1-1066), UBA domain (466- 630), all ZF domains (296-434), ANZF2 ( ⁇ 402-432), AUBA ( ⁇ 558-609), AUBA N-term ( ⁇ 466-549), AUBA C-term ( ⁇ 550-630).
  • hUBA mutant [mHOIP ( 1-473)- hHOIP (480-636)- mHOIP (631 -1066)] was generated from the amplified ORF of human and mouse HOIP.
  • the following proteins were generated from the amplified ORF of mouse HOIL-1L; before NZF (1 -189), UBL domain (1 -140), ⁇ (37-509), UBL ⁇ (37-161 ).
  • the following proteins were generated from the amplified ORF of mouse SHARPIN; ⁇ ( ⁇ 163-197), UBL ⁇ (198-318), mutants of mHOIL-l L and mSHARPIN whose UBL domains are exchanged; S(UBL)-HOIL [SHARPIN ( 163-301 )-HOIL- 1 L (136-509)], S-H(UBL)-S [SHARPIN (1 - 167)-H0IL-1 L (7-135)-SHARPIN (302-380)].
  • Mutants of mHOIP (Q607A/L61 1 A/F614A, M528A/L559A, R479A/Q490A), mHOIL- 1 L (LI 5A, V19A, L 15A/V19A, A18P), mSHARPIN (Y295A, L273A, V268A, L176A, I 180A, L176A/I 180A), hHOIL-l L A18P were generated by two-step PCR.
  • cDNAs were ligated into the appropriate epitope-tag sequences and then cloned into pcDNA3.1, pMAL-c2x (New England Biolabs, Ipswich, MA, USA), pGEX-6pl (GE Healthcare, Little Chalfont, United Kingdom), pMXs-IP, pMXs-neo, pMXs-IRES-Bsr.
  • pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene (Cambridge, MA, USA) plasmid #42230) (Cong et al, Science, 339: 819-823 (2013)) and pSpCas9(BB)-2A-Puro (PX459) (Addgene plasmid #48139) (Cong and Zhang, Methods Mol Biol, 1239: 197-217 (2015)) were gifts from Dr. Feng Zhang.
  • RT-PCR of HOIL-1L +/+ or -/- MEFs were performed using sequence specific primers as follows:
  • Exon4_Fwd 5 ' -GGAATGGAGACGGTGCCTATCTC-3 ' (SEQ ID NO: 81);
  • p-actin_Fwd 5 ' -ATGGATGACGATATCGCTC-3 ' (SEQ ID NO: 85);
  • SHARPIN (ab 125188) (Abeam, Cambridge, United Kingdom); HOIP (ARP43241_P050) (A viva Systems Biology Corp, San Diego, CA, USA); ⁇ (c-21) (Santa Cruz
  • strep-tagged mouse HOIL-1L N-terminus (1 -189) was expressed in Escherichia coli (E. Coli) and then purified using Strep-Tactin Sepharose (IBA). Purified protein was used to immunize rabbits and IgG was purified from their antisera by using Protein A Sepharose (GE).
  • mice chronic dermatitis in mice (cpdm) MEFs were electroporated with pX330 plasmids containing gRNA sequence against mHOIP or mHOIL-lL using NEPA21 electroporator (NEPAGENE, Chiba, Japan). After four days of culture, cells were seeded at a low density. Colonies were picked up and expression level of LUBAC was analyzed by immunoblotting as first screening.
  • genomic regions of HOIL-1L or HOIP were amplified by PCR using the following primers: mHOIL-lL typingJFwd, 5 ' -TTGCC AAC AGGCC AATTTGATG-3 ' (SEQ ID NO: 90) and typing__Rev, 5 ' -TGCGGTGATGC ACAATATCCTG-3 ' (SEQ ID NO: 91).
  • HEK293T HOIP KO cells were transfected with pX459 plasmid containing gRNA sequence of hHOIP by Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA). The following day, cells were selected with puromycin for two days. Then, cells were seeded at a low density and isolated colonies were picked up.
  • expression level of HOIP was analyzed by immunoblotting and genome loci of HOIP amplified by PCR using the following primers:
  • hHOIP typing Fwd 5'- TTCCGGGCAGGCGTTTTCCCTG-3 ' (SEQ ID NO: 94) and typingJRev, 5 ' -CTCTGTGTAGCC ATATAATCG-3 ' (SEQ ID NO: 95) were analyzed by sequence.
  • Fertilized oocytes were microinjected with pX330 containing guide RNA sequence against HOIL-1L (Fig.12). Progeny was genotyped using the following primers: typing_Fwd, 5 ' -TTGCC AACAGGCCAATTTGATG -3' (SEQ ID NO: 90) and typing_Rev, 5' -TGCGGTGATGC ACAATATCCTG-3 ' (SEQ ID NO: 91 ).
  • samples were blocked in 1 % BSA in PBS for 1 hour at room temperature and incubated with anti-CD31 primary antibody diluted in the same blocking buffer at 1 : 100 dilution for 2 days at 4 ° C.
  • Samples were washed with PBS-T for 30 min at 4°C for three times followed by extensive washing at room temperature for 30 min for two times, and then incubated with the Alexa 546-conjugated secondary antibody in the same blocking buffer at 1 : 1000 dilution overnight at 4°C.
  • HEK293T cells MEFs derived from wild-type, HOIL- 1 L-/- or null mice, HEK293T HOIP KO cells and LUBAC TKO MEFs were grown in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum (FBS) with 100 IU/ml of penicillin and 100 ⁇ g/ml of streptomycin. Trans fections were performed using Lipofectamine2000.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • pMXs-IP, pMXs-neo or pMXs-IRES-Bsr containing the LUBAC components were transfected into Plat E packaging cells as described previously (Tokunaga et al, Nature, 471 : 633-636 (201 1)).
  • the resultant viruses were used to infect LUBAC TKO cells or HOIL-1L null MEFs and the stable cells were selected using puromycin, G-418 or Blasticidin.
  • Cells were lysed with lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 % Triton X-100, 2 mM PMSF and protease inhibitor cocktail (Sigma-Aldrich) and lysates were clarified by centrifugation at 15,000 rpm for 20 min at 4°C.
  • Tris-HCl pH 7.5
  • 150 mM NaCl 1 % Triton X-100
  • PMSF protease inhibitor cocktail
  • EMSAs for NF- ⁇ activity were performed using Odyssey Infrared EMSA kit (LI-COR Biosciences, Lincoln, NE, USA) and IRDye 700 NF-KB consensus oligonucleotide (LI-COR Biosciences), and visualized using a Odyssey 9120 Infrared Imaging System (LI-COR Biosciences).
  • RTCA real-time cellular analysis
  • CellTiter-Glo Luminescent Cell Viability Assay kit
  • GST-HOIP UBA (466-630 aa) WT or UBA2 mut (Q607A/L61 1 A/F614A) and MBP-HOIL-1 L UBL (1-140, 37-161 , 1 -189 aa), MBP-SHARPIN UBL (163-301 , 198-318, 163-340 aa) were expressed in ii.coli and purified by using Glutathione Sepharose 4FF (GE Healthcare) or Amylose Resin (BioLabs), respectively. Binding affinities between the UBA and the UBLs were measured by using BIACORE3000 (GE Healthcare).
  • GST-HOIP UBA was immobilized on sensor chip CM5 via anti-GST antibody using GST capture kit (GE Healthcare) in l OmM HEPES buffer (pH 7.4) containing 150 mM NaCl and 0.05% (v/v) surfactant P20 at 25°C. Binding between GST-HOIP UBA and MBP-UBLs was measured in l OmM HEPES buffer (pH 7.4) containing 150 mM NaCl and 0.05% (v/v) surfactant P20 at 25°C.
  • the dissociation constants ( d) of HOIL-IL UBL- HOIP UBA or SHARPIN UBL- HOIP UBA were calculated using steady-state affinity analysis.
  • S100 lysates of Jurkat HOIP KO cells described previously were prepared as follows. Cells were lysed with buffer containing 10 mM Tris-HCl pH7.5, 10 mM KC1, 1.5 mM MgCb, 0.5 mM DTT, 2 mM PMSF, 50 ⁇ g/ml leupeptin, 10 ⁇ g/ml aprotinin.
  • Lysates were centrifuged at 15,000 rpm, 4°C for 15 min and 0.1 1 volume of buffer containing 0.3 mM Tris-HCl pH7.5, 1.4 M KC1, 30 mM MgCk was added to supernatant. Then, centrifuged at 100,000 g, 4°C for 1 hour.
  • SHARPIN UBL (216-302) was homology-modeled by Phyre (or Swiss-Model) program (Kelley et al., Nat Protoc, 10: 845-858 (2015); Schwede et al., Nucleic Acids Res, 31 : 3381 - 3385 (2003)).
  • Phyre or Swiss-Model
  • Created SHARPIN UBL model was superposed onto the HOIL-1 L molecule located at "contact2 " surface of HOIP-UBA structure (PDB: 4DBG).
  • HOIL-SHARPIN N-terminal helical model Initial monomer model of HOIL-1L (2-38) and SHARPIN (173-202) were calculated by swiss-model. Since both HOIL-1L and SHARPIN have sequence similarity to structures in PDB:3ERM, it was chosen as template. Particularly, in 3ERM structure, homodimer was formed. Both of created models were superposed onto either monomer of 3ERM homodimer. Then energy-minimized heterodimer structure was calculated using CNS program.
  • HOIL- 1 L - HOIP - SHARPIN ternary complex model Docking of the HOIL- 1 L- SHARPIN N-terminal heterodimer model to SHARPIN UBL-HOIP UBA-HOIL UBL model was performed using the software ZDOCK (Pierce et al., Bioinformatics, 30: 1771-1773 (2014)). Based on the top-scored docking model, HOIL-1L-HOIP-SHARPIN ternary complex model was constructed. The length of linker polypeptides were sufficient to bridge the each N-terminal segment and UBL.
  • HEK293T cells were transfected with pGL4.32 (Luc2p/NF-KB-RE/Hygro) and pGL4.74 (hRLuc/TK) (Promega) along with WT or mutants of the LUBAC component. 21- 24 hours after transfection, cells were lysed and luciferase activities were measured by using the Dual-Luciferase reporter assay system (Promega) by Lumat Luminometer (Berthold).
  • This example demonstrates SHARPIN and HOIL-1L bind to distinct sites of HOIP UBA.
  • GFP-tagged mouse HOIP (mHOIP) UBA region (466-630 aa) or mHOIP ZF domains (296-434 aa) containing ZF, NZF 1 , and NZF2 was co-introduced with mHOIL-lL or mSHARPIN into HEK293T cells.
  • mSHARPIN did interact with the mHOIP UBA, but not with the ZF domains as was the case with mHOIL-l L ( Figure I B).
  • mSHARPIN bound to mHOIP WT and ANZF2 ( ⁇ 402-432 aa), but not to mHOIP AUBA ( ⁇ 558-609 aa) ( Figures 7A and 7B).
  • mSHARPIN failed to interact with the mHOIP M mut in addition to mHOIP C mut ( Figure I D).
  • the UBL does interact with the M site via a surface that corresponds to the 144 hydrophobic surface of ubiquitin, whereas it does interact with the C site via a distinct surface from 144-like surface.
  • a structural model was generated of the M site of hHOIP and hSHARPIN UBL based on co- crystal structure of the hHOIP UBA and hHOIL- 1 L UBL.
  • Leu273 and Tyr295 of mSHARPIN were corresponding amino acid residues of Leu276 and Tyr298 in hSHARPIN, which are important for HOIP interaction in this model.
  • the UBLs are the interaction domains with HOIP, the mHOIL-l L and mSHARPIN mutants whose UBL domain and N-terminal extension to UBL are exchanged, which are S(UBL)-H0IL-1L and H(UBL)-SHARPIN ( Figure 2B).
  • the accessory subunits having the HOIL-IL UBL (HOIL-IL and H(UBL)-SHARPIN) efficiently activated NF-KB and increased the amount of mHOIP ( Figure 2B), indicating that the mSHARPIN UBL is less effective to stabilize mHOIP as compared with mHOIL-l L UBL.
  • mSHARPIN could increase the amount of mHOIP protein in the presence of mHOIL- 1 L.
  • mSHARPIN could also be efficiently co- immunoprecipitated with the mHOIP UBAl mut in the presence of mHOIL-lL ( Figures 2E- 2G, compare lane 4 to lane 10).
  • SPR analyses confirmed that the addition of mSHARPIN UBL drastically augmented the interaction between mHOIP UBA Q607A/L611 A/F614A (UBA2 mut ) and mHOIL-lL UBL although the UBA2 mutations heavily attenuated the interaction between HOIP UBA and mHOIL-lL UBL ( Figure 8B).
  • Luciferase reporter assays also revealed that additive expression of mSHARPIN WT notably enhanced NF-KB activation induced by mHOIP UBA2 mut and mHOIL-lL.
  • mSHARPIN Y295A and L273A which cannot bind to HOIP UBA1 , failed to activate NF- ⁇ ( Figure 8C).
  • mHOIL-lL could bind to the mSHARPIN mutants even though the mutants failed to interact with mHOIP ( Figures 2H and 21, Myc (HOIL-IL) blot, lanes 11 , 12), which also indicated that direct interaction between mSHARPIN and mHOIL-lL exists.
  • mHOIL-lL UBL (1-140 aa) and mSHARPIN UBL (163-301aa), which effectively bound to HOIP UBA WT and UBA2 mut in SPR analyses ( Figures 2D and 8B), contain additional amino acids at the N-terminus of the UBL domains (1-36 aa or 163-197 aa for HOIL-IL or SHARPIN, respectively).
  • a secondary structure prediction program suggested that both N-terminal regions of HOIL-IL and SHARPIN seem to form ct-helical structure (Not). These Net regions were investigated as to whether they are involved in the HOIL-1 L-SHARPIN interaction.
  • HEK293T cells lacking HOIP were generated using CRISPR/Cas9 system ( Figure 9A and 9B) since HOIP can bind to and bridge the two proteins.
  • HOIP KO CRISPR/Cas9 system
  • mHOIL-lL WT interacted with mHOIL-lL WT and mSHARPIN WT ( Figures 3 A and 3B, lanes 2, 3).
  • N-terminal regions of HOIL-IL and SHARPIN are highly homologous ( Figure 9C), it was inferred that the N-terminal additional regions may be involved in both heterotypic and homotypic interactions of the two accessory subunits of LUBAC.
  • the structural model of the LUBAC ternary complex was generated by docking of HOIL-SHARPIN Na heterodimer (HOIL : 2-38; SHARPIN : 173- 202) on hSHARPIN UBL (216-302 aa)-hHOIP (482-627 aa)-hHOIL UBL (2-133 aa) structure using ZDOCK program (Pierce et al., Bioinformatics, 30: 1771-1773 (2014)).
  • the model shows that both accessory subunits SHARPIN and HOIL-IL can interact with each other via their Na region simultaneously when the UBLs of SHARPIN and HOIL-IL do bind to HOIP UBAl and UBA2, respectively, indicating that three mutual interactions among the three subunits of LUBAC are important for the formation of trimeric LUBAC.
  • the structure modeling confirms the observation that loss of one interaction among the three does not overtly affect the trimeric LUBAC formation, whereas LUBAC is destabilized if two interactions are impaired as a consequence of dissociation of one subunit from the complex.
  • This example demonstrates physiological roles of the interactions between LUBAC subunits and targeting the novel HOIL-1L-SHARPIN interaction by an a-helical stapled polypeptide, in accordance with embodiments of the invention.
  • mice embryonic fibroblasts were established that did not express any of the LUBAC subunits (TKO cells) by knocking out HOIL- 1 L and HOIP in cpdm MEFs using CRISPR/Cas9 technology ( Figures 10- IOC).
  • WT or mutants of three subunits of mouse LUBAC were reconstituted into TKO cells using retroviral expression system, evaluating TNF-a-mediated cytotoxicity by using iCELLigence system ( Figures 4A and 4B).
  • introduction of mSHARPIN did not overtly increase the amount of mHOIP, mHOIL-l L increased mHOIP rather efficiently ( Figure 4A, compare lane 2 to lanes 3, 5).
  • the observation that co-expression of mSHARPIN further potentiated the mHOIL-l L-mediated increase of mHOIP confirmed the important roles of three subunits in stable LUBAC formation ( Figure 4A, lane 6).
  • a hydrocarbon stapled a-helical HOIP polypeptide which inhibits the HOIP-HOIL- 1 L interaction could inhibit the proliferation of ABC-DLBCL cell lines by destabilizing LUBAC (Yang et al., Cancer Discov, 4: 480-493 (2014)). Since simultaneous suppression of both HOIL-1 L-SHARPIN and HOIL-1 L-HOIP interaction destabilize LUBAC complex much more efficiently than suppression of HOIL-1L-HOIP interaction alone ( Figures 4A and 4B), an a-helical stapled polypeptide was developed mimicking the Na region of SHARP IN to inhibit the HOIL-1L- SHARPIN interaction (unstapled: Ala-WEELATRLSQAIA-NH? (SEQ ID NO: 96);
  • LUBAC is involved in the activation of ⁇ kinase. Although addition of purified LUBAC proteins to lysates of Jurkat HOIP KO cells activated IKK, administration of SHARP ⁇ - ⁇ or SHARPIN-Ncc plus HOIP-N polypeptides profoundly inhibited LUBAC- mediated IKK activation (Figure 4H), which indicated that disruption HOIL-IL-SHARPIN interaction attenuated physiological function of LUBAC.
  • This example demonstrates HOIL-1 L null mice exhibit embryonic lethality at midgestational stage as observed in HOIP knockout mice or HOIP mutant mice lacking linear ubiquitin ligase activity.
  • mHOIL-lL increases the amount of mHOIP much more efficiently than mSHARPIN by overexpression study ( Figure 2A).
  • mHOIL-l L or mSHARPIN was co-introduced with mHOIP into TKO cells using the retrovirus expression system.
  • mHOIL-l L, but not mSHARPIN alone increased mHOIP as previously observed in Figure 2A ( Figures 1 1 A and 1 I B).
  • mHOIL-l L could evade TNF-a-mediated cytotoxicity ( Figure 1 1 C) and Caspase-3 activation mediated by TNF-a and cycloheximide (CHX) ( Figure 1 I D) when co-expressed with mHOIP, indicating that loss of mHOIL-l L sensitizes cells to TNF- -mediated cell death as observed in cells lacking mHOIP. Since most genetically engineered mice that are sensitive to TNF-a-mediated cell death are embryonic lethal, mice lacking HOIL- 1 L are expected to embryonic lethal as is the case with HOIP KO mice.
  • N-terminal truncated HOIL-IL may be expressed as an alternative spliced product.
  • the N-terminal truncated HOIL-IL splice variant has been reported as RBCK2.
  • mice homozygous for HOIL- I L null alleles were embryonic lethal and died around embryonic day 10.5, as observed in HOIP knockout mice or HOIP mutant mice lacking linear ubiquitin ligase activity (HOIP ⁇ '""" 7 -- 1 '"" ' "' " ) ( Figures 5B, 5C, 1 1 J and 1 I K).
  • HOIL-I L""" 7 "” mice display intracranial and/or thoracoabdominal hemorrhages and TUNEL-positive cells were significantly increased as compared to its control littermates (Figure 5D). Defects in vasculature of HOIL-I L""" 7 “”" embryos were confirmed in HOIP knockout mice.
  • HOIP and SHARPIN were barely detected in HOIL-lL ra/// / MEFs, as compared with previously described HOIL-1 L " " (RING1 KO) cells ( Figure 5A).
  • Introduction of mHOIL-l L but not m SHARPIN into HOIL-1 L""” 7 TM" MEFs increased the amount of HOIP ( Figure 1 1 L) and protect cells from TNF-ot-mediated cell death (Figure 5E).
  • HBL1 cell viabillity Na induced a decrease in HBL1 cell viabillity at doses of 20 and 40 ⁇ .
  • HBL1 cell were incubated with an unstapled (WT), a negative control (Scramble (SC2)), and a stapled (Na) versions of SHARP IN polypeptide. Viability was measured by CellTiter Glo (Promega, G7570). The results are in Figure 14A.
  • HBL-1 cells were treated with Na during 2 h, then media was exchanged by one with 5 ng/mL of TNF-a, and cells exposed for 4 and 24h.
  • the amount of secreted IL-8 to supernatants was determined using an specific ELISA kit (Peprotech, Rocky Hill, NJ, USA, cat# 900T18) following manufacturer's instructions. The results are in Figure 14C.
  • HBL-1 cells were treated with Na during 2 h, then media was exchanged by one with 5 ng/mL of TNF-a, and exposed to cells for 0, 5, 10, 30, 60 or 240 min. Cell lysates were analyzed for IkBa protein levels by Western blot. The results are in Figures 14D and 14E.
  • Na provokes not only an increase in IkBa levels, but also deactivation of Akt, and ERK.
  • Na treatment reduces the levels of the proapoptotic protein BAX.
  • Cell were treated during 24h and equal amount of protein from cell lysates were analyzed for IkBa, Akt, ERK, BAX, and actin by Western blot. The results are in Figure 14F.
  • HT1080 Fibrosarcoma
  • PANC 1 pancreatic carcinoma
  • HCT1 16 Cold carcinoma
  • PEA1 oval carcinoma
  • SJSA-1 Steosarcoma
  • WT Figures 15A-C
  • Na Figures 15D-F
  • Na showed a wide range of activity in analyzed cell lines, ranging from low (as in HT1080) to high (as in SJSA-1 ) ( Figure 15A (WT) and Figure 15D (Na)).

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Abstract

In embodiments, the invention provides SHARPIN-based polypeptides that inhibit the linear ubiquitin chain assembly complex (LUBAC) and provides methods of treating diseases including activated B-cell like diffuse large B cell lymphoma (ABC DLBCL) and autoimmune or inflammatory disorders.

Description

SHARP IN-BASED POLYPEPTIDES AND THEIR USES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application claims the benefit of U.S. Provisional Patent Application
No. 62/434,227, filed December 14, 2016, and U.S. Provisional Patent Application
No. 62/508,158, filed May 18, 2017, each of which is incorporated herein by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
[0002] This invention was made with Government support under project number ZIA BC 01151 1 awarded by the National Institutes of Health, National Cancer Institute. The Government has certain rights in this invention.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED
ELECTRONICALLY
[0003] Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 54,630 Byte ASCII (Text) file named "735821 ST25.txt," dated December 8, 2017.
BACKGROUND OF THE INVENTION
[0004] Constitutive linear ubiquitin chain assembly complex (LUBAC) activity is associated with several kinds of human malignancies, including ovarian, pancreatic, colon, and lung carcinomas as well as inflammation processes.
[0005] There is a continued need for the development of therapeutic agents which are useful in the treatment of diseases associated with LUBAC activity.
BRIEF SUMMARY OF THE INVENTION
[0006] In an embodiment, the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, the polypeptide comprising at least two non-natural ammo acids that form a covalent cross-link with each other, wherein the non-natural amino acids are the same or are different, and wherein the cross-link is internal to the polypeptide, and (a) wherein the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine; or the polypeptide has from the N- to C-terminus a glutamine one amino acid position before a first valine, and optionally (i) wherein the first valine is three amino acid positions before a second valine, and (ii) wherein the second valine is one amino acid position before a leucine; and (b) wherein the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
[0007] In an embodiment, the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, comprising a modified amino acid sequence of EELAGSLARAI (SEQ ID NO: 1), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO: 4); wherein the modification of the sequence is of at least two of the amino acids within the sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein the non-natural amino acids form a covalent cross-link with each other, wherein the covalent cross-link is internal to the sequence; and wherein the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
[0008] In an embodiment, the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, the polypeptide comprising at least two non-natural amino acids capable of forming an internal cross-link, wherein the non-natural amino acids are the same or are different, wherem each of the non-natural amino acids includes a moiety, wherein the moieties are capable of undergoing a reaction to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the polypeptide; and (a) wherein the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine; or the polypeptide has from the N- to C-terminus (1 ) a glutamine one amino acid position before a first valine, and (2) optionally (i) wherein the first valine is three amino acid positions before a second valine, and (ii) wherein the second valine is one amino acid position before a leucine; and (b) wherein if the non-natural amino acids form an internal cross-link, the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
[0009] In an embodiment, the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, comprising a modified amino acid sequence of EELAGSLARAI (SEQ ID NO: 1 ), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO: 4); wherein the modification of the sequence is of at least two of the amino acids within the sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein each of the non- natural amino acids include a moiety, wherein each moiety is capable of undergoing a reaction with a moiety of one other of the non-natural amino acids to form a covalent crosslink with each other, wherein the covalent cross-link is internal to the sequence; and wherein if the non-natural amino acids form an internal cross-link with each other, the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
[0010] In an embodiment, the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence modified according to the formula (I):
A B
Figure imgf000004_0001
wherein Ri and R? are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl; R3 of formula (I) is alkylene, alkenylene, alkynylene, or [R4-K'-R4']n, each of which is substituted with 0-6 R5; R4 and R^ are independently alkylene, alkenylene, or alkynylene; R5 is independently halo, alkyl, OR6, N(R6)2, SR6, SOR6, SO2R6, CO2R6, R&, a fluorescent moiety, or a radioisotope; K' is independently O, S, SO, S02, CO, CO2, CONR&, or
Figure imgf000004_0002
R-6 is independently H, alkyl, or a therapeutic agent; n is independently an integer from 1-4; [Xaa]x has 2 to 6 amino acids; [Xaa]w, [Xaa]x, and [Xaa]y taken together have the amino acid sequence of EELAGSLARAI (SEQ ID NO: 1), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the amino acids within the sequence are replaced with the residues A and B; and wherein the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
[0011] In an embodiment, the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence modified according to the formula (II):
Figure imgf000005_0001
wherein Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl; R3 and R3' of formula (II) are independently alkenyl, alkynyl, azido, amino, carboxylic acid, or sulfide; [Xaa] has 2 to 6 amino acids; [Xaa]w, [Xaa]x, and [Xaa]y taken together have the amino acid sequence of EELAGSLARAI (SEQ ID NO: 1 ), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the ammo acids within the sequence are replaced with the residues A and B; and wherein if A and B form an internal cross-link, the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC). In certain embodiments, both R3 and R3 are alkenyl. [0012] In an embodiment, the invention also provides a pharmaceutical composition comprising an effective amount of an inventive polypeptide described herein.
[0013] In an embodiment, the invention also provides a method of inhibiting the linear ubiquitin chain assembly complex (LUBAC) in a subject, which method comprises administering an effective amount of an inventive polypeptide described herein or a pharmaceutical composition of an inventive polypeptide described herein to the subject.
[0014] In an embodiment, the invention also provides a method of treating activated B- cell like diffuse large B cell lymphoma (ABC DLBCL) in a subject, which method comprises administering an effective amount of an inventive polypeptide described herein or a pharmaceutical composition of an inventive polypeptide described herein to the subject.
[0015] In an embodiment, the invention also provides a method of treating rheumatoid arthritis in a subject, which method comprises administering an effective amount of an inventive polypeptide described herein or a pharmaceutical composition of an inventive polypeptide described herein to the subject.
[0016] In an embodiment, the invention also provides a method of treating cancer that is resistant to cytotoxic chemotherapy, radiation therapy, vaccine therapy, or cytokine therapy in a subject, which method comprises administering an effective amount of an inventive polypeptide described herein or a pharmaceutical composition of an inventive polypeptide described herein to the subject.
[0017] In an embodiment, the invention also provides a method of treating chronic autoinflammation, systemic lupus erythematosus, Crohn's inflammatory bowel disease, or psoriasis in a subject, which method comprises administering an effective amount of an inventive polypeptide described herein or a pharmaceutical composition of an inventive polypeptide described herein to the subject.
[0018] The description below further provides exemplary embodiments of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 A is a schematic representation of domains of HOIL-1 L, HOIP and SHARP IN.
[0020] Figure I B shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Cell lysates and anti-FLAG immunoprecipitates were immunoblotted with the indicated antibody. [0021] Figure 1C presents a surface plasmon resonance line graph. GST-mHOIP UBA (466-630aa) was immobilized on the sensor chip of surface plasmon resonance via GST antibody. Binding between mHOIP UBA and MBP-mSHARPIN UBL (163-301 aa) was measured.
[0022] Figure ID shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Cell lysates and anti-FLAG immunoprecipitates were immunoblotted with the indicated antibody.
[0023] Figure IE shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Cell lysates and anti-FLAG immunoprecipitates were immunoblotted with the indicated antibody.
[0024] Figure 2 A shows immunoblot gel analysis. Indicated plasmids and 5><NF-KB luciferase reporter were transfected into HEK293T cells. Cell lysates were immunoblotted with the indicated antibody and NF-κΒ activity was measured by luciferase assay (mean ± s.e.m., n=3).
[0025] Figure 2B presents a schematic representation of HOIL-1L and SHARPIN and their mutants and shows immunoblot gel analysis. Indicated plasmids and 5><NF-KB luciferase reporter were transfected into HEK293T cells and analyzed as described in Figure 2A (mean ± s.e.m., n=3).
[0026] Figure 2C shows immunoblot gel analysis. Indicated plasmids and 5><NF-KB luciferase reporter were transfected into HEK293T cells and analyzed as described in Figure 2A (mean ± s.e.m., n=3).
[0027] Figure 2D presents a surface plasmon resonance line graph. GST-tagged mHOIP UBA (466-630 aa) was immobilized on sensor chip of SPR via GST antibody. MBP-tagged HOIL-1 L ( 1 -140 aa) alone (8.5 μΜ), mSHARPIN (163-301 aa) alone (8.5 μΜ) or both (8.5 μΜ each) were used as analytes. See also Figure 8A.
[0028] Figure 2E shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Cell lysates were immunoblotted with the indicated antibody.
|0029] Figure 2F shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Anti-Myc immunoprecipitates were immunoblotted with the indicated antibody. [0030] Figure 2G shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Anti-FLAG immunoprecipitates were immunoblotted with the indicated antibody.
[0031] Figure 2H shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Cell lysates were analyzed by western blotting.
[0032] Figure 21 shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Anti-FLAG immunoprecipitates were analyzed by western blotting.
[0033] Figure 3A shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T HOIP KO cells. Cell lysates were analyzed by immunoblotting.
[0034] Figure 3B shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T HOIP KO cells. Anti-Myc immunoprecipitates were analyzed by immunoblotting.
[0035] Figure 3C presents a surface plasmon resonance line graph. GST-mHOIP UBA WT (466-630 aa) was immobilized on sensor chip of SPR via GST antibody. Binding ability to the UBLs containing or lacking Na region was analyzed as described in Figure 2D.
[0036] Figure 3D shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T HOIP KO cells. Cell lysates were analyzed by immunoblotting.
[0037] Figure 3E shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T HOIP KO cells. Anti-FLAG immunoprecipitates were analyzed by immunoblotting.
[0038] Figure 3F shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Cell lysates were analyzed by immunoblotting.
[0039] Figure 3G shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Anti-FLAG immunoprecipitates were analyzed by immunoblotting.
[0040] Figure 4A presents immunoblot analysis of lysates from LUBAC TKO cells reconstituted with the indicated proteins.
[0041] Figure 4B presents a surface plasmon resonance line graph. TKO cells reconstituted with the indicated proteins were stimulated with TNF-a ( l Ong/ml) and cell viability was measured continuously by using impedance-based real-time cell analyzer (RTCA).
[0042] Figure 4C presents immunoblot analysis of lysates from LUBAC TKO cells reconstituted with the indicated proteins. [0043] Figure 4D presents a surface plasmon resonance line graph. TKO cells reconstituted with the indicated proteins were stimulated with TNF-a (lOng/ml) and analyzed as described in Figure 4B.
[0044] Figure 4E presents sequences of ct-helical SHARPIN polypeptides. The asterisks show the location of the hydrocarbon cross-linker.
[0045] Figure 4F presents a surface plasmon resonance line graph. GST-mHOIP UBA (466-630 aa) was immobilized on the sensor chip of SPR via GST antibody. Mixture of MBP-tagged HOIL-1 L UBL (1 -140 aa) and SHARPIN UBL (163-301 aa) (0.5 μΜ each) in the presence or absence of polypeptides (20 μΜ) were used as analytes.
[0046] Figure 4G shows immunoblot gel analysis. Trimeric LUBAC (0.2 μΜ) was incubated with stapled polypeptides (80 μΜ) on ice for 3 hours. Then, a mixture of El, E2 and ubiquitin was added and incubated at 37°C for 30 min followed by immunoblotting.
[0047] Figure 4H shows immunoblot gel analysis. 10 μg of SI 00 lysate of HOIP KO Jurkat cells and trimeric LUBAC (0.1 μΜ) was incubated with stapled polypeptides (80 μΜ) on ice for 3 hours. Then, a mixture of El , E2 and ubiquitin was added and incubated at 37°C for 30 min followed by immunoblotting.
[0048] Figure 41 shows immunoblot gel analysis. HBL-1 cells were treated with the indicated polypeptides for 24 h. Cell lysates and anti-SHARPIN immunoprecipitates were analyzed by immunoblotting.
[0049] Figure 4J presents a gel. HBL-1 cells were treated with the indicated polypeptides for 24 h. Cell viability was measured by CellTiter-Glo Luminescent Cell Viability assay and NF-KB activity was measured by EMSA assay.
[0050] Figure 5A presents immunoblot analysis of MEF cells from indicated genotypes of mice.
[0051] Figure 5B presents quantification of genotypes of animals obtained after crossing HOIL-l L+/nu" mice.
[0052] Figure 5C presents the numbers of embryos obtained at each embryonic stage (E9.5, 10.5, 1 1.5, and 12.5) after crossing HOIL-l L+/nu" mice.
[0053] Figure 5D presents quantification of the TUNEL-positive cells with the indicated genotypes (n = 3). Scale bars: 200 μηι. Data are given as the means ± the standard error of the mean (SEM). (Significance: **, p < 0.01 ). [0054] Figure 5E presents a line graph showing RTCA results. HOIL-1L null MEFs reconstituted with indicated proteins were stimulated with TNF-a (lOng/ml) and cell viability was measured using RTCA (mean ± s.e.m., n=3).
[0055] Figure 6A shows immunoblot gel analysis. Cell lysates of TKO cells expressing the indicated proteins were analyzed by immunoblotting.
[0056] Figure 6B presents a schematic of sequences of polypeptides used in Figure 6C.
[0057] Figure 6C shows immunoblot gel analysis. Cell lysates of TKO cells
reconstituted with indicated proteins were analyzed by immunoblotting.
[0058] Figure 6D shows immunoblot gel analysis. TKO cells reconstituted with indicated proteins were stimulated with TNF-a (5 ng/ml) for indicated periods and cell lysates were analyzed by immunoblotting.
[0059] Figure 6E shows immunoblot gel analysis. TKO cells reconstituted with indicated proteins were stimulated with TNF-a (1 ng/ml) plus CHX (20 μg/ml) for indicated periods and cell lysates were analyzed by immunoblotting.
[0060] Figure 6F shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T HOIP KO cells. Cell lysates were analyzed by immunoblotting.
[0061] Figure 6G shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T HOIP KO cells. Anti-FLAG immunoprecipitates were analyzed by immunoblotting.
[0062] Figure 7A shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Cell lysates were analyzed by immunoblotting.
[0063] Figure 7B shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Anti-FLAG immunoprecipitates were analyzed by immunoblotting.
[0064] Figure 7C presents a surface plasmon resonance line graph. GST-HOIP UBA (466-630 aa) was immobilized on the sensor chip for SPR via a GST antibody and binding affinity between MBP-HOIL- 1 L UBL (1 -140 aa) was determined.
[0065] Figure 7D shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Cell lysates were analyzed by immunoblotting.
[0066] Figure 7E shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Anti-FLAG immunoprecipitates were analyzed by immunoblotting.
[0067] Figure 7F presents sequences, showing the conserved residues of HOIL-1 L and SHARPIN. Asterisks show the amino acids which are thought to be involved in interactions with HOIP UBA1 or UBA2, respectively. [0068] Figure 8A shows the line graphs of Figure 2D, with responses normalized at the time of stopped applying the UBL.
[0069] Figure 8B presents a surface plasmon resonance line graph. mHOIP UBA UBA2mut (466-630 aa) was immobilized and binding ability to UBLs was analyzed as shown in Figure 2D.
[0070] Figure 8C is a bar graph the results of a luciferase assay: 5><NF-KB luciferase reporter and the indicated plasmids were transfected into HEK293T cells and NF-κΒ activity was measured by luciferase assay (mean ± s.e.m., n=3).
[0071] Figure 9A is a schematic representation of guide R A sequence against hHOIP. 293T HOIP KO cells were homozygous for a 1 bp insertion (depicted as underlined). The protospacer-adjacent motif (P AM) sequence is depicted (GGG at end).
[0072] Figure 9B presents immunoblotting analysis of 293T HOIP KO cells. Cell lysates of parent and HOIP KO 293T cells were immunoblotted with the indicated antibody.
[0073] Figure 9C presents sequences showing conserved residues in Na regions of HOIL-lL and SHARPIN.
[0074] Figure 9D presents a surface plasmon resonance line graph. GST-mHOIP UBA WT (466-630 aa) was immobilized on a sensor chip for SPR via a GST antibody. Binding ability between UBLs containing or lacking Na regions and HOIP UBA were analyzed.
[0075] Figure 9E presents a surface plasmon resonance line graph. GST-HOIP UBA (466-630 aa) was immobilized on sensor chip of SPR via GST antibody and HOIL-1L UBL (1 -189 aa) alone (500 μg/ml) or SHARPIN UBL (500 μ^πιΐ) (163-340 aa) or both (500 g/ml each) were used as analyte.
[0076] Figure 9F shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Cell lysates were analyzed by immunoblotting.
[0077] Figure 9G shows immunoblot gel analysis. Indicated plasmids were transfected into HEK293T cells. Anti-FLAG immunoprecipitates were analyzed by immunoblotting.
[0078] Figure 9H presents sequences showing conserved residues of Na regions of HOIL-1L in various species.
[0079] Figure 91 presents sequences showing conserved residues of Na regions of SHARPIN in various species.
[0080] Figure 1 OA is a schematic representation of guide RNA sequences against mHOIP or mHOIL-l L. TKO cells carry one allele with 1 bp deletion and one allele with 1 1 bp deletion in HOIL- 1 L loci, and one allele with 2 bp deletion and one allele with 1 bp deletion in HOIP loci.
[0081] Figure 1 OB is a schematic representation of guide RNA sequences against mHOIP or mHOIL-lL. TKO cells carry one allele with 1 bp deletion and one allele with 1 1 bp deletion in HOIL-I L loci, and one allele with 2 bp deletion and one allele with 1 bp deletion in HOIP loci.
[0082] Figure 10 C presents immunoblot analysis of lysates from TKO cells.
[0083] Figure 1 1A shows immunoblot gel analysis. Cell lysates of TKO cells reconstituted with the indicated proteins were immunoblotted with the indicated antibody.
[0084] Figure 1 IB shows immunoblot gel analysis. Cell lysates from TKO cells reconstituted with indicated proteins and anti-FLAG immunoprecipitates were
immunoblotted with indicated antibody.
[0085] Figure 1 1C presents a line graph showing RTCA results. TKO cells expressing the indicated proteins were stimulated with TNF- (10 ng/ml) and cell viability were measured continuously by using RTCA (mean ± s.e.m., n=3).
[0086] Figure 1 I D shows immunoblot gel analysis. TKO cells reconstituted with the indicated proteins were stimulated with TNF-a (1 ng/ml) plus CHX (20 μg/ml) for the indicated periods. Cell lysates were immunoblotted with the indicated antibody.
[0087] Figure 1 I E presents RT-PCR analysis of expression levels of N-terminal part or
C-terminal part of HOIL- I L in cells derived from HOIL-I L7" or WT mice.
[0088] Figure 1 I F shows immunoblot gel analysis. Lysates from TKO cells reconstituted with HOIP, HOIL- I L WT or UBL (HOIL-I L 1 -140 aa) in the presence or absence of
SHARP IN were immunoblotted with indicated antibody.
[0089] Figure 1 1 G shows immunoblot gel analysis. TKO cells reconstituted with indicated proteins were stimulated with TNF-a (3 ng/ml) plus CHX (20 μg/ml) for the indicated periods. Lysates were analyzed by immunoblotting.
[0090] Figure 1 1 H is a schematic representation of guide RNA sequences against mHOIL- l L.
[0091] Figure 1 I I presents sequences showing mutated alleles. Mutated allele #1 was obtained using guideRNA# l and mutated allele W2. was obtained using guideRNA#2.
[0092] Figure 1 1 J shows quantification of genotypes of animals obtained after crossing HOIL- l L+/null#2 mice. [0093] Figure 1 IK presents numbers of embryos obtained at each embryonic stage (E9.5, 10.5, 1 1.5, and 12.5) after crossing HOIL-lL+/null#2 mice.
[0094] Figure 1 1L shows immunoblot gel analysis. Immunoblot analysis of HOIL-1L null MEFs reconstituted HOIL-1L or SHARPIN. Lysates were immunoblotted with the indicated antibody.
[0095] Figure 12A presents sequences showing conserved residues of HOIP UBA domain between human and mouse.
[0096] Figure 12B shows immunoblot gel analysis. Indicated plasmids were transfected HEK293T HOIP KO cells. Cell lysates and anti-FLAG immunoprecipitates were analyzed by immunoblotting.
[0097] Figure 12C shows immunoblot gel analysis. Indicated plasmids were transfected HEK293T cells. Cell lysates and anti-HA immunoprecipitates were analyzed by western blotting.
[0098] Figure 12D shows immunoblot gel analysis. Indicated plasmids were transfected HEK293T cells. Cell lysates and anti-HA immunoprecipitates were analyzed by western blotting.
[0099] Figure 13 presents helical wheel diagrams of some of the inventive polypeptides as described herein, in accordance with embodiments of the invention.
[0100] Figures 14A-14F are as described in Example 6.
[0101] Figures 15A-15I are as described in Example 6.
DETAILED DESCRIPTION OF THE INVENTION
[0102] In one aspect, the invention is based on the discovery of SHARPIN polypeptides that can inhibit the linear ubiquitin chain assembly complex (LUBAC), which includes three protein subunits: HOIP (also known as RNF31), HOIL-1L (also known as RBCK1 ), and SHARPIN (also known as SHANK Associated RH domain INteractor). The polypeptides of the invention can be used to disrupt LUBAC activity in vitro or in vivo. Moreover, the polypeptides of the invention can be used to inhibit LUBAC-mediated cell signaling, e.g., NF- B signaling. Since LUBAC-mediated cell signaling is specific to certain cell types, the polypeptide inhibitors of the invention can be used to selectively inhibit LUBAC-mediated signaling, e.g., NF-κΒ signaling in specific cells.
[0103] "Polypeptide" as used herein refers to a polypeptide chain of amino acids having two or more amino acids. The polypeptides of the invention are based on the sequence of wild-type human SHARPIN at position 171-201 :
EREELAGSLARAIAGGDEKGAAQVAAVLAQH (SEQ ID NO: 5) and wild-type mouse SHARPIN at position 168-198: KKEELATRLS Q AI AGGDEKA AAQ V A A VLAQH (SEQ ID NO: 6). These sequences correspond to a portion of an a-helix domain located in the ubiquitin-like (UBL) domain of SHARPIN (accession number for human: NP_112236.3; accession number for mouse: NP_079616).
[0104] In an embodiment, the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, the polypeptide comprising at least two non-natural amino acids that form a covalent cross-link with each other, wherein the non-natural amino acids are the same or are different, and wherein the cross-link is internal to the polypeptide, and (a) wherein the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine; or the polypeptide has from the N- to C-terminus a glutamine one amino acid position before a first valine, and optionally (i) wherein the first valine is three amino acid positions before a second valine, and (ii) wherein the second valine is one amino acid position before a leucine; and (b) wherein the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
[0105] In embodiments, the polypeptide of the invention is a hydrocarbon-stapled - helical polypeptide. In other embodiments, the polypeptide is not stapled but contains amino acids that can be cross-linked to form a stapled polypeptide, e.g., when the polypeptide is contacted with a cross-linking catalyst and/or placed into an environment in which cross- linking can occur. For example, without limitation, for an olefin, a cross-linking catalyst that could be used is Grubbs' ruthenium metathesis catalyst or similar catalysts. Without limitation, other cross-linking catalyst examples include copper for cross-linking alkynes and azides, and oxidizing agents for cross-linking disulfides. Generally, a stapled polypeptide refers to a polypeptide that includes at least one pair of non-natural amino acids that are covalently cross-linked to each other and thereby form an internal hydrocarbon "staple" within the polypeptide. Such internal cross-links can function as "braces'* or "locks" that stabilize the alpha-helical conformation of a polypeptide and/or improves cell penetration, target affinity, proteolytic resistance, or serum half-life of the polypeptide.
[0106] "Non-natural amino acid" as used herein means any amino acid that may form a polypeptide, excluding the "natural amino acids" that form polypeptides in mammals. The "natural amino acids" are alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
[0107] In certain embodiments, the polypeptide can be modified to include one, two, three, or four hydrocarbon staples. In certain embodiments, the stapled polypeptides of the invention are cell-permeable or cell penetrating and therefore, useful for inhibiting LUBAC in vitro (e.g., in cells cultures, tissue cultures, or explants) or in vivo (e.g. in a "subject," as defined herein).
[0108] As understood by those of ordinary skill in the art, the terms "modified," "is modified," "modification," "replaced," "is replaced," etc. are used herein as a way to describe the end resulting polypeptide sequence as if the modification had occurred to a starting polypeptide sequence, without limiting the process by which the modification had or can occur. For example, the phrases "wherein the modification of the sequence is of at least two of the amino acids within the sequence" and "an amino acid sequence modified according to the formula" do not limit the invention such that the modification(s) had to actually occur on the sequence. As understood by one of ordinary skill in the art, the resulting sequence may be, e.g., synthesized such that the modifications are incorporated during the synthesis to produce the end resulting polypeptide such that the unmodified sequence may itself not have been actually obtained/produced and subsequently modified, and/or had amino acids replaced.
[0109] In certain embodiments, a polypeptide according to the invention can include a first non-natural amino acid substitution. The first non-natural amino acid can be cross- linked to a second non-natural amino acid that is substituted or inserted at a position in the polypeptide which is four residues away. The relative positions of the first and second non- natural amino acids in this stapled polypeptide are designated as (z, i + 4). In another embodiment, the first non-natural amino acid can be cross-linked to a second non-natural amino acid located seven residues away (i, i + 7) in the polypeptide. In another embodiment, the first non-natural amino acid can be cross-linked to a second non-natural amino acid located three residues away (/, + 3) in the polypeptide.
[0110] In certain embodiments, the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine, and wherein the cross-link is formed from a non-natural amino acid at position z within the polypeptide and another non-natural amino acid at position i + 3, i + 4, or i + 7 within the polypeptide; and wherein the amino acid at position i + 6 within the polypeptide is the leucine, the amino acid at position i + 9 within the polypeptide is the alanine, and the amino acid at position i + 10 within the polypeptide is the isoleucine.
[0111] In certain embodiments, the polypeptide has from the N- to C-terminus (1) a glutamine one amino acid position before a first valine, and (2) optionally (i) wherein the first valine is three amino acid positions before a second valine, and (ii) wherein the second valine is one amino acid position before a leucine, and wherein the cross-link is formed from a non- natural amino acid at position i within the polypeptide and another non-natural amino acid at position i + 3, i + 4, or i + 7 within the polypeptide; and wherein the amino acid at position i + 1 within the polypeptide is the glutamine, and the amino acid at position i + 2 within the polypeptide is the first valine.
[0112] In certain embodiments, the cross-link of the polypeptide is formed from the amino acid at position i within the polypeptide and another amino acid at position i + 4 within the polypeptide, and the amino acid at position i is (S)-2-(4'-pentenyl)alanine (S5) and the amino acid at position i + 4 is S5; or formed from the amino acid at position i within the polypeptide and another amino acid at position i + 3 within the polypeptide, and the amino acid at position i is (i?)-2-(4'-pentenyl)alanine (R5) or (R)-2-(2'-propenyl)alanine (R3) and the amino acid at position i + 3 is S5; or formed from the amino acid at position i within the polypeptide and another amino acid at position i + 7 within the polypeptide, and the amino acid at position i is (R)-2-(7'-octenyl)alanine (R8) and the amino acid at position i + 7 is S5.
[0113] In certain embodiments, the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine, wherein the polypeptide is a first polypeptide, and wherein the polypeptide further comprises a second polypeptide having non-natural amino acids that form a covalent cross-link internally within the second polypeptide, wherein the second polypeptide has from the N- to C-terminus (1) a glutamine one amino acid position before a first valine, and (2) optionally (i) wherein the first valine is three amino acid positions before a second valine, and (ii) wherein the second valine is one amino acid position before a leucine.
[0114] In certain embodiments, (a) the cross-link in the first polypeptide is formed from a non-natural amino acid at position i within the first polypeptide and another non-natural amino acid at position i + 3, + 4, or i + 7 within the first polypeptide; and wherein the amino acid at position i + 6 within the first polypeptide is the leucine, the amino acid at position i + 9 within the first polypeptide is the alanine, and the amino acid at position i + 10 within the first polypeptide is the isoleucine; and (b) the cross-link in the second polypeptide is formed from a non-natural amino acid at position i within the second polypeptide and another non- natural amino acid at position i + 3, i + 4, or i + 7 within the second polypeptide; and wherein the amino acid at position i + 1 within the second polypeptide is the glutamine, and the amino acid at position i + 2 within the second polypeptide is the first valine.
[0115] In certain embodiments, (a) the cross-link of the first polypeptide is formed from the amino acid at position z within the first polypeptide and another amino acid at position i + 4 within the first polypeptide, and the amino acid at position i is (S)-2-(4'-pentenyl)alanine (S5) and the amino acid at position z + 4 is S5; or formed from the amino acid at position z" within the first polypeptide and another amino acid at position i + 3 within the first polypeptide, and the amino acid at position z is (R)-2-(4'-pentenyl)alanine (R5) or (R)-2-(2'- propenyl)alanine (R3) and the amino acid at position i + 4 is S5; or formed from the amino acid at position z" within the first polypeptide and another amino acid at position z + 7 within the first polypeptide, and the amino acid at position i is (R)-2-(7'-octenyl)alanine (R8) and the amino acid at position z + 7 is S5; and (b) the cross-link of the second polypeptide is formed from the amino acid at position z within the second polypeptide and another amino acid at position z + 4 within the second polypeptide, and the amino acid at position z is (S)-2- (4'-pentenyl)alanine (S5) and the amino acid at position i + 4 is S5; or formed from the amino acid at position z within the second polypeptide and another amino acid at position z + 3 within the second polypeptide, and the amino acid at position i is (R)-2-(4'-pentenyl)alanine (R5) or (R)-2-(2'-propenyl)alanine (R3) and the amino acid at position z + 4 is S5; or formed from the amino acid at position i within the second polypeptide and another amino acid at position z + 7 within the second polypeptide, and the amino acid at position z is (7?)-2-(7'- octenyl)alanine (R8) and the amino acid at position i + 7 is S5.
[0116] In an embodiment, the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, comprising a modified amino acid sequence of EELAGSLARAI (SEQ ID NO: 1), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO: 4); wherein the modification of the sequence is of at least two of the amino acids within the sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein the non-natural amino acids form a covalent cross-link with each other, wherein the covalent cross-link is internal to the sequence; and wherein the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
[0117] In certain embodiments, exactly two of the amino acids are non-natural amino acids. In certain embodiments, two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5.
[0118] In certain embodiments, the modified amino acid sequence is of EELAGSLARAI (SEQ ID NO: 1), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second modified amino acid sequence of
EKGAAQVAAVLAQ (SEQ ID NO: 2); wherein the modification of the second sequence is of at least two of the amino acids within the second sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein the non- natural amino acids form a covalent cross-link with each other, wherein the covalent crosslink is internal to the second sequence.
[0119] In certain embodiments, the modified amino acid sequence is of EELATRLSQAI (SEQ ID NO: 3), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second modified amino acid sequence of
EKAAAQVAAVLAQ (SEQ ID NO: 4); wherein the modification of the second sequence is of at least two of the amino acids within the second sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein the non- natural amino acids form a covalent cross-link with each other, wherein the covalent crosslink is internal to the second sequence.
[0120] In certain embodiments, exactly four of the amino acids are non-natural amino acids. In certain embodiments, two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5, and another two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5.
[0121] In an embodiment, the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, the polypeptide comprising at least two non-natural amino acids capable of forming an internal cross-link, wherein the non-natural amino acids are the same or are different, wherein each of the non-natural amino acids includes a moiety, wherein the moieties are capable of undergoing a reaction to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the polypeptide; and (a) wherein the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine; or the polypeptide has from the N- to C-terminus (1) a glutamine one amino acid position before a first valine, and (2) optionally (i) wherein the first valine is three amino acid positions before a second valine, and (ii) wherein the second valine is one amino acid position before a leucine; and (b) wherein if the non-natural amino acids form an internal cross-link, the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
[0122] An amino acid capable of being cross-linked or having a moiety capable of undergoing a reaction to form a covalent cross-link is a non-natural amino acid that includes a moiety capable of undergoing a reaction with a second moiety on another non-natural amino acid in the polypeptide to form a covalent cross-link formed from the at least two non- natural amino acids. Such amino acids may be the same non-natural amino acids or different non-natural amino acids within the polypeptide, provided they are suitable for cross-linking to each other. These amino acids include, e.g., α,α-disubstituted cross-linking amino acids; a-methyl, a-alkenyl cross-linking amino acids; and a-hydro, a-alkenyl cross-linking amino acids. Such non-natural cross-linking amino acids are commercially available, e.g., from Sigma-Aldrich, St. Louis, MO, USA or EMD Chemicals. Suitable non-natural cross-linking amino acids suitable for use in a stapled polypeptide of the invention as well as methods for cross-linking them are described in U.S. Patent Application Publication 201 1/0144306 Al, which is incorporated by reference herein in its entirety.
[0123] In certain embodiments, the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine, and wherein the non-natural amino acids capable of being cross-linked are at position i within the polypeptide and at position i + 3, / + 4, or i + 7 within the polypeptide; and wherein the amino acid at position i + 6 within the polypeptide is the leucine, the amino acid at position i + 9 within the polypeptide is the alanine, and the amino acid at position i + 10 within the polypeptide is the isoleucine.
[0124] In certain embodiments, the polypeptide has from the N- to C-terminus (1 ) a glutamine one amino acid position before a first valine, and (2) optionally (i) wherein the first valine is three amino acid positions before a second valine, and (ii) wherein the second valine is one amino acid position before a leucine; and wherein the non-natural amino acids capable of being cross-linked are at position i within the polypeptide and at position i + 3, + 4, or + 7 within the polypeptide; and wherein the amino acid at position i + 1 within the polypeptide is the glutamine, and the amino acid at position i + 2 within the polypeptide is the first valine.
[0125] In certain embodiments, the amino acids capable of being cross-linked are at position within the polypeptide and at position i + 4 within the polypeptide, and the amino acid at position i is (S)-2-(4'-pentenyl)alanine (S5) and the amino acid at position i + 4 is S5; or at position i within the polypeptide and another amino acid at position i + 3 within the polypeptide, and the amino acid at position i is (R)-2-(4'-pentenyl)alanine (R5) or (R)-2-(2'~ propenyl)alanine (R3) and the amino acid at position i + 3 is S5; or the amino acids capable of being cross-linked are at position i within the polypeptide and at position i + 7 within the polypeptide, and the amino acid at position i is (R)-2-(7'-octenyl)alanine (R8) and the amino acid at position i + 7 is S5.
[0126] In certain embodiments, the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine, wherein the polypeptide is a first polypeptide, and wherein the polypeptide further comprises a second polypeptide comprising at least two non-natural amino acids capable of forming an internal cross-link, wherein the non-natural amino acids are the same or are different, wherein each of the non-natural amino acids includes a moiety, wherein the moieties are capable of undergoing a reaction to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the second polypeptide, wherein the second polypeptide has from the N- to C-terminus (1 ) a glutamine one amino acid position before a first valine, and (2) optionally (i) wherein the first valine is three amino acid positions before a second valine, and (ii) wherein the second valine is one amino acid position before a leucine.
[0127] In certain embodiments, (a) the non-natural amino acids capable of being cross- linked are at position i within the first polypeptide and at position i + 3, i + 4, or i + 7 within the first polypeptide; and wherein the amino acid at position i + 6 within the first polypeptide is the leucine, the amino acid at position i + 9 within the first polypeptide is the alanine, and the amino acid at position / + 10 within the first polypeptide is the isoleucine; and (b) non- natural the amino acids capable of being cross-linked are at position i within the second polypeptide and at position i + 3, i + 4, or i + 1 within the second polypeptide; and wherein the amino acid at position i + 1 within the second polypeptide is the glutamine, and the amino acid at position i + 2 within the second polypeptide is the first valine. [0128] In certain embodiments, (a) the non-natural amino acids capable of being cross- linked in the first polypeptide are at position i within the first polypeptide and at position i + 4 within the first polypeptide, and the amino acid at position i is (S)-2-(4'-pentenyl)alanine (S5) and the amino acid at position i + 4 is S5; or at position i within the first polypeptide and another amino acid at position i + 3 within the first polypeptide, and the amino acid at position i is (i?)-2-(4'-pentenyl)alanine (R5) or (R)-2-(2'-propenyl)alanine (R3) and the amino acid at position i + 3 is S5; or at position i within the first polypeptide and at position i + 7 within the first polypeptide, and the amino acid at position i is (R)-2-(7'-octenyl)alanine (R8) and the amino acid at position i + 7 is S5; and (b) the non-natural amino acids capable of being cross-linked in the second polypeptide are at position i within the second polypeptide and at position i + 4 within the second polypeptide, and the amino acid at position i is (S)-2- (4'-pentenyl)alanine (S5) and the amino acid at position i + 4 is S5; or at position i within the second polypeptide and another amino acid at position i + 3 within the second polypeptide, and the amino acid at position i is (i?)-2-(4'-pentenyl)alanine (R5) or (R)-2-(2'- propenyl)alanine (R3) and the amino acid at position / + 3 is S5; or at position / within the second polypeptide and at position i + 7 within the second polypeptide, and the amino acid at position i is (i?)-2-(7'-octenyl)alanine (R8) and the amino acid at position i + 7 is S5.
[0129] In an embodiment, the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, comprising a modified amino acid sequence of EELAGSLARAI (SEQ ID NO: 1), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO: 4); wherein the modification of the sequence is of at least two of the amino acids within the sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein each of the non- natural amino acids include a moiety, wherein each moiety is capable of undergoing a reaction with a moiety of one other of the non-natural amino acids to form a covalent crosslink with each other, wherein the covalent cross-link is internal to the sequence; and wherein if the non-natural amino acids form an internal cross-link with each other, the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
[0130] In certain embodiments, e.g., with one polypeptide, exactly two of the amino acids are replaced with non-natural amino acids. In certain embodiments, two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5. [0131] In certain embodiments, the modified amino acid sequence is of EELAGSLARAI (SEQ ID NO: 1), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second modified amino acid sequence of
EKGAAQVAAVLAQ (SEQ ID NO: 2); wherein the modification of the second sequence is of at least two of the amino acids within the second sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein each of the non-natural amino acids include a moiety, wherein each moiety is capable of undergoing a reaction with a moiety of one other of the non-natural amino acids to form a covalent crosslink with each other, wherein the covalent cross-link is internal to the second sequence.
[0132] In certain embodiments, the modified amino acid sequence is of EELATRLSQAI (SEQ ID NO: 3), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second modified amino acid sequence of
EKAAAQVAAVLAQ (SEQ ID NO: 4); wherein the modification of the second sequence is of at least two of the amino acids within the second sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein each of the non-natural amino acids include a moiety, wherein each moiety is capable of undergoing a reaction with a moiety of one other of the non-natural amino acids to form a covalent crosslink with each other, wherein the covalent cross-link is internal to the second sequence.
[0133] In certain embodiments, wherein exactly four of the amino acids are non-natural amino acids. In certain embodiments, two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5, and another two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5.
[0134] In the formula below, the groups for each formula are independently provided for each group. For example, R3 of formula (I) is independently selected of R3 of formula (la).
[0135] In an embodiment, the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence modified according to the formula (I):
Figure imgf000023_0001
wherein Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl; R3 of formula (I) is alkylene, alkenylene, alkynylene, or [R4-K'-R4 ,]n, each of which is substituted with 0-6 R5; R4 and Rr are independently alkylene, alkenylene, or alkynylene; R5 is independently halo, alkyl, OR6, N(R6)2, SR6, SOR6, SO2R6, CO2R6, 6, a fluorescent moiety, or a radioisotope; K' is independently O, S, SO, SO2, CO, C02, CONR6, or
Figure imgf000023_0002
Re is independently H, alkyl, or a therapeutic agent; n is independently an integer from 1-4; [Xaa]x has 2 to 6 amino acids; [Xaa]w, [Xaa]x, and [Xaa]y taken together have the amino acid sequence of EELAGSLARAI (SEQ ID NO: 1), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the amino acids within the sequence are replaced with the residues A and B; and wherein the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex
(LUBAC).
[0136] In certain embodiments, the modified amino acid sequence is of EELAGSLARAI (SEQ ID NO: 1), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second amino acid sequence modified according to the formula (la):
Figure imgf000024_0001
wherein Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl; R3 is alkylene, alkenylene, alkynylene, or [R4-K'-R4']n, each of which is substituted with 0-6 R5; R4 and ^ are independently alkylene, alkenylene, or alkynylene; R5 is independently halo, alkyl, OR6, N(R6)2, SR6, SOR6, SO2R6, CO2R6, R6, a fluorescent moiety, or a radioisotope; K' is independently O, S, SO, SO2, CO, CO2, CONR6, or
Figure imgf000024_0002
R6 is independently H, alkyl, or a therapeutic agent; n is independently an integer from 1 -4; [Xaa]x has 2 to 6 amino acids; [Xaa]w, [Xaa]x, and [Xaa]y taken together have the amino acid sequence of EKGAAQVAAVLAQ (SEQ ID NO: 2), wherein two of the amino acids within the sequence are replaced with the residues A and B.
[0137] In certain embodiments, the modified amino acid sequence is of EELATRLSQAI (SEQ ID NO: 3), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second amino acid sequence modified according to the formula (la):
Figure imgf000025_0001
wherein Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl; R3 is alkylene, alkenylene, alkynylene, or [R4-K'-R4']n, each of which is substituted with 0-6 R5; R4 and R4' are independently alkylene, alkenylene, or alkynylene; R5 is independently halo, alkyl, OR6, N(R6)2, SR6, SOR6, SO2R6, CO2R6, 6, a fluorescent moiety, or a radioisotope; K' is independently O, S, SO, SO2, CO, CO2, CONR6, or
Figure imgf000025_0002
R6 is independently H, alkyl, or a therapeutic agent; n is independently an integer from 1-4; [Xaa]x has 2 to 6 amino acids; [Xaa]w, [Xaa]x, and [Xaa]y taken together have the amino acid sequence of EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the amino acids within the sequence are replaced with the residues A and B.
[0138] In an embodiment, the invention provides a polypeptide, or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence modified according to the formula (II):
Figure imgf000026_0001
wherein Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl; R3 and R of formula (II) are independently alkenyl, alkynyl, azido, amino, carboxylic acid, or sulfide; [Xaa]x has 2 to 6 amino acids; [Xaa]w, [Xaa]x, and [Xaa]y taken together have the amino acid sequence of EELAGSLARAI (SEQ ID NO: 1), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the amino acids within the sequence are replaced with the residues A and B; and wherein if A and B form an internal cross-link, the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex
(LUBAC). In certain embodiments, both R3 and R3' are alkenyl.
[0139] In certain embodiments, the modified amino acid sequence is of EELAGSLARAI (SEQ ID NO: 1), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second amino acid sequence modified according to the formula (Ila):
Figure imgf000027_0001
wherein Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl; R3 and R3' are independently alkenyl, alkynyl, azido, amino, carboxylic acid, or sulfide; [Xaa]x has 2 to 6 amino acids; [Xaa]w, [Xaa]x, and [Xaa]y taken together have the amino acid sequence of EKGAAQVAAVLAQ (SEQ ID NO: 2), wherein two of the amino acids within the sequence are replaced with the residues A and B. In certain embodiments, both R3 and R3* are alkenyl.
[0140] In certain embodiments, the modified amino acid sequence is of EELATRLSQAI (SEQ ID NO: 3), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second amino acid sequence modified according to the formula (Ila):
A
Figure imgf000028_0001
wherein Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl; R3 and R3' are independently alkenyl, alkynyl, azido, amino, carboxylic acid, or sulfide; [Xaa]x has 2 to 6 amino acids; [Xaa]w, [Xaa]x, and [Xaa]y taken together have the amino acid sequence of EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the amino acids within the sequence are replaced with the residues A and B. In certain embodiments, both R3 and R3' are alkenyl.
[0141] In certain embodiments, [Xaa]x has 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, or 6 amino acids.
[0142] Halo includes any halogen, e.g., F, CI, Br, I.
[0143] As used herein, unless otherwise specified, the term "alkyl" means a saturated straight chain or branched non-cyclic hydrocarbon having an indicated number of carbon atoms (e.g., C1 -C20, Ci -Ci o, C1 -C4, C1 -C6, etc.). An alky group may have 1 , 2, 3, 4, 5, 6, 7, 8, or more carbons. Representative saturated straight chain alkyls include -methyl, -ethyl, -n- propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n-nonyl and -n-decyl; while representative saturated branched alkyls include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, - isopentyl, 2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2- methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylbutyl, 2,3- dimethylpentyl, 2,4-dimethylpentyl, 2,3 -dimethylhexyl, 2,4-dimethylhexyl, 2,5- dimethylhexyl, 2,2-dimethylpentyl, 2,2-dimethylhexyl, 3,3-dimtheylpentyl, 3,3- dimethylhexyl, 4,4-dimethylhexyl, 2-ethylpentyl, 3-ethylpentyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, 2-methyl-4-ethylpentyl, 2- methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-methyl-4-ethylhexyl, 2,2-diethylpentyl, 3,3- diethylhexyl, 2,2-diethylhexyl, 3,3-diethylhexyl and the like. "Alkenyl" means an unsaturated straight chain or branched non-cyclic hydrocarbon having an indicated number of carbon atoms (e.g., C1 -C20, C1 -C10, C1-C4, C1 -C6, etc.), where at least one carbon-carbon bond is a double bond. An alkenyl group may have 1, 2, 3, 4, 5, 6, 7, 8, or more carbons.
"Alkynyl" means an unsaturated straight chain or branched non-cyclic hydrocarbon having an indicated number of carbon atoms (e.g., C1 -C20, C1 -C10, C1-C4, C1-C6, etc.), where at least one carbon-carbon bond is a triple bond. An alkynyl group may have 1 , 2, 3, 4, 5, 6, 7, 8, or more carbons. As understood in the art, "alkylene," "alkenylene," and "alkynylene" are the bivalent radical forms of alkyl, alkenyl, and alkynyl, respectively.
[0144] The term "cycloalkyl," as used herein, means a cyclic alkyl moiety containing from, for example, 3 to 6 carbon atoms, preferably from 5 to 6 carbon atoms. Examples of such moieties include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
"Cycloalkylalkyl" is a cycloalkyl as defined above substituted with an alkyl as defined above.
[0145] The term "heterocyclyl" means a cycloalkyl moiety having one or more heteroatoms selected from nitrogen, sulfur, and/or oxygen. Preferably, a heterocyclyl is a 5 or 6-membered monocyclic ring and contains one, two, or three heteroatoms selected from nitrogen, oxygen, and/or sulfur. The heterocyclyl can be attached to the parent structure through a carbon atom or through any heteroatom of the heterocyclyl that results in a stable structure. Examples of such heterocyclic rings are pyrrolinyl, pyranyl, piperidyl,
tetrahydrofuranyl, tetrahydrothiopheneyl, and morpholinyl. "Heterocyclylalkyl" is a heterocyclyl as defined above substituted with an alkyl as defined above.
[0146] As used herein, unless otherwise specified, the term "alkylamino" means
-NH(alkyl) or -N(alkyl)(alkyl), wherein alkyl is defined above. As used herein, unless otherwise specified, the term "cycloalkylamino" means -NH(cycloalkyl) or
-N(cycloalkyl)(cycloalkyl), wherein cycloalkyl is defined above.
[0147] The term "aryl" refers to an unsubstituted or substituted aromatic carbocyclic moiety, as commonly understood in the art, and includes monocyclic and polycyclic aromatics such as, for example, phenyl, biphenyl, naphthyl, anthracenyl, pyrenyl, and the like. An aryl moiety generally contains from, for example, 6 to 30 carbon atoms, preferably from 6 to 18 carbon atoms, more preferably from 6 to 14 carbon atoms and most preferably from 6 to 10 carbon atoms. It is understood that the term aryl includes carbocyclic moieties that are planar and comprise 4n+2 π electrons, according to Huckel's Rule, wherein n = 1, 2, or 3. "Arylalkyl" means an aryl as defined above substituted with an alkyl as defined above.
[0148] The term "heteroaryl" refers to aromatic 4, 5, or 6 membered monocyclic groups, 9 or 10 membered bicyclic groups, and 11 to 14 membered tricyclic aryl groups having one or more heteroatoms (O, S, or N). Each ring of the heteroaryl group containing a heteroatom can contain one or two oxygen or sulfur atoms and/or from one to four nitrogen atoms provided that the total number of heteroatoms in each ring is four or less and each ring has at least one carbon atom. The nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen atoms may optionally be quatemized. Illustrative examples of heteroaryl groups are pyridinyl, pyridazinyl, pyrimidyl, pyrazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1 ,2,3,)- and (l ,2,4)-triazolyl, pyrazinyl, pyrimidinyl, tetrazolyl, furyl, thiophenyl, isothiazolyl, thiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrrolo[2,3-c]pyridinyl, pyrrolo[3,2- c]pyridinyl, pyrrolo[2,3-b]pyridinyl, pyrrolo[3,2-b]pyridinyl, pyrrolo[3,2-d]pyrimidinyl, and pyrrolo[2,3-d]pyrimidinyl. "Heteroarylalkyl" is a heteroaryl as defined above substituted with an alkyl as defined above.
[0149] Whenever a range of the number of atoms in a structure is indicated (e.g., a Ci -Cs, Ci-C6, C1 -C4, or C1 -C3 alkyl, haloalkyl, alkylamino, alkenyl, etc.), it is specifically contemplated that any sub-range or individual number of carbon atoms falling within the indicated range also can be used. Thus, for instance, the recitation of a range of 1-8 carbon atoms (e.g., Ci -Cs), 1-6 carbon atoms (e.g., C1 -C6), 1-4 carbon atoms (e.g., C1-C4), 1-3 carbon atoms (e.g., C1 -C3), or 2-8 carbon atoms (e.g., C2-Cg) as used with respect to any chemical group (e.g., alkyl, haloalkyl, alkylamino, alkenyl, etc.) referenced herein encompasses and specifically describes 1 , 2, 3, 4, 5, 6, 7, or 8 carbon atoms, as appropriate, as well as any sub-range thereof (e.g., 1-2 carbon atoms, 1 -3 carbon atoms, 1 -4 carbon atoms,
1 - 5 carbon atoms, 1 -6 carbon atoms, 1 -7 carbon atoms, 1-8 carbon atoms, 2-3 carbon atoms,
2- 4 carbon atoms, 2-5 carbon atoms, 2-6 carbon atoms, 2-7 carbon atoms, 2-8 carbon atoms,
3- 4 carbon atoms, 3-5 carbon atoms, 3-6 carbon atoms, 3-7 carbon atoms, 3-8 carbon atoms,
4- 5 carbon atoms, 4-6 carbon atoms, 4-7 carbon atoms, 4-8 carbon atoms, 5-6 carbon atoms,
5- 7 carbon atoms, 5-8 carbon atoms, 6-7 carbon atoms, or 6-8 carbon atoms, as appropriate).
[0150] R3 and R3 described in the formulas herein are capable of forming a cross-link. The following provides exemplary reactions for forming a cross-link between R3 and R3 . Alkene-alkene, alkene-alkyne, and alkyne-alkyne pairs can react by olefin metathesis. A sulfide-sulfide pair can react under oxidizing conditions. Azide-alkyne pairs can react to form triazoles. Carboxylic acid-amine pairs can react to form lactams. Carboxylic acid- sulfide pairs could react to form thioesters.
[0151] Any of the above embodiments of the inventive polypeptides, as appropriate, may have a sequence such that a polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine. Any of the above embodiments of the inventive polypeptides, as appropriate, may have a sequence such that a polypeptide has from the N- to C-terminus a glutamine one amino acid position before a first valine, optionally wherein the first valine is three amino acid positions before a second valine, wherein the second valine is one amino acid position before a leucine. These amino acids may be positioned within the polypeptide such that the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
[0152] Tables 1 and 2 below present certain polypeptide sequences of the invention compared to wild-type SHARPIN sequences, showing the amino acids that are conserved among the inventive polypeptides, and including possible locations for cross-linkages. Non- natural amino acids are shown. "R5/R3" indicates that either R5 or R3 may be used at that amino acid position. In embodiments, the polypeptides with non-natural amino acids may include any amino acids shown to be N-terminal to the amino acids of the polypeptide with non-natural amino acids and in the natural sequence. For example, in Table 1 A, any polypeptide with non-natural amino acids can include the arginine and/or glutamic acid- arginine directly N-terminal to the amino acids shown in the SEQ ID NOs.
Table 1A
Figure imgf000032_0001
Table IB
Polypeptides Based on Human SHARPIN sequence
AA SEQ ID ]
conserved 7 14 15 16 17 18 19 20 21
E R E E E E E E E E E
E R8 i E S5 i E R5/R3i E E E
L L L L L L R5/R3i L L
A A A A A A A A A
G G R8 i G S5 i S5 i + 3 G G G
S S S S5 i+4 S S S5 i + 3 S R5/R3i
L L L L L L L L L L
A A A A A A A A A
R S5 i + 7 R R S5 i + 4 R R R5/R3i SS i + 3
A A A A A A A A A A
I I I I I I I I I I
A A S5 i + 7 A A A A S5 i + 3 A Table 1C
Figure imgf000033_0002
Table ID
Figure imgf000033_0001
Table IE
Figure imgf000034_0001
[0153] Any sequence of Tables 1C-1E can have a histidine added directly to its C- terminus.
[0154] The C-terminus of any of the polypeptides of Table 1A or IB may be linked to the N-terminus of any of the polypeptides of Tables 1C-1E, directly or with the use of one or more spacer amino acids. Such a polypeptide may have 0, 1 , or 2 cross-links.
Table 2A
Figure imgf000035_0002
Table 2B
Figure imgf000035_0001
Table 2C
Figure imgf000036_0002
Table 2D
Figure imgf000036_0001
Table 2E
Figure imgf000037_0001
[0155] Any sequence of Tables 2C-2E can have a histidine added directly to its C- terminus.
[0156] The C-terniinus of any of the polypeptides of Table 2A or 2B may be linked to the N-tenninus of any of the polypeptides of Tables 2C-2E, directly or with the use of one or more spacer amino acids. Such a polypeptide may have 0, 1 , or 2 cross-links.
[0157] Any of the inventive polypeptides described herein can have a tryptophan added directly to the N- or C- terminus or through the use of one or more spacer amino acids.
[0158] The polypeptides may be synthesized using any suitable method. For example, the process may include synthesis, ring closing metathesis (RCM) and capping for a single helix and partial synthesis, RCM, hydrogenation, synthesis, RCM, and capping for two helices. In embodiments, the side chains of non-natural amino acids, wherein each non-natural ammo acid includes a moiety, wherein each moiety is capable of undergoing a reaction with a moiety of one other of the non-natural amino acids to form a covalent cross-link, can be covalently linked (e.g., R3 to S5, S5 to S5, R5 to S5, or R8 to S5) in the presence of a catalyst to produce the "staple" of the polypeptide. Methods of synthesis that may be used are described in Bird et al., Methods in Enzymology, 446: 369-386 (2008), incorporated herein by reference, and Shim et al., Chem. Biol. Drug Des., 82: 635-642 (2013), incorporated herein by reference. Specifically, methods of making hydrocarbon stapled polypeptides are known in the art and have been described (see, e.g., the Examples and Verdme et al., "Stapled Peptides for Intracellular Drug Targets" in Methods in Enzymology, 503: 3-23 (2012), which is incorporated herein by reference in its entirety).
[0159] In certain embodiments, the polypeptide includes a capping group, a linker group, or both a capping group and linker group. Capping groups include fluorescein thiourea (FITC) and biotin (Bt). An example of an N-terminal capping group is acetyl. An example of a C-terminal capping group is the amino group, such that the C-terminus is amidated. The linker group can be one or more naturally occurring a-amino acids (e.g., A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V), non-natural a-amino acids, natural β-amino acids (e.g., β-alanine or in the form of N-P-Fmoc-P-alanine), and/or non-natural β-amino acids. In certain embodiments, the polypeptide includes a linker group, wherein the linker group is β- alanine or N^-Fmoc^-alanine. In certain embodiments, the polypeptide includes an N- terminal amino protecting group, a C-terminal carboxyl protecting group, or both an N- terminal amino protecting group and a C-terminal carboxyl protecting group. Protecting groups include fluorenylmethoxy-carbonyl (Fmoc) and any suitable amino protecting group disclosed in Greene et al., Protecting Groups in Organic Synthesis, 3rd ed., (John Wiley & Sons, 1999), the entirety of which is incorporated herein by reference. Carboxylic acid protecting groups include groups that form an amino-, silyl-, alkyl-, alkenyl-, aryl-, or arylalkyl-protected carboxylic acid. Other suitable carboxylic acid protecting groups are disclosed in Greene et al., supra. In certain embodiments, the polypeptide includes a linker group, wherein the linker group is β-alanine or N-P-Fmoc^-alanine located between an N- terminus protective group or capping group and the first amino acid position corresponding to SHARPIN-derived sequence. In certain embodiments, the polypeptide includes a linker group, wherein the linker group is β-alanine or
Figure imgf000038_0001
located between a C- terminus protective group or capping group and the last amino acid position corresponding to SHARPIN-derived sequence.
[0160] In the inventive polypeptides, one or more peptide bonds may be replaced by a different bond that may increase the stability of the polypeptide in the body. Peptide bonds can be replaced by: a retro-inverso bond (C(O)-NFI); a reduced amide bond (NH-CHb); a thiomethylene bond (S-CH? or CFb-S); an oxomethylene bond (O-CH? or Cff^O); an ethylene bond (CH2-CH2); a thioamide bond (C(S) -NH); a trans-olefin bond (CH=CH); a fluoro substituted trans-olefin bond (CF=CH); a ketomethylene bond (C(O)-CHR) or CHR-C(O) wherein R is H or CH3; and a fluoro-ketomethylene bond (C(O)-CFR or CFR-C(O) wherein R is H or F or CH3.
[0161] The inventive polypeptides can be modified by: acetylation, amidation, biotinylation, cinnamoylation, farnesylation, fluoresceination, formylation, myristoylation, palmitoylation, phosphorylation (Ser, Tyr or Thr), stearoylation, succinylation and sulfurylation. The polypeptides of the invention may also be conjugated to, for example, polyethylene glycol (PEG); alkyl groups (e.g., C1-C20 straight or branched alkyl groups); fatty acid radicals; and combinations thereof.
[0162] Each of U.S. Patent No. 8,889,632 and U.S. Patent Pre-Grant Publication No. US 2016/0031957 is incorporated herein by reference in its entirety, including with respect to the disclosure regarding capping groups, linker groups, protecting groups, other moieties of modifying amino acids, modified amino acids, etc.
[0163] Amino acids of the inventive polypeptides that are not identified specifically, any non-natural amino acid capable of forming a cross-link that is recited is specifically recited, may be substituted using amino acid substitutions, so long as the polypeptides are capable of inhibiting LUBAC. Such substitutions are preferably not amoung the conserved amino acids of the inventive polypeptides as shown herein. Such substitutions may be conservative substitutions. Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same or similar chemical or physical properties. For instance, the conservative amino acid substitution can be an acidic/negatively charged polar amino acid substituted for another acidic/negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic/positively charged polar amino acid substituted for another basic/positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain substituted for another uncharged amino acid with a polar side chain (e.g., Gly, Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid with a beta-branched side-chain substituted for another amino acid with a beta-branched side-chain (e.g., He, Thr, and Val), an amino acid with an aromatic side-chain substituted for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc. Also, substitution of amino acids that are not shown as conserved herein may be accomplished using amino acids having high helical propensity, such as a-aminoisobutyric acid (Aib). [0164] In an embodiment, the invention also provides a substance, the substance comprising a polypeptide, the polypeptide having a sequence consisting essentially of or consisting of ELAGSLARAIA (SEQ ID NO: 78), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAIA (SEQ ID NO: 79), or EKAAAQVAAVLAQ (SEQ ID NO: 4). In an embodiment, the invention also provides a polypeptide, the polypeptide consisting essentially of or consisting of the sequence ELAGSLARAIA (SEQ ID NO: 78), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAIA (SEQ ID NO: 79), or EKAAAQVAAVLAQ (SEQ ID NO: 4). Any of these may be isolated, purified, modified, or any combination thereof, as described elsewhere herein.
[0165] Table 3 below presents certain polypeptide sequences of the invention compared to wild-type SHARPIN sequences.
Table 3
Figure imgf000040_0001
[0166] The inventive polypeptides can be any suitable length of amino acids. For example, any of the inventive sequences can have an additional 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids on either the N-terminus or C-terminus or both, as long as when cross- linked the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex.
[0167] Any of the inventive polypeptides may be isolated. Any of the inventive polypeptides may be purified. By "isolated" is meant the removal of a substance (e.g., a polypeptide) from its natural environment. By "purified" is meant that a given substance (e.g., a polypeptide), whether one that has been removed from nature (e.g., a protein enzymatically cleaved into polypeptides) or synthesized (e.g., by polypeptide synthesis), has been increased in purity, wherein "purity" is a relative term, not "absolute purity." It is to be understood, however, that polypeptides may be formulated with diluents or adjuvants and still for practical purposes be isolated. For example, polypeptides can be mixed with an acceptable earner or diluent when used for introduction into cells. [0168] The inventive polypeptides described herein may be provided in the form of a salt, e.g., a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts, for example, include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example, -toluenesulphonic acid.
[0169] In an embodiment, the invention also provides a pharmaceutical composition comprising an effective amount of an inventive polypeptide described herein. Thus, one or more inventive polypeptides described herein can be administered alone or in a composition (e.g., formulated in a pharmaceutically acceptable composition). Such a composition comprises a carrier (e.g., a pharmaceutically acceptable carrier), such as those known in the art. A pharmaceutically acceptable carrier (or excipient) preferably is chemically inert to the inventive polypeptide and has few or no detrimental side effects or toxicity under the conditions of use. The choice of earner is determined, in part, by the particular method used to administer the composition.
[0170] Carrier formulations suitable for parenteral, oral, nasal (and otherwise inhaled), topical, and other administrations can be found in Remington 's Pharmaceutical Scie?ices 17th ed., Mack Publishing Co., Easton, PA (2000), which is incorporated herein in its entirety by reference thereto. Requirements for effective pharmaceutical carriers in parenteral and injectable compositions are well known to those of ordinary skill in the art. See, e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 ( 1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 ( 1986). Accordingly, there is a wide variety of suitable formulations of the composition.
[0171] The composition can contain suitable buffering agents, including, for example, acetate buffer, citrate buffer, borate buffer, or a phosphate buffer. The pharmaceutical composition also, optionally, can contain suitable preservatives, such as benzalkonium chloride, chlorobutanol, parabens, and thimerosal.
[0172] The composition can be presented in unit dosage form and can be prepared by any suitable method, many of which are well known in the art of pharmacy. Such methods include the step of bringing the inventive polypeptide into association with a carrier that constitutes one or more accessory ingredients. In general, the composition is prepared by uniformly and intimately bringing the inventive polypeptide into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
[0173] The composition can be administered using any suitable method including, but not limited to parenteral, oral, nasal (or otherwise inhaled), and topical administration. Delivery systems useful in the context of the invention include time-released, delayed-release, and sustained-release delivery systems.
[0174] A composition suitable for parenteral administration conveniently comprises a sterile aqueous preparation of the inventive polypeptide, which may be isotonic with the blood of the recipient. This aqueous preparation can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
[0175] Sterile powders for sterile injectable solutions can be prepared by vacuum drying and/or freeze-drying to yield a powder of the inventive polypeptide, optionally, in association with a filler or diluent.
[0176] A composition suitable for oral administration can be formulated in discrete units such as capsules, cachets, tablets, or lozenges, each containing a predetermined amount of the inventive polypeptide as a powder or granules. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine, with the inventive polypeptide being in a free-flowing form, such as a powder or granules, which optionally is mixed with a binder, disintegrant, lubricant, inert diluent, surface inventive polypeptide, or discharging agent. Molded tablets comprised of a mixture of the inventive polypeptide with a suitable carrier may be made by molding in a suitable machine.
[0177] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active inventive polypeptide, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In certain embodiments for parenteral administration, the proteins, polypeptides, and polypeptides of the invention are mixed with solubilizing agents such a Cremophor, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, or any combination thereof.
[0178] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution, and 1 ,3-butanediol. In addition, sterile, fixed oils can be employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.
[0179] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
[0180] Topical formulations comprise at least one inventive polypeptide dissolved or suspended in one or more media, such as mineral oil, petroleum, polyhydroxy alcohols, or other bases used for topical pharmaceutical formulations. Transdermal formulations may be prepared by incorporating the inventive polypeptide in a thixotropic or gelatinous carrier such as a cellulosic medium, e.g., methyl cellulose or hydroxyethyl cellulose, with the resulting formulation then being packed in a transdermal device adapted to be secured in dermal contact with the skin of a wearer.
[0181] The amount (e.g., therapeutically effective amount) of inventive polypeptide suitable for administration depends on the specific inventive polypeptide used and the particular route of administration. In certain embodiments, for example, inventive polypeptide can be administered in a dose of about 0.5 ng to about 900 ng (e.g., about 1 ng, 25 ng, 50 ng, 100, ng, 200 ng, 300 ng, 400 ng, 500, ng, 600 ng, 700 ng, 800 ng, or any range bounded by any two of the aforementioned values), in a dose of about 1 μg to about 900 μg (e.g., about 1 μg, 2 g, 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500, μg, 600 μg, 700 μg, 800 μg, or any range bounded by any two of the aforementioned values), or in a dose of about 1 mg to about 200 mg (e.g., about 1 mg, 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, or any range bounded by any two of the aforementioned values) per kilogram body weight of the subject. Several doses can be provided over a period of days or weeks.
[0182] In an embodiment, the invention also provides a method of inhibiting the linear ubiquitin chain assembly complex (LUBAC) in a subject, which method comprises administering an effective amount of an inventive polypeptide described herein or a pharmaceutical composition of an inventive polypeptide described herein to the subject.
[0183] In an embodiment, the invention also provides a method of treating activated B- cell like diffuse large B cell lymphoma (ABC DLBCL) in a subject, which method comprises administering an effective amount of an inventive polypeptide described herein or a pharmaceutical composition of an inventive polypeptide described herein to the subject.
[0184] In an embodiment, the invention provides a method of killing ABC DLBCL that comprises administering a therapeutically effective amount of an inventive polypeptide described herein to ABC DLBCL and thereby killing (or inhibiting proliferation) of ABC DLBCL. The method can include administering a therapeutically effective amount of the inventive polypeptide to ABC DLBCL in vitro or in a subject (i.e., in vivo). The inventive polypeptide can be administered to ABC DLBCL that is in, for example, a primary cell culture or an animal model of ABC DLBCL.
[0185] In an embodiment, the invention provides a method of treating a subject that is suffering from ABC DLBCL or at risk for ABC DLBCL. The method can include administering a pharmaceutical composition comprising a therapeutically effective amount of one or more inventive polypeptides described herein to the subject. As used herein, a subject that is suffering from or at risk for ABC DLBCL can be a subject diagnosed with ABC DLBCL, a subject undergoing treatment for ABC DLBCL, a subject suspected to have ABC DLBCL, or a subject at risk for having ABC DLBCL (for example, a subject at risk for recurrence of ABC DLBCL).
[0186] The methods of using the inventive polypeptides as described herein can each further include the co-administration a second therapeutic agent. For example, the method of killing ABC DLBCL or the method of treating ABC DLBCL by administration of an inventive polypeptide can further include the co-administration of a cytotoxic, cystostatic, or antiangiogenic agent suitable for use against DLBCL. Such a method can include, for example, the co-administration of SAH-RNF31 -N, polypeptides described in US
2016/0031957 (incorporated herein by reference in its entirety), rituximab, alemtuzumab, bortezomib, dasatinib, BTK Kinase inhibitors (e.g., PCI-32765), a chemotherapeutic agent, a radiotherapeutic agent, or a combination of the foregoing. In an embodiment, the invention provides, for example, methods that include co-administration of an inventive polypeptide described herein and one or more cytotoxic agents used in CHOP, EPOCH, R-CHOP, therapeutic regimens. Such cytotoxic agents include cyclophosphamide, doxorubicin, vincristine, prednisone, etoposide, and derivatives thereof.
[0187] In an embodiment, the invention also provides a method of treating rheumatoid arthritis in a subject, which method comprises administering an effective amount of an inventive polypeptide described herein or a pharmaceutical composition of an inventive polypeptide described herein to the subject.
[0188] In an embodiment, the invention also provides a method of treating cancer that is resistant to cytotoxic chemotherapy, radiation therapy, vaccine therapy, or cytokine therapy in a subject, which method comprises administering an effective amount of an inventive polypeptide described herein or a pharmaceutical composition of an inventive polypeptide described herein to the subject.
[0189] In an embodiment, the invention also provides a method of sensitizing ABC DLBCL to cytotoxic agents. The method includes administering an inventive polypeptide described herein to ABC DLBCL and thereby reducing or inactivating resistance to cytotoxic therapy in the ABC DLBCL.
[0190] In another aspect, the invention provides a method of treating a disease or pathological condition mediated by LUBAC activity. In one embodiment, the invention provides a method of treating a subject with cancer that is resistant to cytotoxic
chemotherapy, radiation therapy, vaccine therapy, or cytokine therapy due to LUBAC activation of NF-KB-signaling. This method of the invention comprises administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an inventive polypeptide described herein. In certain embodiments, the method further includes co-administering a second therapeutic agent which is an agent for cytotoxic chemotherapy, radiation therapy, cytokine therapy, or vaccine therapy.
[0191] In an embodiment, the invention also provides a method for screening whether a subject suffering from cancer that is resistant to cytotoxic chemotherapy, radiation therapy, or cytokine therapy is a candidate for treatment with a LUBAC inhibitor. The method includes obtaining a sample (biopsy) from the subject that includes cancer cells, then administering to the sample an inventive polypeptide as described herein, and co-administering to the sample one or more second therapeutic agents for cytotoxic chemotherapy, radiation therapy, or cytokine therapy. The cancer cells in the sample are assayed for viability before the inventive polypeptide and the second therapeutic agent(s) can exert any cytotoxic effect (e.g., just before administration of the inventive polypeptide and second therapeutic agent(s)). The cells are subsequently assayed for viability at one or more times after the co-administration of the inventive polypeptide and the second therapeutic agent(s) to determine whether there is a significant decrease in the number of viable cancer cells. Such a decrease indicates that the subject is a candidate for treatment with a LUBAC inhibitor.
[0192] In some embodiments, the method of screening can further include treating a second control sample from the subject. An equivalent dose or amount of the second therapeutic agent(s), without inventive polypeptide, is administered to the control sample. Cancer cells in the sample are assayed for viability before the second therapeutic agent(s) can exert any effect and after administration of the second therapeutic agent(s). If the number of viable cells remaining in the control sample is greater than the number of viable cells in the sample treated by co-administration with an inventive polypeptide and the second therapeutic agent, then the inventive polypeptide has sensitized the cancer cells to the cytotoxic chemotherapy, radiation therapy, or cytokine therapy, and the subject is a candidate for treatment with a LUBAC inhibitor.
[0193] In an embodiment, the invention also provides a method of treating chronic autoinflammation, systemic lupus erythematosus, Crohn's inflammatory bowel disease, or psoriasis in a subject, which method comprises administering an effective amount of an inventive polypeptide described herein or a pharmaceutical composition of an inventive polypeptide described herein to the subject.
[0194] In another embodiment, the invention provides a method of treating a condition associated with a dysregulated inflammatory response, such as rheumatoid arthritis, chronic autoinflammation, systemic lupus erythematosus, Crohn's inflammatory bowel disease, and psoriasis. The method can include administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an inventive polypeptide described herein.
[0195] The terms "treat," "treating," "treatment," and "therapeutically effective" used herein do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment, which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive method can provide any amount of any level of treatment. Furthermore, the treatment provided by the inventive method can include the treatment of one or more conditions or symptoms of the disease being treated. [0196] The terms "treat," "treating," "treatment," "therapeutically effective," "inhibit," etc. used herein do not necessarily imply 100% or complete treatment/inhibition/reduction. Rather, there are varying degrees, which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive polypeptides and methods can provide any amount of any level of treatment/inhibition/reduction. Furthermore, the treatment provided by the inventive method can include the treatment of one or more conditions or symptoms of the disease being treated.
[0197] "Inhibiting the linear ubiquitin chain assembly complex (LUBAC)" as used herein means at least a 20% reduction in LUBAC activity as assayed using the in vitro ubiquitin assay as described in the Example.
[0198] The terms "co-administering," "co-administration" and "co-administered" used herein refer to the administration of an inventive polypeptide described herein and one or more additional therapeutic agents sufficiently close in time to (i) enhance the effectiveness of the inventive polypeptide or the one or more additional therapeutic agents and/or (ii) reduce an undesirable side effect of the inventive polypeptide or the one or more additional therapeutic agents. In this regard, the inventive polypeptide can be administered first, and the one or more additional therapeutic agents can be administered second, or vice versa.
Alternatively, the inventive polypeptide and the one or more additional therapeutic agents can be co-administered simultaneously.
[0199] The term "subject" is used herein, for example, in connection with therapeutic and screening methods, to refer to human or animal subjects (e.g., mammals). Animal subjects include, but are not limited to, animal models, such as, mammalian models of conditions or disorders associated dysregulated LUBAC-signaling. For example, the subject can be an animal model of ABC DLBCL, or a cancer that is resistant to cytotoxic chemotherapy, radiation therapy, or cytokine therapy. A subject can also be an animal model of an autoimmune disorder associated with dysregulated innate immune response. Alternatively, a subject can be a human patient suffering from or at risk for (i) ABC DLBCL, (ii) a cancer that is resistant to cytotoxic chemotherapy, radiation therapy, or cytokine therapy, or (iii) an autoimmune disorder associated with dysregulated innate immune response.
[0200] In an embodiment, the invention also provides kits suitable for carrying out the methods of the invention. Typically, a kit comprises two or more components required for performing a therapeutic or screening method of the invention. Kit components include, but are not limited to, one or more inventive polypeptides of the invention, appropriate reagents, and/or equipment. A kit can comprise one or more inventive polypeptides of the invention and a second therapeutic agent, e.g., a cytotoxic, cystostatic, or antiangiogenic agent.
Generally, the kit includes inventive polypeptide of the invention suitably packaged, e.g., in a vial, pouch, ampoule, and/or any container appropriate for a therapeutic or screening method. Kit components can be provided as concentrates (including lyophilized compositions), which may be further diluted prior to use, or the kit components can be provided at the
concentration intended for use. When a polypeptide of the invention is intended to be used in vivo, single dosages may be provided in sterilized containers having the desired amount and concentration of agents.
[0201] In another aspect, the invention provides a method for identifying and optimizing LUBAC inhibitors. Generally, the method includes binding an inventive polypeptide described herein to the HOIL- 1 L UBL domain and evaluating the ability of a test compound to disrupt or inhibit the binding reaction. The UBL domain can be provided in context of the entire HOIL-1L protein or as a fragment thereof that includes the portion of the UBL that binds to the LUBAC inhibitor of the invention.
[0202] In one embodiment, the method can include tagging an inventive polypeptide described herein (or tagging the UBL domain) to a fluorescent moiety. The inventive polypeptide or the UBL domain can be fixed (e.g., immobilized or covalently bound) to a substrate and contacted to its fluorescently tagged partner to thereby create a substrate -bound dimer. The amount of fluorescently tagged polypeptide or UBL domain that binds to substrate can be determined, e.g., by detecting the strength of signal from the fluorescent moiety that is bound to the substrate. A fluorescence binding curve can be generated. A test compound can be added to the reaction and the ability of the test compound to disrupt or inhibit the fluorescently tagged polypeptide or UBL domain from forming a substrate-bound dimer can be measured, for example, by detecting a reduction in the signal produced by the fluorescent moiety that is bound to the substrate. A test compound that significantly reduces the fluorescence signal is a candidate inhibitor or a candidate optimized inhibitor of LUBAC.
[0203] Variations of the foregoing method can be performed. For example, a fluorescent moiety can be attached to either the polypeptide of the invention or the UBL domain, and a quencher moiety can be attached to the other. After allowing the polypeptide of the invention to bind to the UBL domain, a baseline signal of the fluorescent moiety is measured. A test compound can be added and the ability of the test compound to disrupt or inhibit the binding reaction of the polypeptide inhibitor and the UBL domain can be measured, for example, by detecting an increase in the signal produced by the fluorescent moiety. A test compound that significantly increases the fluorescence signal in this assay is a candidate inhibitor or a candidate optimized inhibitor of LUBAC. Other techniques for detecting the ability of test compounds to disrupt protein binding can be used. These include, for example, surface plasmon resonance (SPR) binding assays, co-immunoprecipitation, affinity chromatography, and the like.
[0204] As used herein a test compound can be a small molecule compound.
Alternatively, the test compound can be any polypeptide of the invention which has been modified to include one, two, three, or four substitutions, deletions, or insertions.
[0205] The following includes certain aspects of the invention.
[0206] 1. A polypeptide, or a pharmaceutically acceptable salt thereof, the polypeptide comprising:
at least two non-natural amino acids that form a covalent cross-link with each other, wherein the non-natural amino acids are the same or are different, and wherein the cross-link is internal to the polypeptide, and
(a) wherein
the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine; or the polypeptide has from the N- to C-terminus a glutamine one amino acid position before a first valine, and optionally
(i) wherein the first valine is three amino acid positions before a second valine, and
(ii) wherein the second valine is one amino acid position before a leucine;
and
(b) wherein the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
[0207] 2. The polypeptide of aspect 1 , wherein the polypeptide has from the N- to C- terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine, and
wherein the cross-link is formed from a non-natural amino acid at position i within the polypeptide and another non-natural amino acid at position z + 3, + 4, or i + 7 within the polypeptide; and wherein the amino acid at position i + 6 within the polypeptide is the leucine, the amino acid at position i + 9 within the polypeptide is the alanine, and the amino acid at position i + 10 within the polypeptide is the isoleucine.
[0208] 3. The polypeptide of aspect 1, wherein the polypeptide has from the N- to C- terminus (1) a glutamine one amino acid position before a first valine, and (2) optionally
(i) wherein the first valine is three amino acid positions before a second valine, and
(ii) wherein the second valine is one amino acid position before a leucine, and
wherein the cross-link is formed from a non-natural amino acid at position i within the polypeptide and another non-natural amino acid at position i + 3, i + 4, or i + 7 within the polypeptide; and
wherein the amino acid at position /' + 1 within the polypeptide is the glutamine, and the amino acid at position i + 2 within the polypeptide is the first valine.
[0209] 4. The polypeptide of aspect 2 or 3, wherein the cross-link of the polypeptide is: formed from the amino acid at position i within the polypeptide and another amino acid at position i + 4 within the polypeptide, and the amino acid at position i is (5)-2-(4'- pentenyl)alanine (S5) and the amino acid at position i + 4 is S5; or
formed from the amino acid at position i within the polypeptide and another amino acid at position i + 3 within the polypeptide, and the amino acid at position i is (R)-2- (4'-pentenyl)alanine (R5) or (i?)-2-(2'-propenyl)alanine (R3) and the amino acid at position i + 3 is S5; or
formed from the amino acid at position i within the polypeptide and another amino acid at position i + 1 within the polypeptide, and the amino acid at position i is (R)-2-(7'- octenyl)alanine (R8) and the amino acid at position i + 7 is S5.
[0210] 5. The polypeptide of aspect 1 , wherein the polypeptide has from the N- to C- terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine, wherein the polypeptide is a first polypeptide, and wherein the polypeptide further comprises a second polypeptide having non-natural amino acids that form a covalent cross-link internally within the second polypeptide, wherein the second polypeptide has from the N- to C-terminus (1 ) a glutamine one amino acid position before a first valine, and (2) optionally
(i) wherein the first valine is three amino acid positions before a second valine, and
(ii) wherein the second valine is one amino acid position before a leucine. [0211] 6. The polypeptide of aspect 5, wherein
(a) the cross-link in the first polypeptide is formed from a non-natural amino acid at position i within the first polypeptide and another non-natural amino acid at position i + 3, i + 4, or i + 7 within the first polypeptide; and
wherein the amino acid at position i + 6 within the first polypeptide is the leucine, the amino acid at position i + 9 within the first polypeptide is the alanine, and the amino acid at position / + 10 within the first polypeptide is the isoleucine;
and
(b) the cross-link in the second polypeptide is formed from a non-natural amino acid at position i within the second polypeptide and another non-natural amino acid at position i + 3, i + 4, or i + 7 within the second polypeptide; and
wherein the amino acid at position i + 1 within the second polypeptide is the glutamine, and the amino acid at position i + 2 within the second polypeptide is the first valine.
[0212] 7. The polypeptide of aspect 6, wherein
(a) the cross-link of the first polypeptide is:
formed from the amino acid at position i within the first polypeptide and another amino acid at position i + 4 within the first polypeptide, and the amino acid at position i is (S)-2-(4?-pentenyl)alanine (S5) and the amino acid at position + 4 is S5; or
formed from the amino acid at position i within the first polypeptide and another ammo acid at position i + 3 within the first polypeptide, and the amino acid at position is (7?)-2-(4'-pentenyl)alanine (R5) or (i?)-2-(2'-propenyl)alanine (R3) and the amino acid at position + 4 is S5; or
formed from the amino acid at position within the first polypeptide and another amino acid at position i + 7 within the first polypeptide, and the amino acid at position i is (7?)-2-(7'-octenyl)alanine (R8) and the amino acid at position + 7 is S5;
and
(b) the cross-link of the second polypeptide is:
formed from the amino acid at position i within the second polypeptide and another amino acid at position i + 4 within the second polypeptide, and the amino acid at position is (S)-2-(4'-pentenyl)alanine (S5) and the amino acid at position /' + 4 is S5; or
formed from the amino acid at position i within the second polypeptide and another ammo acid at position /' + 3 within the second polypeptide, and the amino acid at position is (R)-2-(4'-pentenyl)alanine (R5) or (R)-2-(2'-propenyl)alanine (R3) and the amino acid at position ;' + 4 is S5; or
formed from the amino acid at position i within the second polypeptide and another amino acid at position i + 7 within the second polypeptide, and the amino acid at position i is (R)-2-(7'-octenyl)alanine (R8) and the amino acid at position + 7 is S5.
[0213] 8. A polypeptide, or a pharmaceutically acceptable salt thereof, comprising a modified amino acid sequence of EELAGSLARAl (SEQ ID NO: 1), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO:
4);
wherein the modification of the sequence is of at least two of the amino acids within the sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids;
wherein the non-natural amino acids form a covalent cross-link with each other, wherein the covalent cross-link is internal to the sequence; and
wherein the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
[0214] 9. The polypeptide of aspect 8, wherein exactly two of the amino acids are non- natural amino acids.
[0215] 10. The polypeptide of aspect 8 or 9, wherein two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5.
[0216] 1 1. The polypeptide of aspect 8, wherein the modified amino acid sequence is of EELAGSLARAl (SEQ ID NO: 1), wherein the sequence is a first modified amino acid sequence, and
wherein the polypeptide further comprises a second modified amino acid sequence of EKGAAQVAAVLAQ (SEQ ID NO: 2);
wherein the modification of the second sequence is of at least two of the amino acids within the second sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids;
wherein the non-natural amino acids form a covalent cross-link with each other, wherein the covalent cross-link is internal to the second sequence. [0217] 12. The polypeptide of aspect 8, wherein the modified amino acid sequence is of EELATRLSQAI (SEQ ID NO: 3), wherein the sequence is a first modified amino acid sequence, and
wherein the polypeptide further comprises a second modified amino acid sequence of EKAAAQVAAVLAQ (SEQ ID NO: 4);
wherein the modification of the second sequence is of at least two of the amino acids within the second sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids;
wherein the non-natural amino acids form a covalent cross-link with each other, wherein the covalent cross-link is internal to the second sequence.
[0218] 13. The polypeptide of aspect 11 or 12, wherein exactly four of the amino acids are non-natural amino acids.
[0219] 14. The polypeptide of any one of aspects 1 1-13, wherein two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5, and another two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5.
[0220] 15. A polypeptide, or a pharmaceutically acceptable salt thereof, the polypeptide comprising:
at least two non-natural amino acids capable of forming an internal cross-link, wherein the non-natural amino acids are the same or are different,
wherein each of the non-natural amino acids includes a moiety, wherein the moieties are capable of undergoing a reaction to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the polypeptide; and
(a) wherein
the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine; or the polypeptide has from the N- to C-terminus (1) a glutamine one amino acid position before a first valine, and (2) optionally
(i) wherein the first valine is three amino acid positions before a second valine, and
(ii) wherein the second valine is one amino acid position before a leucine;
and
(b) wherein if the non-natural amino acids form an internal cross-link, the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC). [0221] 16. The polypeptide of aspect 15, wherein the polypeptide has from the N- to C- terminus a leucine tliree amino acid positions before an alanine, wherem the alanine is one amino acid position before an isoleucine, and
wherein the non-natural amino acids capable of being cross-linked are at position i within the polypeptide and at position i + 3, i + 4, or i + 7 within the polypeptide; and
wherein the amino acid at position i + 6 within the polypeptide is the leucine, the amino acid at position i + 9 within the polypeptide is the alanine, and the amino acid at position i + 10 within the polypeptide is the isoleucine.
[0222] 17. The polypeptide of aspect 15, wherein the polypeptide has from the N- to C- terminus (1) a glutamine one amino acid position before a first valine, and (2) optionally
(i) wherein the first valine is three amino acid positions before a second valine, and
(ii) wherein the second valine is one amino acid position before a leucine; and wherein the non-natural amino acids capable of being cross-linked are at position /' within the polypeptide and at position i + 3, i + 4, or i + 7 within the polypeptide; and
wherein the amino acid at position i + 1 within the polypeptide is the glutamine, and the amino acid at position i + 2 within the polypeptide is the first valine.
[0223] 18. The polypeptide of aspect 16 or 17, wherein the amino acids capable of being cross-linked are:
at position within the polypeptide and at position i + 4 within the polypeptide, and the amino acid at position i is (S)-2-(4'-pentenyl)alanine (S5) and the amino acid at position i + 4 is S5; or
at position i within the polypeptide and another amino acid at position i + 3 within the polypeptide, and the amino acid at position / is (R)-2-(4'-pentenyl)alanine (R5) or ( ?)-2-(2'- propenyl)alanine (R3) and the amino acid at position i + 3 is S5; or
the amino acids capable of being cross-linked are at position i within the polypeptide and at position i + 7 within the polypeptide, and the amino acid at position i is (K)-2-(T- octenyl)alanine (R8) and the amino acid at position i + 7 is S5.
[0224] 19. The polypeptide of aspect 15, wherein the polypeptide has from the N- to C- terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine, wherein the polypeptide is a first polypeptide, and wherein the polypeptide further comprises a second polypeptide comprising at least two non-natural amino acids capable of forming an internal cross-link, wherein the non- natural amino acids are the same or are different, wherein each of the non-natural amino acids includes a moiety, wherein the moieties are capable of undergoing a reaction to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the second polypeptide,
wherein the second polypeptide has from the N- to C-terminus (1) a glutamine one amino acid position before a first valine, and (2) optionally
(i) wherein the first valine is three amino acid positions before a second valine, and
(ii) wherein the second valine is one amino acid position before a leucine.
[0225] 20. The polypeptide of aspect 19, wherein
(a) the non-natural amino acids capable of being cross-linked are at position i within the first polypeptide and at position + 3, i + 4, or /' + 7 within the first polypeptide; and
wherein the amino acid at position i + 6 within the first polypeptide is the leucine, the amino acid at position i + 9 within the first polypeptide is the alanine, and the amino acid at position i + 10 within the first polypeptide is the isoleucine;
and
(b) the non-natural amino acids capable of being cross-linked are at position i within the second polypeptide and at position i + 3, i + 4, or i + 7 within the second polypeptide; and
wherein the amino acid at position i + 1 within the second polypeptide is the glutamine, and the amino acid at position + 2 within the second polypeptide is the first valine.
[0226] 21. The polypeptide of aspect 19 or 20, wherein
(a) the non-natural amino acids capable of being cross-linked in the first polypeptide are:
at position within the first polypeptide and at position i + 4 within the first polypeptide, and the amino acid at position i is (5)-2-(4'-pentenyl)alanine (S5) and the amino acid at position i + 4 is S5; or
at position i within the first polypeptide and another ammo acid at position + 3 within the first polypeptide, and the amino acid at position is (R)-2-(4'-pentenyl)alanine (R5) or ( ?)-2-(2'-propenyl)alanine (R3) and the amino acid at position / + 3 is S5; or
at position i within the first polypeptide and at position i + 7 within the first polypeptide, and the amino acid at position i is (R)-2-(7'-octenyl)alanine (R8) and the amino acid at position z + 7 is S5; and
(b) the non-natural amino acids capable of being cross-linked in the second polypeptide are:
at position i within the second polypeptide and at position i + 4 within the second polypeptide, and the amino acid at position i is (S)-2-(4'-pentenyl)alanine (S5) and the amino acid at position i + 4 is S5; or
at position i within the second polypeptide and another amino acid at position i + 3 within the second polypeptide, and the amino acid at position i is (R)-2-(4'-pentenyl)alanine (R5) or (R)-2-(2'-propenyl)alanine (R3) and the amino acid at position i + 3 is S5; or
at position i within the second polypeptide and at position + 7 within the second polypeptide, and the amino acid at position is (R)-2-(7'-octenyl)alanine (R8) and the amino acid at position i + 7 is S5.
[0227] 22. A polypeptide, or a pharmaceutically acceptable salt thereof, comprising a modified amino acid sequence of EELAGSLARAI (SEQ ID NO: 1), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAl (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO:
4);
wherein the modification of the sequence is of at least two of the amino acids within the sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids;
wherein each of the non-natural amino acids include a moiety, wherein each moiety is capable of undergoing a reaction with a moiety of one other of the non-natural amino acids to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the sequence; and
wherein if the non-natural amino acids form an internal cross-link with each other, the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
[0228J 23. The polypeptide of aspect 22, wherein exactly two of the amino acids are replaced with non-natural amino acids.
[0229] 24. The polypeptide of aspect 22 or 23, wherein two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5.
[0230] 25. The polypeptide of aspect 22, wherein the modified amino acid sequence is of EELAGSLARAI (SEQ ID NO: 1 ), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second modified amino acid sequence of EKGAAQVAAVLAQ (SEQ ID NO: 2);
wherein the modification of the second sequence is of at least two of the amino acids within the second sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids;
wherein each of the non-natural amino acids include a moiety, wherein each moiety is capable of undergoing a reaction with a moiety of one other of the non-natural amino acids to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the second sequence.
[0231] 26. The polypeptide of aspect 22, wherein the modified amino acid sequence is of EELATRLSQAI (SEQ ID NO: 3), wherein the sequence is a first modified amino acid sequence, and
wherein the polypeptide further comprises a second modified amino acid sequence of EKAAAQVAAVLAQ (SEQ ID NO: 4);
wherein the modification of the second sequence is of at least two of the amino acids within the second sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids;
wherein each of the non-natural amino acids include a moiety, wherein each moiety is capable of undergoing a reaction with a moiety of one other of the non-natural amino acids to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the second sequence.
[0232] 27. The polypeptide of aspect 25 or 26, wherein exactly four of the amino acids are non-natural amino acids.
[0233] 28. The polypeptide of any one of aspects 25-27, wherein two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5, and another two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5
[0234] 29. A polypeptide, or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence modified according to the formula (I): A B
Figure imgf000058_0001
wherein:
Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
R3 is alkylene, alkenylene, alkynylene, or
Figure imgf000058_0002
each of which is substituted with 0-6 R5;
R4 and R4' are independently alkylene, alkenylene, or alkynylene;
R5 is independently halo, alkyl, OR6, N(R6)2, SR6, SOR6, S02R6, CO?R6, Re, a fluorescent moiety, or a radioisotope;
K' is independently O, S, SO, S02, CO, C02, CONR6, or
Figure imgf000058_0003
R6 is independently H, alkyl, or a therapeutic agent;
n is independently an integer from 1 -4;
[Xaa]x has 2 to 6 amino acids;
[Xaa]w, [Xaa]x, and [Xaa]y taken together have the amino acid sequence of
EELAGSLARAI (SEQ ID NO: 1), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAl (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the amino acids within the sequence are replaced with the residues A and B; and
wherein the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
[0235] 30. The polypeptide of aspect 29, wherein the modified amino acid sequence is of EELAGSLARAI (SEQ ID NO: 1 ), wherein the sequenceis a first modified amino acid sequence, and wherein the polypeptide further comprises a second amino acid sequence modified according to the formula la):
Figure imgf000059_0001
wherein:
Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
R3 is alkylene, alkenylene, alkynylene, or [R4-K'-R4']n, each of which is substituted with 0-6 R5;
R4 and R4' are independently alkylene, alkenylene, or alkynylene;
R5 is independently halo, alkyl, OR6, N(R6)2, SR6, SOR6, SO2R6, C02R6, ¾>, a fluorescent moiety, or a radioisotope;
K' is independently O, S, SO, SO2, CO, CO2, CONR6, or
Figure imgf000059_0002
R6 is independently H, alkyl, or a therapeutic agent;
n is independently an integer from 1-4;
[Xaa]x has 2 to 6 amino acids;
[Xaa]w, [Xaa]x, and [Xaa]y taken together have the amino acid sequence of
EKGAAQVAAVLAQ (SEQ ID NO: 2), wherein two of the amino acids within the sequence are replaced with the residues A and B.
[0236] 31. The polypeptide of aspect 29, wherein the modified amino acid sequence is of EELATRLSQAI (SEQ ID NO: 3), wherein the sequenceis a first modified amino acid sequence, and
wherein the polypeptide further comprises a second amino acid sequence modified according to the formula (la): A B
Figure imgf000060_0001
wherein:
Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
R3 is alkylene, alkenylene, alkynylene, or [R4-K'-R4<]n, each of which is substituted with 0-6 R5;
R4 and R4' are independently alkylene, alkenylene, or alkynylene;
R5 is independently halo, alkyl, OR6, N(R6)2, SR6, SOR6, S02R6, C02R6, R&, a fluorescent moiety, or a radioisotope;
K' is independently O, S, SO, S02, CO, CO2, CONR6, or
Figure imgf000060_0002
R6 is independently H, alkyl, or a therapeutic agent;
n is independently an integer from 1 -4;
[Xaa]x has 2 to 6 amino acids;
[Xaa]w, [Xaa]x, and [Xaa]y taken together have the amino acid sequence of
EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the amino acids within the sequence are replaced with the residues A and B.
[0237] 32. A polypeptide, or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence modified according to the formula (II): A B
Figure imgf000061_0001
wherein:
Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
R3 and R3' are independently alkenyl, alkynyl, azido, amino, carboxylic acid, or sulfide;
[Xaa]x has 2 to 6 amino acids;
[Xaa]w, [Xaa]x, and [Xaa]y taken together have the amino acid sequence of
EELAGSLARAI (SEQ ID NO: 1 ), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the amino acids within the sequence are replaced with the residues A and B; and
wherein if A and B form an internal cross-link, the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
[0238] 33. The polypeptide of aspect 32, wherein the modified amino acid sequence is of EELAGSLARAI (SEQ ID NO: 1 ), wherein the sequenceis a first modified amino acid sequence, and
wherein the polypeptide further comprises a second amino acid sequence modified according to the formula (Ila): B
Figure imgf000062_0001
wherein:
Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
R3 and R3' are independently alkenyl, alkynyl, azido, amino, carboxylic acid, or sulfide;
[Xaa]x has 2 to 6 amino acids;
[Xaa]w, [Xaa]x, and [Xaa]y taken together have the amino acid sequence of
EKGAAQVAAVLAQ (SEQ ID NO: 2), wherein two of the amino acids within the sequence are replaced with the residues A and B.
[0239] 34. The polypeptide of aspect 32, wherein the modified amino acid sequence is of EELATRLSQAI (SEQ ID NO: 3), wherein the sequenceis a first modified amino acid sequence, and
wherein the polypeptide further comprises a second amino acid sequence modified according to the formula (Ila): A B
Figure imgf000063_0001
wherein:
Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
R3 and R3' are independently alkenyl, alkynyl, azido, amino, carboxylic acid, or sulfide;
[Xaa]x has 2 to 6 amino acids;
[Xaa]w, [Xaa]x, and [Xaa]y taken together have the amino acid sequence of
EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the amino acids within the sequence are replaced with the residues A and B.
[0240] 35. The polypeptide of any one of aspects 29-34, wherein [Xaa]x has 2 amino acids.
[0241] 36. The polypeptide of any one of aspects 29-34, wherein [Xaa]x has 3 amino acids.
[0242] 37. The polypeptide of any one of aspects 29-34, wherein [Xaa]x has 6 amino acids.
[0243] 38. A polypeptide comprising the amino acid sequence of any one of SEQ ID NOS: 8-21 , 23-41 , 43-57, or 59-77.
[0244] 39. The polypeptide of aspect 38, wherein the non-natural amino acids are not cross-linked.
[0245] 40. The polypeptide of aspect 38, wherein the non-natural amino acids are cross- linked. [0246] 41. The polypeptide of any one of aspects 1-40, wherein the polypeptide includes a capping group, a linker group, or both a capping group and linker group.
[0247] 42. The polypeptide of aspect 41, wherein the polypeptide includes a linker group, wherein the linker group is beta-alanine.
[0248] 43. The polypeptide of any one of aspects 1-40, wherein the polypeptide includes an N-terminal amino protecting group, a C-terminal carboxyl protecting group, or both an N- terminal amino protecting group and a C-terminal carboxyl protecting group.
[0249] 44. The polypeptide of any one of aspects 1-40, wherein the polypeptide includes an N-terminal acetyl.
[0250] 45. The polypeptide of any one of aspects 1-40, wherein the polypeptide C- terminus is amidated.
[0251] 46. A pharmaceutical composition comprising an effective amount of a polypeptide of any one of aspects 1-45.
[0252] 47. A method of inhibiting the linear ubiquitin chain assembly complex (LUBAC) in a subject, which method comprises administering an effective amount of a polypeptide of any one of aspects 1-45 or a pharmaceutical composition of aspect 46 to the subject.
[0253] 48. A method of treating activated B-cell like diffuse large B cell lymphoma (ABC DLBCL) in a subject, which method comprises administering an effective amount of a polypeptide of any one of aspects 1-45 or a pharmaceutical composition of aspect 46 to the subject.
[0254] 49. A method of treating rheumatoid arthritis in a subject, which method comprises administering an effective amount of a polypeptide of any one of aspects 1-45 or a pharmaceutical composition of aspect 46 to the subject.
[0255] 50. A method of treating cancer that is resistant to cytotoxic chemotherapy, radiation therapy, vaccine therapy, or cytokine therapy in a subject, which method comprises administering an effective amount of a polypeptide of any one of aspects 1-45 or a pharmaceutical composition of aspect 46 to the subject.
[0256] 51. A method of treating chronic autoinflammation, systemic lupus
erythematosus, Crohn's inflammatory bowel disease, or psoriasis in a subject, which method comprises administering an effective amount of a polypeptide of any one of aspects 1 -45 or a pharmaceutical composition of aspect 46 to the subject.
[0257] It shall be noted that the preceding are merely examples of embodiments. Other exemplary embodiments are apparent from the entirety of the description herein. It will also be understood by one of ordinary skill in the art that each of these embodiments may be used in various combinations with the other embodiments provided herein.
[0258] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
EXAMPLES
[0259] A discussion of the results of Examples 1-5 is provided below.
[0260] The LUBAC ubiquitin ligase, which is composed of three subunits, the catalytic HOIP and two accessory HOIL-IL and SHARPIN subunits, is important in various biological phenomena such as NF-κΒ activation and cell death protection (Iwai et al., Nat Rev Mol Cell Biol, 15: 503-508 (2014)). Using structural modeling based on the reported structures of homologous proteins and mutational analyses, it is shown there are important roles for the three interactions between each subunit of LUBAC in stabilization of the complex. It was also found that mHOIP and mSHARPIN cannot form the stable complex, which indicated the important role of mHOIL-lL in formation of the mouse LUBAC complex. HOIL-IL"""7""" mice were established, which has the loss of function mutation in the exon encoding the UBL, and it was found that HOIL-IL""" """ mice are embryonic lethal and exhibit phenotypes almost identical to mice lacking LUBAC ligase activity although HOIL-I L"7" mice established previously do not exhibit any overt phenotypes. Small N-terminal truncated alternative splicing product of HOIL-IL (RBCK2-like) that contains intact Not and UBL exist in the previously described HOIL-I L 7", but not HOIL-I L"""""" mice. Since the RBCK2-like product is able to form a complex with HOIP and SHARPIN, small amount of functional trimeric LUBAC exist in HOIL-I L7" mice and mice appeared to be non-phenotype. HOIL- 1 L deficiency has been separately reported in humans: one report showed that patients exhibit immunodeficiency and autoinfiammation and polyglucosan body myopathy in skeletal muscle and heart (Boisson et al., Nat Immunol, 13: 1 178-1 186 (2012)), whereas patients in other reports showed polyglucosan body myopathy without having immunological symptoms (Nilsson et al., Ann Neurol, 74: 2246-2257 (2013)). Most of the genomic mutations found in patients who exhibit only polyglucosan body myopathy seem not to affect expression of RBCK2-like product (Nilsson et al., Ann Neurol, 74: 2246-2257 (2013)).
Presence of the RBCK2-like product, corresponding mRNA of which is reported to exist in human, may determine the phenotypical difference of patients with HOIL-I L mutations. The A 1 8P mutation in the Na-region of HOIL-I L impaired HOIL-1 L-SHARP1N interaction likely because the A18P mutation may disrupt a-helix of the Na region as it is known that proline breaks a-helix. Although disruption of SHARPIN-HOIL-1L interaction profoundly destabilized LUBAC, tiny amount of trimeric LUBAC may still exist because HOIL-I L A18P could bind to HOIP. It is suspected that small amount of LUBAC containing HOIL-IL (full or RBCK2-like) may exist in patients with polyglucosan myopathy, however, the HOIL- IL mutation found in patients having immunodeficiency and autoinflamrnation appears to abolish expression of RBCK2-like product as well as HOIL-I L (Boisson et al., Nat
Immunol, 13: 1 178-1186 (2012)). Complete loss of HOIL-IL (HOIL-I L and RBCK2-like) in mice (HOIL-IL""'7'""77 mice) results in embryonic lethality with symptoms of which are distinct from human patients. It may be that differential role of HOIP-SHARPIN interaction between mouse and human in LUBAC stabilization underlies the phenotypical difference between mouse and human. It was unexpectedly found that differential involvement of SHARPIN-HOIP interaction in stabilization of LUBAC between mouse and human:
SHARPIN can stabilize HOIP in human, but not in mouse. Thus, trace amounts of LUBAC composed of SHARPIN and HOIP might exist in immunodeficiency and autoinflamrnation patients. However, it cannot be ruled out the possibility completely that in patients with immunodeficiency and autoinflamrnation do express very trace amount of product containing Na and UBL of HOIL-IL. It is noted that SHARPIN could barely be detected in MEFs of HOIL-IL""'"""" but not HOIL-IL7" mice. Considering that HOIL-IL can dimerize with SHARPIN via the Na region in the absence of HOIP, a LUBAC-independent function of SHARPIN as indicated by several previous reports (Pouwels et al., Cell Rep, 5: 619-628 (2013); Rantala et al., Nat Cell Biol, 13 : 13 15-1324 (201 1)) might be exerted by the HOIL- 1 L-SHARPIN complex. HOIL-1 , which is a shorter alternative product of HOIL-I L, lacks part of Na and the spacer region between Na and UBL (Yamanaka et al., Nat Cell Biol, 5: 336-340 (2003)). It was found that HOIL-1 is resistant to incorporation into the LUBAC complex (Kirisako et al., EMBO J, 25: 4877-4887 (2006)), possibly because HOIL-1 cannot bind to SHARPIN on account of the lack of these regions. It might be plausible that HOIL-1 exert LUBAC-independent functions (Donley et al, Oncogene, 33 : 3441 -3450 (2014);
Queisser et al., Am J Respir Crit Care Med, 190: 688-698 (2014)). Since the amino acid sequences of Na are highly conserved among HOIL-I L and SHARPIN from different species including human, it is likely that Na-mediated interactions between HOIL- I L and SHARPIN are general mechanisms for robust LUBAC complex formation. The stapled polypeptide mimicking the Na of SHARPIN (SHARPIN-Na) effectively suppressed the formation of the trimetric complex composed of the HOIP ubiquitin-associated (UBA) region and the two UBLs. The SPR analyses revealed that deletion of Na of either HOIL-1L or SHARPIN attenuated trimeric complex formation much more profoundly than mutations in the UBA2 of HOIP, which impairs HOIP-HOIL-1L interaction. Thus, it might be plausible that inhibition of SHARPIN-HOIL-IL interaction is superior to that of HOIL-1L-HOIP. The SHARPIN- Na polypeptide suppresses LUBAC mediated linear ubiquitination and IKK activation more effectively than the HOIP-N polypeptide, which inhibits the HOIL-1L-HOIP interaction, and kills the HBL-1 cells more efficiently than previously reported HOIP polypeptides (Yang et al., Cancer Discov, 4: 480-493 (2014)). Loss of LUBAC ligase activity is embryonic lethal (Shimizu et al, Mol Cell Biol, 36: 1569-1583 (2016)), however, the LUBAC ligase activity cannot be completely lost by inhibition of SHARPIN-HOIL-IL interaction because the inhibition does not affect the other interactions between the LUBAC components and LUBAC composed of HOIL-1L-HOIP or SHARPIN-HOIP can exist in human cells.
[0261] The below methods were followed for the Examples.
RT-PCR, plasmids, antibodies and reagents:
[0262] cDNA used in this study were described previously (Fujita et al., 2014; Tokunaga et al., Nature, 471 : 633-636 (201 1 ); Tokunaga et al, Nat Cell Biol, 1 1 : 123-132 (2009)). The following full-length proteins, deletion mutants, and fragments were generated from the amplified ORF of mouse HOIP; wild type (WT) (amino acids 1-1066), UBA domain (466- 630), all ZF domains (296-434), ANZF2 (Δ402-432), AUBA (Δ558-609), AUBA N-term (Δ466-549), AUBA C-term (Δ550-630). hUBA mutant [mHOIP ( 1-473)- hHOIP (480-636)- mHOIP (631 -1066)] was generated from the amplified ORF of human and mouse HOIP. The following proteins were generated from the amplified ORF of mouse HOIL-1L; before NZF (1 -189), UBL domain (1 -140), ΔΝα (37-509), UBL ΔΝα (37-161 ). The following proteins were generated from the amplified ORF of mouse SHARPIN; ΔΝα (Δ163-197), UBL ΔΝα (198-318), mutants of mHOIL-l L and mSHARPIN whose UBL domains are exchanged; S(UBL)-HOIL [SHARPIN ( 163-301 )-HOIL- 1 L (136-509)], S-H(UBL)-S [SHARPIN (1 - 167)-H0IL-1 L (7-135)-SHARPIN (302-380)]. Mutants of mHOIP (Q607A/L61 1 A/F614A, M528A/L559A, R479A/Q490A), mHOIL- 1 L (LI 5A, V19A, L 15A/V19A, A18P), mSHARPIN (Y295A, L273A, V268A, L176A, I 180A, L176A/I 180A), hHOIL-l L A18P were generated by two-step PCR. cDNAs were ligated into the appropriate epitope-tag sequences and then cloned into pcDNA3.1, pMAL-c2x (New England Biolabs, Ipswich, MA, USA), pGEX-6pl (GE Healthcare, Little Chalfont, United Kingdom), pMXs-IP, pMXs-neo, pMXs-IRES-Bsr. pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene (Cambridge, MA, USA) plasmid #42230) (Cong et al, Science, 339: 819-823 (2013)) and pSpCas9(BB)-2A-Puro (PX459) (Addgene plasmid #48139) (Cong and Zhang, Methods Mol Biol, 1239: 197-217 (2015)) were gifts from Dr. Feng Zhang.
[0263] RT-PCR of HOIL-1L +/+ or -/- MEFs were performed using sequence specific primers as follows:
Exon4_Fwd, 5 ' -GGAATGGAGACGGTGCCTATCTC-3 ' (SEQ ID NO: 81);
Exon5_Fwd, 5 ' -CGAAGCCC AGGACC AACCAGGAG-3 ' (SEQ ID NO: 82);
Exon6_Fwd, 5 ' -TGTGAGATGTGCTGTCGTGC AAG-3 ' (SEQ ID NO: 83);
Exon8_Fwd, 5'-CATTGACAGCACCTACTCATGCC-3' (SEQ ID NO: 84);
p-actin_Fwd, 5 ' -ATGGATGACGATATCGCTC-3 ' (SEQ ID NO: 85);
Exon5_Rev, 5 ' -CTGGGCTTCGGAAGGAC AGGTTC-3 ' (SEQ ID NO: 86);
Exon9_Rev, 5 ' -CTCAAGGTGCTTCGGTTCTCTG-3 ' (SEQ ID NO: 87);
ExonlO Rev, 5 ' -TCCCTGC AATTC ATGTGCTC ATG-3 ' (SEQ ID NO: 88);
β-actinj ev, 5 ' -G ATTCC ATACCCAGGAAGG-3 ' (SEQ ID NO: 89).
[0264] The following antibodies were used: FLAG (M2), Actin (A2228) (Sigma, St. Louis, MO, USA); DDDDK (PM020), HA (M180-3), HA (561) (MBL); Myc (4A6)
(Millipore, Billenca, MA, USA); GFP (JL-8) (Clontech, Mountain View, CA, USA);
SHARPIN (ab 125188) (Abeam, Cambridge, United Kingdom); HOIP (ARP43241_P050) (A viva Systems Biology Corp, San Diego, CA, USA); ΙκΒα (c-21) (Santa Cruz
Biotechnology, Dallas, TX, USE). For generating anti-mouse HOIL-1L N-term antibody, strep-tagged mouse HOIL-1L N-terminus (1 -189) was expressed in Escherichia coli (E. Coli) and then purified using Strep-Tactin Sepharose (IBA). Purified protein was used to immunize rabbits and IgG was purified from their antisera by using Protein A Sepharose (GE
Healthcare). Other antibodies and reagents were described previously (Fujita et al., Mol Cell Biol, 34: 1322-1335 (2014); Tokunaga et al., Nature, 471 : 633-636 (201 1 ); Tokunaga et al., Nat Cell Biol, 1 1 : 123-132 (2009))
[0265] Polypeptide synthesis, reverse-phase high-performance liquid chromatography purification, and amino acid analysis were performed as described previously (Bernal et al., J Am Chem Soc, 129: 2456-2457 (2007), incorporated by reference). Generation of KO cells and HOIL-1L null mice by CRISPR/Cas9:
[0266] To generate the HOIP-HOIL- I L-SHARPIN TKO MEFs, chronic dermatitis in mice (cpdm) MEFs were electroporated with pX330 plasmids containing gRNA sequence against mHOIP or mHOIL-lL using NEPA21 electroporator (NEPAGENE, Chiba, Japan). After four days of culture, cells were seeded at a low density. Colonies were picked up and expression level of LUBAC was analyzed by immunoblotting as first screening. Then, genomic regions of HOIL-1L or HOIP were amplified by PCR using the following primers: mHOIL-lL typingJFwd, 5 ' -TTGCC AAC AGGCC AATTTGATG-3 ' (SEQ ID NO: 90) and typing__Rev, 5 ' -TGCGGTGATGC ACAATATCCTG-3 ' (SEQ ID NO: 91).
mHOIP typing_Fwd, 5 ' - AGCGCCCTGAGGTGGGATT-3 ' (SEQ ID NO: 92) and typing Rev, GCGCTCCTCAGTATAGCCATACAACC-3 ' (SEQ ID NO: 93).
The PCR products were cloned into TOPO cloning vector to identify the mutations by sequence. To generate HEK293T HOIP KO cells, HEK293T cells were transfected with pX459 plasmid containing gRNA sequence of hHOIP by Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA). The following day, cells were selected with puromycin for two days. Then, cells were seeded at a low density and isolated colonies were picked up. To identify the HOIP KO cells, expression level of HOIP was analyzed by immunoblotting and genome loci of HOIP amplified by PCR using the following primers:
hHOIP typing Fwd, 5'- TTCCGGGCAGGCGTTTTCCCTG-3 ' (SEQ ID NO: 94) and typingJRev, 5 ' -CTCTGTGTAGCC ATATAATCG-3 ' (SEQ ID NO: 95) were analyzed by sequence.
[0267] Fertilized oocytes were microinjected with pX330 containing guide RNA sequence against HOIL-1L (Fig.12). Progeny was genotyped using the following primers: typing_Fwd, 5 ' -TTGCC AACAGGCCAATTTGATG -3' (SEQ ID NO: 90) and typing_Rev, 5' -TGCGGTGATGC ACAATATCCTG-3 ' (SEQ ID NO: 91 ).
[0268] Numbers of HOIL- 1 L null embryos at each embryonic stage were analyzed as described previously (Shimizu et al., Mol Cell Biol, 36: 1569- 1583 (2016)). TUNEL assay and Immunohistochemistry were performed as described previously (Shimizu et al., Mol Cell Biol, 36: 1569-1583 (2016)). For whole-mount immunohistochemical analyses on CD31 , embryos at embryonic day 10.5 were fixed with 4% paraformaldehyde in PBS for 2-4 hours at 4°C, followed by washing with PBS-T (PBS added with 0.2% Triton-X) for 30 min at 4°C for three times. Then, samples were blocked in 1 % BSA in PBS for 1 hour at room temperature and incubated with anti-CD31 primary antibody diluted in the same blocking buffer at 1 : 100 dilution for 2 days at 4°C. Samples were washed with PBS-T for 30 min at 4°C for three times followed by extensive washing at room temperature for 30 min for two times, and then incubated with the Alexa 546-conjugated secondary antibody in the same blocking buffer at 1 : 1000 dilution overnight at 4°C. After extensive washing with PBS-T at room temperature for 30 min for five times, samples were dehydrated with graded concentration of methanol, and then, incubated in the graded concentration of methyl salicylate and benzyl-alcohol-benzyl-benzoate overnight at 4°C for optimized optical clearing. Samples were analyzed under fluorescent stereoscopic microscope. Primary MEFs from HOIL-1L null or littermate WT were immobilized with SV-40 large T antigen.
Cell cultures, and transfection and retroviral expression:
[0269] HEK293T cells, MEFs derived from wild-type, HOIL- 1 L-/- or null mice, HEK293T HOIP KO cells and LUBAC TKO MEFs were grown in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum (FBS) with 100 IU/ml of penicillin and 100 μg/ml of streptomycin. Trans fections were performed using Lipofectamine2000. For retroviral expression, pMXs-IP, pMXs-neo or pMXs-IRES-Bsr containing the LUBAC components were transfected into Plat E packaging cells as described previously (Tokunaga et al, Nature, 471 : 633-636 (201 1)). The resultant viruses were used to infect LUBAC TKO cells or HOIL-1L null MEFs and the stable cells were selected using puromycin, G-418 or Blasticidin.
Immunoprecipitation, immunoblotting and EMSA:
[0270] Cells were lysed with lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 % Triton X-100, 2 mM PMSF and protease inhibitor cocktail (Sigma-Aldrich) and lysates were clarified by centrifugation at 15,000 rpm for 20 min at 4°C. For
immunoprecipitations, lysates were incubated with the appropriate antibodies for 90 min on ice, and then immobilized on rmp-Protem A Sepharose beads (GE Healthcare). The beads were washed five times with lysis buffer. Samples were separated by SDS-PAGE and then transferred to PVDF membranes. After blocking in Tris-buffered saline (TBS) containing 0.1 % Tween-20 containing 5% (w/v) nonfat dry milk, the membrane was incubated with the appropriate primary antibodies, followed by incubation with secondary antibodies. The membranes were visualized using enhanced chemiluminescence and analyzed by
LAS4000mini or LAS3000 (GE Healthcare). EMSAs for NF- Β activity were performed using Odyssey Infrared EMSA kit (LI-COR Biosciences, Lincoln, NE, USA) and IRDye 700 NF-KB consensus oligonucleotide (LI-COR Biosciences), and visualized using a Odyssey 9120 Infrared Imaging System (LI-COR Biosciences).
Cell viability assay using real-time cellular analysis (RTCA) technology or CellTiter-Glo Luminescent Cell Viability Assay kit:
[0271] Cell viability was continuously monitored as impedance-based cell index by using the iCELLigence system (ACEA Bioscience, Inc., San Diego, CA, USA). 20,000 cells were plated onto E-Plate L8 plate. Next day cells were treated with TNF-a (lOng/ml) and cell index were continuously monitored. The plots, which were normalized cell indices at the time point of TNF-a treatment, were shown. Cell viability of HBL-1 cells in Figure 4 J were evaluated using CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA).
SPR analysis:
[0272] GST-HOIP UBA (466-630 aa) WT or UBA2mut (Q607A/L61 1 A/F614A) and MBP-HOIL-1 L UBL (1-140, 37-161 , 1 -189 aa), MBP-SHARPIN UBL (163-301 , 198-318, 163-340 aa) were expressed in ii.coli and purified by using Glutathione Sepharose 4FF (GE Healthcare) or Amylose Resin (BioLabs), respectively. Binding affinities between the UBA and the UBLs were measured by using BIACORE3000 (GE Healthcare). GST-HOIP UBA was immobilized on sensor chip CM5 via anti-GST antibody using GST capture kit (GE Healthcare) in l OmM HEPES buffer (pH 7.4) containing 150 mM NaCl and 0.05% (v/v) surfactant P20 at 25°C. Binding between GST-HOIP UBA and MBP-UBLs was measured in l OmM HEPES buffer (pH 7.4) containing 150 mM NaCl and 0.05% (v/v) surfactant P20 at 25°C. The dissociation constants ( d) of HOIL-IL UBL- HOIP UBA or SHARPIN UBL- HOIP UBA were calculated using steady-state affinity analysis. In Figures 2 and 3, binding abilities of HOIL-I L UBL alone (8.5μΜ), SHARPIN UBL alone (8.5μΜ) or the mixture of the HOIL-I L and SHARPIN UBL (8.5μΜ each) to HOIP UBA were measured. In Figure 4F, mixture of the mHOIL-lL and mSHARPIN UBL (0.5 μΜ each) with dimethyl sulfoxide (DMSO) or HOIP-N polypeptide (20 μΜ) or SHARPIN polypeptide (20 μΜ) or both (20 μΜ) were used as analyte. Ubiquitin assay and kinase assay:
[0273] Recombinant His6-HOIP/HOIL-lL/SHARPIN, E l , UbcH7 were prepared as described previously (Tokunaga et al., Nature, 471 : 633-636 (201 1)). Trimeric LUBAC (0.2 μΜ) was incubated with DMSO or HOIP-N polypeptide (80 μΜ) or SHARPIN polypeptides (80 μΜ) or both (80 μΜ each) in buffer containing 20 mM Tris Tris-HCl (pH 7.5), 1 mM DTT on ice for 3h. Then, mixture of El ( 100 ng), UbcH7 (400 ng), Ub (5 μg) in the presence or absence of 2 mM ATP was added with buffer containing 20 mM Tris Tris-HCl (pH 7.5), 5 mM MgCb, 1 mM DTT and incubated at 37°C for 30 min.
[0274] S100 lysates of Jurkat HOIP KO cells described previously (Sakamoto et al., ACS Chem Biol, 10: 675-681 (2015)) were prepared as follows. Cells were lysed with buffer containing 10 mM Tris-HCl pH7.5, 10 mM KC1, 1.5 mM MgCb, 0.5 mM DTT, 2 mM PMSF, 50 μg/ml leupeptin, 10 μg/ml aprotinin. Lysates were centrifuged at 15,000 rpm, 4°C for 15 min and 0.1 1 volume of buffer containing 0.3 mM Tris-HCl pH7.5, 1.4 M KC1, 30 mM MgCk was added to supernatant. Then, centrifuged at 100,000 g, 4°C for 1 hour.
Finally, supernatant was dialyzed against buffer containing 20 mM Tris-HCl pH 7.2, 1 mM DTT. 10 μg of S I 00 lysates of HOIP KO Jurkat cells and trimeric LUBAC (0.1 μΜ) were incubated with DMSO or HOIP-N (80 μΜ) or SHARPIN polypeptides (80 μΜ) or both (80 μΜ each) in buffer containing 20 mM Tris Tris-HCl (pH 7.5), 1 mM DTT, 10 μg/ml leupeptin on ice for 3h. Then, mixture of El (100 ng), UbcH7 (400 ng), Ub (5 μg), creatine phospho kinase ( Sigma- Aldrich) with buffer containing 20mM Tris-HCl pH7.5, 10 mM MgC12, 1 mM DTT, 10 mM creatine phosphate, phosphatase inhibitor cocktail (Nacalai Tesque, San Diego, CA, USA) and incubated at 37°C for 30 min.
Structure modeling:
[0275] HOIP UBA-SHARPIN UBL complex model: Initial structural model of
SHARPIN UBL (216-302) was homology-modeled by Phyre (or Swiss-Model) program (Kelley et al., Nat Protoc, 10: 845-858 (2015); Schwede et al., Nucleic Acids Res, 31 : 3381 - 3385 (2003)). Created SHARPIN UBL model was superposed onto the HOIL-1 L molecule located at "contact2" surface of HOIP-UBA structure (PDB: 4DBG). Then SHARPIN UBL model was energy-minimized using CNS program (Brunger, Nat Protoc, 2: 2728-2733 (2007); Brunger et al., Acta Crystallogr D Biol Crystallogr, 54: 905-921 ( 1998)) while HOIP- UBA was fixed. [0276] HOIL-SHARPIN N-terminal helical model: Initial monomer model of HOIL-1L (2-38) and SHARPIN (173-202) were calculated by swiss-model. Since both HOIL-1L and SHARPIN have sequence similarity to structures in PDB:3ERM, it was chosen as template. Particularly, in 3ERM structure, homodimer was formed. Both of created models were superposed onto either monomer of 3ERM homodimer. Then energy-minimized heterodimer structure was calculated using CNS program.
[0277] HOIL- 1 L - HOIP - SHARPIN ternary complex model: Docking of the HOIL- 1 L- SHARPIN N-terminal heterodimer model to SHARPIN UBL-HOIP UBA-HOIL UBL model was performed using the software ZDOCK (Pierce et al., Bioinformatics, 30: 1771-1773 (2014)). Based on the top-scored docking model, HOIL-1L-HOIP-SHARPIN ternary complex model was constructed. The length of linker polypeptides were sufficient to bridge the each N-terminal segment and UBL.
Luciferase assays:
[0278] HEK293T cells were transfected with pGL4.32 (Luc2p/NF-KB-RE/Hygro) and pGL4.74 (hRLuc/TK) (Promega) along with WT or mutants of the LUBAC component. 21- 24 hours after transfection, cells were lysed and luciferase activities were measured by using the Dual-Luciferase reporter assay system (Promega) by Lumat Luminometer (Berthold).
EXAMPLE 1
[0279] This example demonstrates SHARPIN and HOIL-1L bind to distinct sites of HOIP UBA.
[0280] The UBL domains of EIOIL- 1 L and SHARPIN are involved in the interaction with the catalytic HOIP subunit of LUBAC. Although both UBLs are highly homologous, HOIL- 1L interacts with the UBA region of HOIP, whereas it has been reported that the UBL of SHARPIN interacts with the NZF2 domain of HOIP. However, since it was found that the SHARPIN UBL can form a complex with HOIP lacking NZF2, the interaction between HOIP and the SHARPIN UBL was first re-evaluated using LUBAC subunits of mouse origin (Figure 1A).
[0281] GFP-tagged mouse HOIP (mHOIP) UBA region (466-630 aa) or mHOIP ZF domains (296-434 aa) containing ZF, NZF 1 , and NZF2 was co-introduced with mHOIL-lL or mSHARPIN into HEK293T cells. mSHARPIN did interact with the mHOIP UBA, but not with the ZF domains as was the case with mHOIL-l L (Figure I B). Furthermore, mSHARPIN bound to mHOIP WT and ANZF2 (Δ402-432 aa), but not to mHOIP AUBA (Δ558-609 aa) (Figures 7A and 7B).
[0282] Surface plasmon resonance (SPR) analyses revealed that mSHARPIN UBL (163- 301 aa) directly binds to mHOIP UBA (466-630 aa) and calculated dissociation constant (Kd) is 12.4 μΜ (Figure 1C), which is approximately ten-fold lower than that of the mHOIP UBA- mHOIL-lL UBL (1-140 aa) interaction (¾ = 0.90 μΜ) (Figure 7C). These results indicated that the SHARPIN UBL indeed interacts with the UBA region of HOIP.
[0283] It was previously shown that the human HOIL- 1 L (hHOIL- 1 L) UBL binds to the hHOIP UBA at three different surfaces (N-terminal:N, Middle:M, and C-terminal:C) in the co-crystal structure of the hHOIL- 1L UBL and the hHOIP UBA and that the C site is the hHOIL- 1L UBL binding site by mutation analyses. Considering the high sequence similarity between SHARPIN UBL and HOIL-1L UBL, it was suspected that the SHARPIN UBL may interact with the HOIP UBA via one of the remaining two putative interaction sites. mHOIP mutants were prepared in which corresponding amino acids for three interaction sites of hHOIP were substituted for Ala. These mutants are mHOIP R479A/Q490A (Nmut),
M528A/L559A (Mmut), and Q607A/L61 1A/F614A (Cmut). Expressing them along with mHOIL-lL or mSHARPIN into HEK293T cells, it was confirmed that mHOIL-lL interacted with the C site since mHOIL-lL failed to bind to mHOIP Cmut (Figures 7D and 7E).
[0284] mSHARPIN failed to interact with the mHOIP Mmut in addition to mHOIP Cmut (Figure I D). In the co-crystal structure of hHOIL-lL UBL and hHOIP UBA region, the UBL does interact with the M site via a surface that corresponds to the 144 hydrophobic surface of ubiquitin, whereas it does interact with the C site via a distinct surface from 144-like surface. A structural model was generated of the M site of hHOIP and hSHARPIN UBL based on co- crystal structure of the hHOIP UBA and hHOIL- 1 L UBL. Leu273 and Tyr295 of mSHARPIN were corresponding amino acid residues of Leu276 and Tyr298 in hSHARPIN, which are important for HOIP interaction in this model.
[0285] To verify the model, these amino acids were mutated to Ala (L273A and Y295A). The mSHARPIN mutants failed to interact with mHOIP (Figure I E), confirming that mSHARPIN UBL binds to the M site in HOIP UBA. Since both M (523-571 aa) and C interaction sites (573-622 aa) exhibits UBA-like structure, the M and C sites can be designated as UBA1 and UBA2 of HOIP, respectively (Figure 1A). EXAMPLE 2
[0286] This example demonstrates differential roles of UBL domains of mHOIL-lL and mSHARPIN in LUBAC stabilization.
[0287] Since mHOIL-lL could stabilize mHOIP much more efficiently than mSHARPIN (Figures 1 and 7), the roles played by two accessory subunits of mouse LUBAC in NF-KB activation were evaluated using the luciferase reporter assay (Figure 2A). mHOIL-lL efficiently activated NF-κΒ in a dose dependent manner as observed in human LUBAC subunits. However, surprisingly, in contrast to previous observation using human LUBAC, mSHARPIN could not induce NF-κΒ activation at any amount of plasmids (Figure 2A). Immunoblot analyses revealed that mSHARPIN failed to increase the amount of mHOIP efficiently although mSHARPIN does interact with mHOIP (Figures ID and 2A).
[0288] Since the UBLs are the interaction domains with HOIP, the mHOIL-l L and mSHARPIN mutants whose UBL domain and N-terminal extension to UBL are exchanged, which are S(UBL)-H0IL-1L and H(UBL)-SHARPIN (Figure 2B). The accessory subunits having the HOIL-IL UBL (HOIL-IL and H(UBL)-SHARPIN) efficiently activated NF-KB and increased the amount of mHOIP (Figure 2B), indicating that the mSHARPIN UBL is less effective to stabilize mHOIP as compared with mHOIL-l L UBL. Since co-introduction of mSHARPIN with mHOIP and mHOIL-l L enhanced the NF-κΒ activation and increased the amount mHOIP in a dose-dependent fashion (Figure 2C), mSHARPIN could increase the amount of mHOIP protein in the presence of mHOIL- 1 L.
[0289] To probe the mechanism underlying the stabilization of the LUBAC complex in the presence of both UBLs, the mHOIP UBA region (466-630 aa) was fixed on the sensor chip of SPR. Surprisingly, when appling both HOIL-I L UBL and SHARPIN UBL together, both UBLs interacted with the mHOIP UBA region much more tightly than either single UBL alone (Figure 2D). Since determination of a precise binding constant does not appear possible in the case of the interactions between three proteins, dissociation rates were compared between two UBLs applied together or UBL alone applied (Figure 8A). The HOIL-I L or SHARPIN UBL alone rapidly dissociated from HOIP UBA after stopped applying the UBL. However, when applied together, both UBLs remained bound to HOIP UBA (Figure 8A), indicating that both UBLs bind to HOIP UBA very tightly in a coordinated manner. [0290] To dissect the mechanisms underlying the cooperative binding of HOIL-1L and SHARPEN to HOIP, mHOIP WT, UBAlmut (M528A/L559A) or UBA2mut
(Q607A/L611 A/F614A) were introduced into HEK293T cells together with either mHOIL- 1L, mSHARPIN, or both (Figures 2E-2G). mHOIL-lL could not interact with mHOIP UBA2mut in the absence of mSHARPIN. However, in the presence of mSHARPIN, mHOIP UBA2mut could interact with mHOIL-lL at the comparable level to mHOIP WT (Figures 2E- 2G, compare lane 8 to lanes 9, 1 1). mSHARPIN could also be efficiently co- immunoprecipitated with the mHOIP UBAlmut in the presence of mHOIL-lL (Figures 2E- 2G, compare lane 4 to lane 10). SPR analyses confirmed that the addition of mSHARPIN UBL drastically augmented the interaction between mHOIP UBA Q607A/L611 A/F614A (UBA2mut) and mHOIL-lL UBL although the UBA2 mutations heavily attenuated the interaction between HOIP UBA and mHOIL-lL UBL (Figure 8B). Luciferase reporter assays also revealed that additive expression of mSHARPIN WT notably enhanced NF-KB activation induced by mHOIP UBA2mut and mHOIL-lL. However, mSHARPIN Y295A and L273A, which cannot bind to HOIP UBA1 , failed to activate NF-κΒ (Figure 8C).
[0291] The role of mSHARPIN-mHOIP interaction was evaluated in the formation of the trimeric LUBAC complex. mSHARPIN Y295A and L273A failed to bind to HOIP WT as described above. However, in the presence of mHOIL-lL WT, mSHARPIN mutants could bind to HOIP WT (Figures 2H and 21, compare lanes 3, 4 to lanes 7, 8). Furthermore, when co-introduced with HOIP UBA2mm and mHOIL-lL, mSHARPIN Y295A and L273A but not WT failed to stabilize nor co-immunoprecipitate HOIP UBA2mut (Figures 2H and 21, compare lane 10 to lanes 1 1 , 12).
[0292] These results indicate that the trimeric LUBAC complex can be formed even if interaction of mHOIP to either HOIL-1 L or SHARPIN is impaired. However, when both HOIL-IL-HOIP and SHARPIN-HOIP interactions are impaired, functional trimeric LUBAC is not formed.
EXAMPLE 3
[0293] This example demonstrates HOIL-1 L-SHARPIN interaction via their a-helical region located at N-terminal regions of UBLs is important for LUBAC stabilization, in accordance with embodiments of the invention. [0294] The observation that trimeric LUBAC is formed even if either mHOIL-lL- mHOIP or mSHARPIN-mHOIP interaction is impaired suggests that mHOIL-lL and mSHARPIN can bind each other (Figure 2). In cells expressing mHOIP UBA2mut, mHOIL- 1L and mSHARPIN Y295A or L273A, mHOIL-lL could bind to the mSHARPIN mutants even though the mutants failed to interact with mHOIP (Figures 2H and 21, Myc (HOIL-IL) blot, lanes 11 , 12), which also indicated that direct interaction between mSHARPIN and mHOIL-lL exists. mHOIL-lL UBL (1-140 aa) and mSHARPIN UBL (163-301aa), which effectively bound to HOIP UBA WT and UBA2mut in SPR analyses (Figures 2D and 8B), contain additional amino acids at the N-terminus of the UBL domains (1-36 aa or 163-197 aa for HOIL-IL or SHARPIN, respectively). A secondary structure prediction program suggested that both N-terminal regions of HOIL-IL and SHARPIN seem to form ct-helical structure (Not). These Net regions were investigated as to whether they are involved in the HOIL-1 L-SHARPIN interaction.
[0295] HEK293T cells lacking HOIP (HOIP KO) were generated using CRISPR/Cas9 system (Figure 9A and 9B) since HOIP can bind to and bridge the two proteins. When introduced into HEK293T HOIP KO cells, mHOIL-lL WT interacted with mHOIL-lL WT and mSHARPIN WT (Figures 3 A and 3B, lanes 2, 3). However, mHOIL-lL
ΔΝ (Δ1-36 aa) or mSHARPIN ΔΝ (Al 63-197 aa) impaired the interaction between the two LUBAC subunits (Figures 3A and 3B, lanes 4, 7). Since N-terminal regions of HOIL-IL and SHARPIN are highly homologous (Figure 9C), it was inferred that the N-terminal additional regions may be involved in both heterotypic and homotypic interactions of the two accessory subunits of LUBAC.
[0296] SPR analyses were performed using mHOIL-lL UBL ΔΝ (37-161 aa) and mSHARPIN UBL ΔΝα (198-318 aa) to investigate the role of the Net region in trimeric LUBAC complex formation. Co-administration of mHOIL-lL UBL and mSHARPIN UBL greatly enhanced the affinity to HOIP UBA as described above (Figures 2D and 3C).
However co-administration of mHOIL-l L UBL ΔΝα and mSHARPIN UBL completely abrogated the coordinated binding of both UBLs to mHOIP UBA (Figure 3C) as is also the case with the mSHARPIN UBL ΔΝα (Figure 9D). Since co-administration of longer forms of UBLs that contained the Not region, HOIL-IL (1 -189 aa) and SHARPIN ( 163-340 aa), could bind to the mHOIP UBA region tightly (Figure 9E), the Net regions appear to be important for formation of the trimeric LUBAC complex. [0297] It was found that the Na of HOIL-1L and SHARPIN are homologous to a protein named conserved protein with unknown function from Pseudomonas syringae PV, which forms dimer (4-helix bundle like structure) in the reported crystal-structure (PDB code:
3ERM). Structure modeling of the HOIL-IL-SHARPIN interaction was perfonned based on the reported structure. Since Leul 5 and Vall9 of mHOIL-lL, and Leul76 and Ilel 80 of mSHARPIN are likely to be involved in HOIL-IL-SHARPIN interaction, mutants of mHOIL-lL and mSHARPIN were generated, in which these amino acids are mutated to Ala (mHOIL-lL L15A, L19A, and L15A/L19A, and mSHARPIN L176A, I180A, and
L176A/I180A). When introduced into HEK293T HOIP KO cells, mHOIL-lL WT binds to mSHARPIN WT efficiently (Figures 3D and 3E). However, mHOIL-lL L15A, L19A, and L15A/L19A mutants failed to interact with mSHARPIN WT as observed in mHOIL-lL ΔΝ , and this was also the case with the mSHARPIN mutants (Figures 3D and 3E). The role of the SHARPIN-HOIL-1L interaction was assessed in the formation of trimeric LUBAC. The Na regions appeared dispensable for the mHOIL-lL-mHOIP interaction because mHOIL-lL L15A/V19A and ΔΝα could bind to mHOIP WT as efficiently as mHOIL-lL WT (Figures 3F and 3G, compare lane 2 to lanes 3, 4).
[0298] In cells that expressed both mHOIL- 1 L WT and mSHARPIN WT along with the mHOIP UBA2mut, mutations in Na region of either mHOIL- 1 L or mSHARPIN heavily attenuated the trimeric LUBAC formation although mHOIP UBA2mut was efficiently stabilized and co-immunoprecipitated in cells expressing WT of mSHARPIN and mHOIL- 1L, (Figures 3F and 3G, compare lane 6 to lanes 7-10).
[0299] Collectively, these results indicate the importat role of the mHOIL- 1 L- mSHARPIN interaction in stabilization of trimeric LUBAC.
[0300] Since Tyr92 and Phe94 in the UBL of mHOIL- 1 L are important in the interaction with mHOIP UBA2, the two amino acids were mutated to Ala in mHOIL- 1 L WT (mHOIL- 1 L Y92A/F94A (UBLmut)) or mHOIL- 1 L L 15 A/V 19 A (mHOIL- 1 L
L15A/V19A/Y92A/F94A (Na+UBLniul)). Involvement of Tyr92 and Phe94 of mHOIL- 1 L in mHOIP interaction was confirmed as both mHOIL- 1 L UBL'™1 and Na+UBLmut failed to interact with mHOIP (Figures 9F and 9G, compare lane 2 to lanes 3, 4). However, in the presence of mSHARPIN WT, mHOIL- 1 L UBLmul but not Na+UBL1™' could bind to and stabilize mHOIP (Figures 9F and 9G, compare lane 6 to lane 7). Therefore, these results confirmed that trimeric LUBAC can be formed even if one of three interactions among the three LUBAC subunits is impaired.
[0301] Based on the findings, the structural model of the LUBAC ternary complex was generated by docking of HOIL-SHARPIN Na heterodimer (HOIL : 2-38; SHARPIN : 173- 202) on hSHARPIN UBL (216-302 aa)-hHOIP (482-627 aa)-hHOIL UBL (2-133 aa) structure using ZDOCK program (Pierce et al., Bioinformatics, 30: 1771-1773 (2014)). The model shows that both accessory subunits SHARPIN and HOIL-IL can interact with each other via their Na region simultaneously when the UBLs of SHARPIN and HOIL-IL do bind to HOIP UBAl and UBA2, respectively, indicating that three mutual interactions among the three subunits of LUBAC are important for the formation of trimeric LUBAC. The structure modeling confirms the observation that loss of one interaction among the three does not overtly affect the trimeric LUBAC formation, whereas LUBAC is destabilized if two interactions are impaired as a consequence of dissociation of one subunit from the complex.
EXAMPLE 4
[0302] This example demonstrates physiological roles of the interactions between LUBAC subunits and targeting the novel HOIL-1L-SHARPIN interaction by an a-helical stapled polypeptide, in accordance with embodiments of the invention.
[0303] With precise molecular mechanisms underlying formation of trimeric LUBAC in hand, the roles of the three interactions among LUBAC subunits were assessed in
physiological settings. To this end, mouse embryonic fibroblasts (MEFs) were established that did not express any of the LUBAC subunits (TKO cells) by knocking out HOIL- 1 L and HOIP in cpdm MEFs using CRISPR/Cas9 technology (Figures 10- IOC). WT or mutants of three subunits of mouse LUBAC were reconstituted into TKO cells using retroviral expression system, evaluating TNF-a-mediated cytotoxicity by using iCELLigence system (Figures 4A and 4B). Although introduction of mSHARPIN did not overtly increase the amount of mHOIP, mHOIL-l L increased mHOIP rather efficiently (Figure 4A, compare lane 2 to lanes 3, 5). However, the observation that co-expression of mSHARPIN further potentiated the mHOIL-l L-mediated increase of mHOIP confirmed the important roles of three subunits in stable LUBAC formation (Figure 4A, lane 6).
[0304] In TKO cells expressing three LUBAC subunits, mutations in mHOIL-l L UBL or in mHOIP UBA2, which impair the HOIL-1 L-HOIP interaction, mildly decreased the amount of mHOIP and sensitized cells to TNF-a-mediated cell death (Figure 4A, lanes 7, 8 and 4B), indicating that loss of one out of three interactions does not affect function of LUBAC significantly. However, mHOIL-l L UBLmut or mHOIP UBA2mut failed to increase the amount of mHOIP or to protect cells from TNF-a-mediated death almost completely when mSHARPIN was omitted (Figures 4C and 4D). In cells expressing the mHOIL-l L mutant having mutations in both UBL and Na-region, which impair both the mHOIP-mHOIL-lL and the mHOIL-lL-mSHARPIN interaction, mSHARPIN failed to increase the amount of mHOIP and to protect cells from TNF-a-mediated death (Figures 4A, lane 9 and 4B). These results indicated that dissociation of mHOIL-lL from mHOIP, which is achieved by the impairment of the mHOIP-mHOIL-lL and the mHOIL-lL-mSHARPIN interactions, profoundly decreases the amount of LUBAC and attenuates its function in physiological settings.
[0305] It has been reported that augmented LUBAC ubiquitin ligase activity is involved in the pathogenesis of activated B cell-like type of diffuse large B-cell lymphoma (ABC DLBCL), and attenuated expression of LUBAC subunits suppresses the proliferation of cells derived from ABC DLBCL. It has also been reported that increased expression of LUBAC rendered cells to be resistant to the widely used anti-cancer drug, cisplatin. Therefore, destabilization of LUBAC has therapeutics potential. A hydrocarbon stapled a-helical HOIP polypeptide (HOIP-N) which inhibits the HOIP-HOIL- 1 L interaction could inhibit the proliferation of ABC-DLBCL cell lines by destabilizing LUBAC (Yang et al., Cancer Discov, 4: 480-493 (2014)). Since simultaneous suppression of both HOIL-1 L-SHARPIN and HOIL-1 L-HOIP interaction destabilize LUBAC complex much more efficiently than suppression of HOIL-1L-HOIP interaction alone (Figures 4A and 4B), an a-helical stapled polypeptide was developed mimicking the Na region of SHARP IN to inhibit the HOIL-1L- SHARPIN interaction (unstapled: Ala-WEELATRLSQAIA-NH? (SEQ ID NO: 96);
stapled: Ala-W(R8)ELATRL(S5)QAIA-NH2 (SEQ ID NO: 97). SPR analyses revealed that the SHARPIN stapled polypeptide (SHARPIN-Na) could inhibit the formation of the tnmeric complex composed of HOIP UBA, HOIL-1 L UBL and SHARPIN UBL (Figure 4F). Furthermore, using both stapled polypeptides in combination, but not HOIP-N plus
SHARPIN unstapled polypeptide, profoundly inhibited the formation of the trimeric complex (Figure 4F). [0306] In order to examine the impacts of polypeptides on the LUBAC activity, in vitro ubiquitin assay was performed using the purified trimeric human LUBAC (Figure 4G).
Addition of SHARPIN-Να inhibited the activity of trimeric LUBAC more effectively than HOIP-N, suggesting that inhibition of HOIL-IL-SHARPIN interaction is superior to that of HOIL-1L-HOIP interaction in suppressing LUBAC. In addition, the ligase activity was greatly attenuated in the presence of both HOIP-N and SHARPIN-Not staple polypeptides.
[0307] LUBAC is involved in the activation of ΙκΒ kinase. Although addition of purified LUBAC proteins to lysates of Jurkat HOIP KO cells activated IKK, administration of SHARP ΓΝ-Να or SHARPIN-Ncc plus HOIP-N polypeptides profoundly inhibited LUBAC- mediated IKK activation (Figure 4H), which indicated that disruption HOIL-IL-SHARPIN interaction attenuated physiological function of LUBAC.
[0308] ABC DLBCL cell line cells, HBL-1 cells, were treated with SHARPIN-N or SHARPIN-unstapled polypeptide. SHARPIN-Na, but not SHARPIN-unstapled polypeptide disnipted the interaction between LUBAC subunits in HBL-1 cells (Figure 41). Importantly, SHARPIN-Να efficiently killed the HBL-1 cells, for which proliferation LUBAC is important, in a dose dependent manner (Figure 4J) and NF-κΒ activity was efficiently attenuated by the SHARPIN-Να polypeptide (Figure 4J).
[0309] These results demonstrated the therapeutic potential targeting HOIL-IL- SHARPIN interaction to inhibit formation and function of the LUBAC ligase complex.
EXAMPLE 5
[0310] This example demonstrates HOIL-1 L null mice exhibit embryonic lethality at midgestational stage as observed in HOIP knockout mice or HOIP mutant mice lacking linear ubiquitin ligase activity.
[0311] mHOIL-lL increases the amount of mHOIP much more efficiently than mSHARPIN by overexpression study (Figure 2A). To dissect the roles of the subunits more precisely, mHOIL-l L or mSHARPIN was co-introduced with mHOIP into TKO cells using the retrovirus expression system. mHOIL-l L, but not mSHARPIN alone, increased mHOIP as previously observed in Figure 2A (Figures 1 1 A and 1 I B). In accordance with the amount of HOIP, mHOIL-l L, but not mSHARPIN, could evade TNF-a-mediated cytotoxicity (Figure 1 1 C) and Caspase-3 activation mediated by TNF-a and cycloheximide (CHX) (Figure 1 I D) when co-expressed with mHOIP, indicating that loss of mHOIL-l L sensitizes cells to TNF- -mediated cell death as observed in cells lacking mHOIP. Since most genetically engineered mice that are sensitive to TNF-a-mediated cell death are embryonic lethal, mice lacking HOIL- 1 L are expected to embryonic lethal as is the case with HOIP KO mice. However, HOIL-IL7" mice described previously appeared not to exhibit any overt pheno types except deposition of PAS positive substances in muscles when aged. The RT- PCR analysis revealed that mRNA containing sequence encoding the N-terminal regions of HOIL-IL was expressed in cells from HOIL-IL7" mice (Figure 1 IE). Immunoblotting using anti-mHOIL- 1 L detecting the UBL region (1-189 aa) also revealed that anti-HOIL-lL reactive material, whose molecular weight is around 30 kDa, could be detected in both WT and HOIL-IL 7- MEFs although full length HOIL-IL is not expressed in HOIL-IL7" MEFs (Figure 5 A). Since the exon encoding the RING1 domain is deleted but exons encoding the N-terminal HOIL-IL region containing UBL to NZF domains are intact in the previously described HOIL-IL7" mice (Tokunaga et al., Nat Cell Biol, 1 1 : 123-132 (2009)), N-terminal truncated HOIL-IL may be expressed as an alternative spliced product. The N-terminal truncated HOIL-IL splice variant has been reported as RBCK2. It has been observed that co- introduction of mHOIL-l L UBL (1 -140 aa) together with mHOIP and mSHARPIN WT into LUBAC TKO MEFs could increase the expression level of mHOIP and profoundly attenuated caspase-3 activation induced by TNF-a and CHX (Figures 1 IF and 1 1 G).
[0312] These results raised the possibility that the expression of the N-terminal truncated HOIL-I L gene product rendered previously described HOIL-IL7" mice to not overt phenotypes. To probe this possibility, two HOIL- IL mutant mice were generated using two guide RNAs (mutated allele #1 and #2) targeting its UBL domain using CRISPR/Cas9 system (Figure 1 1H and 1 II). Both mouse strains exhibited the same phenotypes and mice having mutated allele #1 (null) were used for further study. The mice homozygous for HOIL- I L null alleles (HOIL-I L"""7'"'") were embryonic lethal and died around embryonic day 10.5, as observed in HOIP knockout mice or HOIP mutant mice lacking linear ubiquitin ligase activity (HOIP^'"""7--1'""'"'") (Figures 5B, 5C, 1 1 J and 1 I K).
[0313] HOIL-I L"""7""" mice display intracranial and/or thoracoabdominal hemorrhages and TUNEL-positive cells were significantly increased as compared to its control littermates (Figure 5D). Defects in vasculature of HOIL-I L"""7""" embryos were confirmed in HOIP knockout mice. 30 kD anti-mHOIL-lL N-term ( 1 -189 aa) reactive protein did not exist in MEFs from HOIL-I L""'"""", but did express in littermate control MEFs, confirming that 30 kD anti-mHOIL- 1 L ( 1 - 189 aa) reactive protein is a N-terminal truncated alternative splicing product of mHOIL-l L (RBCK2-like) and does contain the UBL domain, the binding site to mHOIP (Figure 5A). HOIP and SHARPIN were barely detected in HOIL-lLra/// / MEFs, as compared with previously described HOIL-1 L" " (RING1 KO) cells (Figure 5A). Introduction of mHOIL-l L but not m SHARPIN into HOIL-1 L"""7™" MEFs increased the amount of HOIP (Figure 1 1 L) and protect cells from TNF-ot-mediated cell death (Figure 5E).
[0314] These results collectively indicate that loss of HOIL- 1 L profoundly reduced the amount of functional LUBAC and exhibits the comparable effects as loss of LUBAC catalytic activity in mice, which robustly support the ex vivo data described above.
[0315] It has been shown that hSHARPIN-hHOIP, but not mSHARPIN-mHOIP, induces NF-KB activation effectively. The difference between hLUBAC and mLUBAC was examined in TKO MEF cells using retroviral expression system (Figure 6A). Although both mouse and human HOIL-1 L could increase the amount of HOIP in both species, mouse and human SHARPIN could increase hHOIP but not mHOIP, suggesting that the inability of SHARPIN to increase the amount of mHOIP can be attributed to the differences between mHOIP and hHOIP. A mHOIP mutant was prepared whose UBA domain (Figure 12) is substituted for hHOIP UBA (hUBA) (Figures 6B and 6C). When co-introduced with mSHARPIN into LUBAC TKO cells, the amount of hHOIP or hUBA, but not mHOIP, was increased (Figures 6B and 6C). TNF-ot-mediated phosphorylation and degradation of ΙκΒα, which are hallmarks of NF-κΒ activation, were augmented by expression of hHOIP or hUBA along with mSHARPIN (Figure 6D). Co-expression of hHOIP or hUBA but not mHOIP with mSHARPIN could attenuate the caspase-3 activation by TNF-a and CHX (Figure 6E). These results indicate that differences of the UBA domain between mouse and human appears to be important for HOIP stabilization and function by SHARPIN.
[0316] It has been reported that patients having mutations in HOIL- 1 L exhibited polyglucosan myopathy or immunodeficiency and autoinflammation. Among patients having polyglucosan myopathy, homozygous mutation of Ala 18 of HOIL- 1 L to Pro was reported. Since Ala 18 is located within the N -region, the role of the A 1 8P mutation in HOIL- 1 L- SHARPIN interaction was dissected. The A l 8P mutation of both mouse and human HOIL- 1 L heavily attenuated HOIL- 1 L-SHARPIN interaction when co-expressed in HEK293T HOIP KO cells (Figure 6F and 12B). Moreover, mHOIL- l L A1 8P also heavily decreased the complex formation with mSHARPIN and mHOIP UBA2mul, which cannot interact with HOIL- 1 L UBL, as observed with HOIL- 1 L L15A/V 19A, an N mutant (Figures 12C and 12D, compare lane 6 to lane 8). These results demonstrate the involvement of the Noc- mediate I IOIL-1 L-SHARPI interaction in preventing polyglucosan myopathy.
EXAMPLE 6
[0317] This example provides additional data for the Na polypeptide, in accordance with embodiments of the invention.
[0318] Na induced a decrease in HBL1 cell viabillity at doses of 20 and 40 μΜ. HBL1 cell were incubated with an unstapled (WT), a negative control (Scramble (SC2)), and a stapled (Na) versions of SHARP IN polypeptide. Viability was measured by CellTiter Glo (Promega, G7570). The results are in Figure 14A.
[0319] After 2 hour of polypeptide treatment, Na (20 μΜ) decreased NF-κΒ activity. 30 μg of protein from cell lysates were analyzed for NF-kB activity using a TransAM® NFKB Transcription Factor ELISA Kit (Active Motif, Carlsbad, CA, USA, cat# 40596) following manufacturer's instructions. The results are in Figure 14B.
[0320] Na efficiently reduced the amount of secreted IL-8, a known NF-KB gene target, in response to TNF-a stimulation. HBL-1 cells were treated with Na during 2 h, then media was exchanged by one with 5 ng/mL of TNF-a, and cells exposed for 4 and 24h. The amount of secreted IL-8 to supernatants was determined using an specific ELISA kit (Peprotech, Rocky Hill, NJ, USA, cat# 900T18) following manufacturer's instructions. The results are in Figure 14C.
[0321] Na delays TNF-a stimulated IkBa degradation in HBL1 cells. HBL-1 cells were treated with Na during 2 h, then media was exchanged by one with 5 ng/mL of TNF-a, and exposed to cells for 0, 5, 10, 30, 60 or 240 min. Cell lysates were analyzed for IkBa protein levels by Western blot. The results are in Figures 14D and 14E.
[0322] Na provokes not only an increase in IkBa levels, but also deactivation of Akt, and ERK. Na treatment reduces the levels of the proapoptotic protein BAX. Cell were treated during 24h and equal amount of protein from cell lysates were analyzed for IkBa, Akt, ERK, BAX, and actin by Western blot. The results are in Figure 14F.
[0323] HT1080 (Fibrosarcoma), PANC 1 (pancreatic carcinoma), HCT1 16 (Colon carcinoma), PEA1 (Ovaric carcinoma), and SJSA-1 (Osteosarcoma) cells were treated with WT (Figures 15A-C) or Na (Figures 15D-F) for 6h. Na showed a wide range of activity in analyzed cell lines, ranging from low (as in HT1080) to high (as in SJSA-1 ) (Figure 15A (WT) and Figure 15D (Na)). Also, the pattern of cytotoxicity (Figure 15B (WT) anf Figure 15E (Να)), and caspase 3/7 activity (Figure 15C (WT) and Figure 15F (Na)) suggest that, except for HT1080 ceils, Na induced necroptotic death, instead of apoptosis, which becomes important because it is known that lack of SHARPIN, as part of LUBAC, negatively regulates necrosis. To test the hypothesis that Na induces necrosis, PANCl cells were co- treated with Na and Nec-1. The cotreatment could prevent Na cell death without affecting caspase3/7 activity, strongly supporting the idea that Na induces necroptotic cell death in PANCl (Figures 15G-15I; PANCl cells were treated with Na (20 μΜ) and/or Nec-1 (50 μΜ) for 6h -cCell viability, cytotoxicity, and caspase 3/7 activity was measured by the ApoTox Glo kit (Promega, cat# G6321)).
[0324] Nuclei of HT1080 and SJSA-1 cells were stained with Hoechst 33342, and the media was exchanged with fresh media with 10 μΜ of FITC-Na. After 2h of incubation with polypeptide, cells were observed and microphotographed under an LSM 710 confocal microscope (Zeiss, Oberkochen, Germany). Live cell microscopy showed that, after 2 h of treatment with a FITC labeled version of Να, HT1080 seems to have a lower permeability to this Na than SJSA-1.
[0325] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
[0326] The use of the terms "a" and "an" and "the" and "at least one" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (for example, "at least one of A and B") is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
[0327] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):
1. A polypeptide, or a pharmaceutically acceptable salt thereof, the polypeptide comprising: at least two non-natural amino acids that form a covalent cross-link with each other, wherein the non-natural amino acids are the same or are different, and wherein the cross-link is internal to the polypeptide, and
(a) wherein the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine; or the polypeptide has from the N- to C-terminus a glutamine one amino acid position before a first valine, and optionally
(i) wherein the first valine is three amino acid positions before a second valine, and
(ii) wherein the second valine is one amino acid position before a leucine; and
(b) wherein the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
2. The polypeptide of claim 1 , wherein the polypeptide has from the N- to C- terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine, and wherein the cross-link is formed from a non-natural amino acid at position i within the polypeptide and another non-natural amino acid at position i + 3, i + 4, or i + 7 within the polypeptide; and wherein the amino acid at position i + 6 within the polypeptide is the leucine, the amino acid at position i + 9 within the polypeptide is the alanine, and the amino acid at position i + 10 within the polypeptide is the isoleucine.
3. The polypeptide of claim 1 , wherein the polypeptide has from the N- to C- terminus (1) a glutamine one amino acid position before a first valine, and (2) optionally
(i) wherein the first valine is three amino acid positions before a second valine, and
(ii) wherein the second valine is one amino acid position before a leucine, and wherein the cross-link is formed from a non-natural amino acid at position i within the polypeptide and another non-natural amino acid at position i + 3, i + 4, or i + 7 within the polypeptide; and wherein the amino acid at position i + 1 within the polypeptide is the glutamine, and the amino acid at position i + 2 within the polypeptide is the first valine.
4. The polypeptide of claim 2 or 3, wherein the cross-link of the polypeptide is: formed from the amino acid at position / within the polypeptide and another amino acid at position i + 4 within the polypeptide, and the amino acid at position is (S)-2-(4'- pentenyl)alanine (S5) and the amino acid at position / + 4 is S5; or formed from the amino acid at position i within the polypeptide and another amino acid at position i + 3 withm the polypeptide, and the amino acid at position /' is (R)-2- (4'-pentenyl)alanine (R5) or (R)-2-(2'-propenyl)alanine (R3) and the amino acid at position i + 3 is S5; or formed from the amino acid at position within the polypeptide and another amino acid at position + 7 within the polypeptide, and the amino acid at position i is (R)-2-(7'- octenyl)alanine (R8) and the amino acid at position i + 7 is S5.
5. The polypeptide of claim 1 , wherein the polypeptide has from the N- to C- terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine, wherein the polypeptide is a first polypeptide, and wherein the polypeptide further comprises a second polypeptide having non-natural amino acids that form a covalent cross-link internally within the second polypeptide, wherein the second polypeptide has from the N- to C-tenninus (1) a glutamine one amino acid position before a first valine, and (2) optionally
(i) wherein the first valine is three amino acid positions before a second valine, and
(ii) wherein the second valine is one amino acid position before a leucine.
6. The polypeptide of claim 5, wherein
(a) the cross-link in the first polypeptide is formed from a non-natural amino acid at position i within the first polypeptide and another non-natural amino acid at position i + 3, i + 4, or i + 7 within the first polypeptide; and wherein the amino acid at position i + 6 within the first polypeptide is the leucine, the amino acid at position i + 9 within the first polypeptide is the alanine, and the amino acid at position i + 10 within the first polypeptide is the isoleucine; and
(b) the cross-link in the second polypeptide is formed from a non-natural amino acid at position i within the second polypeptide and another non-natural amino acid at position i + 3, i + 4, or i + 7 within the second polypeptide; and wherein the amino acid at position i + 1 within the second polypeptide is the glutamine, and the amino acid at position + 2 within the second polypeptide is the first valine.
7. The polypeptide of claim 6, wherein (a) the cross-link of the first polypeptide is: formed from the amino acid at position /' within the first polypeptide and another amino acid at position i + 4 within the first polypeptide, and the amino acid at position i is (S)-2-(4'-pentenyl)alanine (S5) and the amino acid at position + 4 is S5; or formed from the amino acid at position i within the first polypeptide and another amino acid at position i + 3 within the first polypeptide, and the amino acid at position i is (R)-2-(4'-pentenyl)alanine (R5) or (R)-2-(2'-propenyl)alanine (R3) and the amino acid at position i + 4 is S5; or formed from the amino acid at position i within the first polypeptide and another amino acid at position i + 7 within the first polypeptide, and the amino acid at position i is (i?)-2-(7'-octenyl)alanine (R8) and the amino acid at position i + 7 is S5; and
(b) the cross-link of the second polypeptide is: formed from the amino acid at position /' within the second polypeptide and another amino acid at position i + 4 within the second polypeptide, and the amino acid at position i is (S)-2-(4'-pentenyl)alanine (S5) and the amino acid at position i + 4 is S5; or formed from the amino acid at position i within the second polypeptide and another amino acid at position i + 3 within the second polypeptide, and the amino acid at position i is (R)-2-(4'-pentenyl)alanine (R5) or (R)-2-(2'-propenyl)alanine (R3) and the amino acid at position i + 4 is S5; or formed from the amino acid at position i within the second polypeptide and another amino acid at position i + 7 within the second polypeptide, and the amino acid at position i is (R)-2-(7'-octenyl)alanine (R8) and the amino acid at position i + 7 is S5.
8. A polypeptide, or a pharmaceutically acceptable salt thereof, comprising a modified amino acid sequence of EELAGSLARAl (SEQ ID NO: 1), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO:
4); wherein the modification of the sequence is of at least two of the amino acids within the sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein the non-natural amino acids form a covalent cross-link with each other, wherein the covalent cross-link is internal to the sequence; and wherein the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
9. The polypeptide of claim 8, wherein exactly two of the amino acids are non- natural amino acids.
10. The polypeptide of claim 8 or 9, wherein two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5.
1 1. The polypeptide of claim 8, wherein the modified amino acid sequence is of EELAGSLARAl (SEQ ID NO: 1), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second modified amino acid sequence of EKGAAQVAAVLAQ (SEQ ID NO: 2); wherein the modification of the second sequence is of at least two of the amino acids within the second sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein the non-natural amino acids form a covalent cross-link with each other, wherein the covalent cross-link is internal to the second sequence.
12. The polypeptide of claim 8, wherein the modified amino acid sequence is of EELATRLSQAI (SEQ ID NO: 3), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second modified amino acid sequence of EKAAAQVAAVLAQ (SEQ ID NO: 4); wherein the modification of the second sequence is of at least two of the amino acids within the second sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein the non-natural amino acids form a covalent cross-link with each other, wherein the covalent cross-link is internal to the second sequence.
13. The polypeptide of claim 1 1 or 12, wherein exactly four of the amino acids are non-natural amino acids.
14. The polypeptide of any one of claims 11-13, wherein two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5, and another two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5.
15. A polypeptide, or a pharmaceutically acceptable salt thereof, the polypeptide comprising: at least two non-natural amino acids capable of forming an internal cross-link, wherein the non-natural amino acids are the same or are different, wherein each of the non-natural amino acids includes a moiety, wherein the moieties are capable of undergoing a reaction to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the polypeptide; and
(a) wherein the polypeptide has from the N- to C-terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine; or the polypeptide has from the N- to C-terminus (1 ) a glutamine one amino acid position before a first valine, and (2) optionally
(i) wherein the first valine is three amino acid positions before a second valine, and
(li) wherein the second valine is one amino acid position before a leucine; and (b) wherein if the non-natural amino acids form an internal cross-link, the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
16. The polypeptide of claim 15, wherein the polypeptide has from the N- to C- terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine, and wherein the non-natural amino acids capable of being cross-linked are at position within the polypeptide and at position i + 3, i + 4, or i + 7 within the polypeptide; and wherein the amino acid at position i + 6 within the polypeptide is the leucine, the amino acid at position i + 9 within the polypeptide is the alanine, and the amino acid at position i + 10 within the polypeptide is the isoleucine.
17. The polypeptide of claim 15, wherein the polypeptide has from the N- to C- terminus (1) a glutamine one amino acid position before a first valine, and (2) optionally
(i) wherein the first valine is three amino acid positions before a second valine, and
(ii) wherein the second valine is one amino acid position before a leucine; and wherein the non-natural amino acids capable of being cross-linked are at position / within the polypeptide and at position i + 3, i + 4, or i + 7 within the polypeptide; and wherein the amino acid at position i + 1 within the polypeptide is the glutamine, and the amino acid at position i + 2 within the polypeptide is the first valine.
18. The polypeptide of claim 16 or 17, wherein the amino acids capable of being cross-linked are: at position i within the polypeptide and at position i + 4 within the polypeptide, and the amino acid at position i is (5)-2-(4'-pentenyl)alanine (S5) and the amino acid at position i + 4 is S5; or at position within the polypeptide and another amino acid at position + 3 within the polypeptide, and the amino acid at position i is (i?)-2-(4'-pentenyl)alanine (R5) or ( ?)-2-(2'- propenyl)alanine (R3) and the amino acid at position i + 3 is S5; or the amino acids capable of being cross-linked are at position i within the polypeptide and at position i + 7 within the polypeptide, and the amino acid at position is (R)-2-(7'- octenyl)alanine (R8) and the amino acid at position i + 7 is S5.
19. The polypeptide of claim 15, wherein the polypeptide has from the N- to C- terminus a leucine three amino acid positions before an alanine, wherein the alanine is one amino acid position before an isoleucine, wherein the polypeptide is a first polypeptide, and wherein the polypeptide further comprises a second polypeptide comprising at least two non-natural amino acids capable of forming an internal cross-link, wherein the non- natural amino acids are the same or are different, wherein each of the non-natural amino acids includes a moiety, wherein the moieties are capable of undergoing a reaction to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the second polypeptide, wherein the second polypeptide has from the N- to C-terminus (1) a glutamine one amino acid position before a first valine, and (2) optionally
(i) wherein the first valine is three amino acid positions before a second valine, and
(h) wherein the second valine is one amino acid position before a leucine.
20. The polypeptide of claim 19, wherein
(a) the non-natural amino acids capable of being cross-linked are at position i within the first polypeptide and at position i + 3, z + 4, or i + 7 within the first polypeptide; and wherem the amino acid at position + 6 within the first polypeptide is the leucine, the amino acid at position i + 9 within the first polypeptide is the alanine, and the amino acid at position / + 10 within the first polypeptide is the isoleucine; and
(b) the non-natural amino acids capable of being cross-linked are at position i within the second polypeptide and at position i + 3, /' + 4, or / + 7 within the second polypeptide; and wherein the amino acid at position i + 1 within the second polypeptide is the glutamine, and the amino acid at position i + 2 within the second polypeptide is the first valine.
21. The polypeptide of claim 19 or 20, wherein
(a) the non-natural amino acids capable of being cross-linked in the first polypeptide are: at position /' within the first polypeptide and at position i + 4 within the first polypeptide, and the amino acid at position i is (5)-2-(4'-pentenyl)alanine (S5) and the amino acid at position / + 4 is S5; or at position i within the first polypeptide and another amino acid at position i + 3 within the first polypeptide, and the amino acid at position i is (R)-2-(4'-pentenyl)alanine (R5) or (R)-2-(2'-propenyl)alanine (R3) and the amino acid at position i + 3 is S5; or at position i within the first polypeptide and at position i + 7 within the first polypeptide, and the amino acid at position i is (R)-2-(7!-octenyl)alanine (R8) and the amino acid at position / + 7 is S5; and
(b) the non-natural amino acids capable of being cross-linked in the second polypeptide are: at position i within the second polypeptide and at position i + 4 within the second polypeptide, and the amino acid at position i is (S)-2-(4'-pentenyl)alanine (S5) and the amino acid at position i + 4 is S5; or at position i within the second polypeptide and another amino acid at position i + 3 within the second polypeptide, and the amino acid at position / is (R)-2-(4'-pentenyl)alanine (R5) or (i?)-2-(2'-propenyl)alanine (R3) and the amino acid at position + 3 is S5; or at position i within the second polypeptide and at position i + 7 within the second polypeptide, and the amino acid at position i is (R)-2-(7'-octenyl)alanine (R8) and the amino acid at position i + 7 is S5.
22. A polypeptide, or a pharmaceutically acceptable salt thereof, comprising a modified amino acid sequence of EELAGSLARAI (SEQ ID NO: 1), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO:
4); wherein the modification of the sequence is of at least two of the amino acids within the sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein each of the non-natural amino acids include a moiety, wherein each moiety is capable of undergoing a reaction with a moiety of one other of the non-natural amino acids to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the sequence; and wherein if the non-natural amino acids form an internal cross-link with each other, the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
23. The polypeptide of claim 22, wherein exactly two of the amino acids are replaced with non-natural amino acids.
24. The polypeptide of claim 22 or 23, wherein two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5.
25. The polypeptide of claim 22, wherein the modified amino acid sequence is of EELAGSLARAI (SEQ ID NO: 1 ), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second modified amino acid sequence of EKGAAQVAAVLAQ (SEQ ID NO: 2); wherein the modification of the second sequence is of at least two of the amino acids within the second sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein each of the non-natural amino acids include a moiety, wherein each moiety is capable of undergoing a reaction with a moiety of one other of the non-natural amino acids to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the second sequence.
26. The polypeptide of claim 22, wherein the modified amino acid sequence is of EELATRLSQAI (SEQ ID NO: 3), wherein the sequence is a first modified amino acid sequence, and wherein the polypeptide further comprises a second modified amino acid sequence of EKAAAQVAAVLAQ (SEQ ID NO: 4); wherein the modification of the second sequence is of at least two of the amino acids within the second sequence, wherein the at least two amino acids are replaced by non-natural amino acids, wherein the non-natural amino acids are all the same or are different for the at least two of the non-natural amino acids; wherein each of the non-natural amino acids include a moiety, wherein each moiety is capable of undergoing a reaction with a moiety of one other of the non-natural amino acids to form a covalent cross-link with each other, wherein the covalent cross-link is internal to the second sequence.
27. The polypeptide of claim 25 or 26, wherein exactly four of the ammo acids are non-natural amino acids.
28. The polypeptide of any one of claims 25-27, wherein two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5, and another two non-natural amino acids are R3 and S5, S5 and S5, R5 and S5, or R8 and S5
29. A polypeptide, or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence modified according to the formula (I):
A B
Figure imgf000098_0001
wherein:
Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
R3 is alkylene, alkenylene, alkynylene, or [R4-K'-R4']n, each of which is substituted with 0-6 R5;
R4 and R4' are independently alkylene, alkenylene, or alkynylene;
R5 is independently halo, alkyl, OR6, N(R6)2, SR6, SOR6, S02R6, C02R6, Re, a fluorescent moiety, or a radioisotope;
K' is independently O, S, SO, SO2, CO, CO2, CONR6, or
Figure imgf000098_0002
R6 is independently H, alkyl, or a therapeutic agent; n is independently an integer from 1 -4;
[Xaa]x has 2 to 6 amino acids; [Xaa]w, [Xaa]x, and [Xaa]y taken together have the amino acid sequence of
EELAGSLARAI (SEQ IE) NO: 1), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the amino acids within the sequence are replaced with the residues A and B; and wherein the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
30. The polypeptide of claim 29, wherein the modified amino acid sequence is of EELAGSLARAI (SEQ ID NO: 1), wherein the sequenceis a first modified amino acid sequence, and wherein the polypeptide further comprises a second amino acid sequence modified according to the formula (la):
Figure imgf000099_0001
wherein:
Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
R3 is alkylene, alkenylene, alkynylene, or [R4-K'-R4 ,]n, each of which is substituted with 0-6 R5;
R4 and R ' are independently alkylene, alkenylene, or alkynylene;
R5 is independently halo, alkyl, OR6, N(R6)2, SR6, SOR6, S02R6, C02R6, R&, a fluorescent moiety, or a radioisotope; independently O, S, SO, SO2, CO, CO2, CONR&,
Figure imgf000100_0001
R6 is independently H, alkyl, or a therapeutic agent; n is independently an integer from 1-4; [Xaa]x has 2 to 6 amino acids;
[Xaa]w, [Xaa]x, and [Xaa]y taken together have the amino acid sequence of
EKGAAQVAAVLAQ (SEQ ID NO: 2), wherein two of the amino acids within the sequence are replaced with the residues A and B.
31. The polypeptide of claim 29, wherein the modified amino acid sequence is of EELATRLSQAI (SEQ ID NO: 3), wherein the sequenceis a first modified amino acid sequence, and wherein the polypeptide further comprises a second amino acid sequence modified according to the formula (la):
Figure imgf000100_0002
wherein:
Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl; R3 is alkylene, alkenylene, alkynylene, or [ILt-K'-R^n, each of which is substituted with 0-6 R5;
R4 and R4' are independently alkylene, alkenylene, or alkynylene;
Rs is independently halo, alkyl, OR6, N(R6)2, SR6, SOR6, S02R6, C02R6, R&, a fluorescent moiety, or a radioisotope;
K' is independently O, S, SO, SO2, CO, CO2, CONR6, or
Figure imgf000101_0001
R6 is independently H, alkyl, or a therapeutic agent; n is independently an integer from 1 -4; [Xaa]x has 2 to 6 amino acids;
[Xaa]w, [Xaa]x, and [Xaa]y taken together have the amino acid sequence of
EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the amino acids within the sequence are replaced with the residues A and B.
32. A polypeptide, or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence modified according to the formula (II):
Figure imgf000102_0001
wherein:
Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
R3 and R3' are independently alkenyl, alkynyl, azido, amino, carboxylic acid, or sulfide;
[Xaa]x has 2 to 6 amino acids;
[Xaa]w, [Xaa]x, and [Xaa]y taken together have the amino acid sequence of
EELAGSLARAI (SEQ ID NO: 1), EKGAAQVAAVLAQ (SEQ ID NO: 2), EELATRLSQAI (SEQ ID NO: 3), or EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the amino acids within the sequence are replaced with the residues A and B; and wherein if A and B form an internal cross-link, the polypeptide is capable of inhibiting the linear ubiquitin chain assembly complex (LUBAC).
33. The polypeptide of claim 32, wherein the modified amino acid sequence is of EELAGSLARAI (SEQ ID NO: 1 ), wherein the sequenceis a first modified amino acid sequence, and wherein the polypeptide further comprises a second amino acid sequence modified according to the formula (Ila):
A B
Figure imgf000103_0001
wherein:
Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
R3 and Ry are independently alkenyl, alkynyl, azido, amino, carboxylic acid, or sulfide:
[Xaa]x has 2 to 6 amino acids;
[Xaa]w, [Xaa]x, and [Xaa]y taken together have the amino acid sequence of
EKGAAQVAAVLAQ (SEQ ID NO: 2), wherein two of the amino acids within the sequence are replaced with the residues A and B.
34. The polypeptide of claim 32, wherein the modified amino acid sequence is of EELATRLSQAI (SEQ ID NO: 3), wherein the sequenceis a first modified amino acid sequence, and wherein the polypeptide further comprises a second amino acid sequence modified according to the formula (Ila):
Figure imgf000104_0001
wherein:
Ri and R2 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;
R3 and R3- are independently alkenyl, alkynyl, azido, amino, carboxylic acid, or sulfide;
[Xaa]x has 2 to 6 amino acids;
[Xaa]w, [Xaa]x, and [Xaa]y taken together have the amino acid sequence of
EKAAAQVAAVLAQ (SEQ ID NO: 4), wherein two of the amino acids within the sequence are replaced with the residues A and B.
35. The polypeptide of any one of claims 29-34, wherein [Xaa]x has 2 amino acids.
36. The polypeptide of any one of claims 29-34, wherein [Xaa]x has 3 amino acids.
37. The polypeptide of any one of claims 29-34, wherein [Xaa]x has 6 amino acids.
38. A polypeptide comprising the amino acid sequence of any one of SEQ ID NOS: 8-21, 23-41, 43-57, or 59-77.
39. The polypeptide of claim 38, wherein the non-natural amino acids are not cross-linked.
40. The polypeptide of claim 38, wherein the non-natural amino acids are cross- linked.
41. The polypeptide of any one of claims 1 -40, wherein the polypeptide includes a capping group, a linker group, or both a capping group and linker group.
42. The polypeptide of claim 41, wherein the polypeptide includes a linker group, wherein the linker group is beta-alanine.
43. The polypeptide of any one of claims 1 -40, wherein the polypeptide includes an N-terminal amino protecting group, a C-terminal carboxyl protecting group, or both an N- terminal amino protecting group and a C-terminal carboxyl protecting group.
44. The polypeptide of any one of claims 1-40, wherein the polypeptide includes an N-terminal acetyl.
45. The polypeptide of any one of claims 1-40, wherein the polypeptide C- terminus is amidated.
46. A pharmaceutical composition comprising an effective amount of a polypeptide of any one of claims 1 -45.
47. A polypeptide of any one of claims 1 -45 or a pharmaceutical composition of claim 46 for use in inhibiting the linear ubiquitin chain assembly complex (LUBAC) in a subject.
48. A polypeptide of any one of claims 1 -45 or a pharmaceutical composition of claim 46 for use in treating activated B-cell like diffuse large B cell lymphoma (ABC DLBCL) in a subject.
49. A polypeptide of any one of claims 1-45 or a pharmaceutical composition of claim 46 for use in treating rheumatoid arthritis in a subject.
50. A polypeptide of any one of claims 1-45 or a pharmaceutical composition of claim 46 for use in treating cancer that is resistant to cytotoxic chemotherapy, radiation therapy, vaccine therapy, or cytokine therapy in a subject,.
51. A polypeptide of any one of claims 1-45 or a pharmaceutical composition of claim 46 for use in treating chronic autoinflammation, systemic lupus erythematosus, Crohn's inflammatory bowel disease, or psoriasis in a subject.
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