WO2005014798A2 - Novel compositions and methods for promoting, inhibiting, and detecting protein entry into cells - Google Patents

Novel compositions and methods for promoting, inhibiting, and detecting protein entry into cells Download PDF

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WO2005014798A2
WO2005014798A2 PCT/US2004/009829 US2004009829W WO2005014798A2 WO 2005014798 A2 WO2005014798 A2 WO 2005014798A2 US 2004009829 W US2004009829 W US 2004009829W WO 2005014798 A2 WO2005014798 A2 WO 2005014798A2
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lys
glu
compound
thr
translocation
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PCT/US2004/009829
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French (fr)
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WO2005014798A3 (en
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John R. Murphy
Ryan Ratts
Daniel A. Pearson
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Boston Medical Center Corporation
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Publication of WO2005014798A2 publication Critical patent/WO2005014798A2/en
Publication of WO2005014798A3 publication Critical patent/WO2005014798A3/en
Priority to US11/214,997 priority Critical patent/US7517667B2/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
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention features methods and compositions that relate to the cytosolic translocation factor complex (CTL) responsible for the translocation of the catalytic domain of diphtheria toxin from the lumen of endosomes to the cytosol.
  • CTL cytosolic translocation factor complex
  • Diphtheria toxin (58 kDa) is a typical single chain AB toxin composed of three functional domains: the amino terminal catalytic (C) domain corresponds to fragment A (21 kDa), and the transmembrane (T) and carboxy terminal receptor binding (R) domains comprise fragment B (37 kDa) of the toxin (Choe et al., Nature 357: 216-22, 1992).
  • C amino terminal catalytic
  • T transmembrane
  • R carboxy terminal receptor binding
  • Cysl86 and Cys201 subtends a protease sensitive loop and connects fragment A with fragment B. Furin mediated cleavage within this loop and retention of the disulf ⁇ de bond have been shown to be pre-requisites for intoxication of eukaryotic cells (Tsuneoka et al., J iol. Chem. 268:26461-5, 1993; Ariansen et al., Biochem. 32:83-90, 1993).
  • Anthrax toxin is a binary complex that may be assembled from three distinct protein chains: protective antigen (PA), lethal factor (LF), and edema factor (EF) (Mourez et al, Trends in Microbiology 10: 287-293, 2002).
  • PA protective antigen
  • LF lethal factor
  • EF edema factor
  • Protective antigen (p83) binds to a universal cell surface receptor and a 20 kDa fragment is removed by digestion with the endoprotease furin (MoUoy et al, J.
  • bacterial toxins such as, for example, diphtheria, anthrax lethal factor, anthrax lethal edema factor, and the seven serotypes of botulinum toxin, as well as viral transcription factors, such as, for example, HIV-1 reverse transcriptase and Tat, and that that process requires both a cytosolic translocation factor (CTF) complex and components of the outer surface of endocytic vesicles.
  • CTF cytosolic translocation factor
  • the invention features a compound of formula I: X - AA 210 - AA 211 - AA 212 - AA 213 - AA 214 - AA 215 - AA 16 - AA 217 - AA 218 - AA 219 - AA 220 - AA 221 - AA 222 -Y (I), where X is H or a chain of amino acids of from 1 to 5 residues substituted at the N- terminus with R 1 -C(0)-, a nitrogen protecting group, or H; Y is OH, NH 2 , NHR 2 , NHR 2 R 3 , OR 4 , or a chain of amino acids of from 1 to 76 residues substituted at the C-terminus with OH, NH 2 , NHR 2 , NHR 2 R 3 , OR 4 , where R 1 is a Ci- 6 alkyl, C 6 or C 10 aryl, - 9 heterocyclyl, - 6 alkoxy,
  • AA is Arg or Lys
  • AA is Asp or Glu
  • AA is Lys or Arg
  • AA 213 is Thr, Ser, Ala, Gly, Val, Asn, or Gin
  • AA 214 is Lys or Arg
  • AA 215 is Thr, Ser, Ala, Gly, Val, Asn, or Gin
  • AA 216 is Lys or Arg
  • AA 217 is lie, Leu, or Val
  • AA 218 is Glu or Asp
  • AA 219 is Ser, Ala, or Gly
  • AA 219 is Ser, Ala, or Gly
  • AA 218 is Glu or Asp
  • AA 219 is Ser, Ala, or Gly
  • AA 219 is Ser, Ala, or Gly
  • AA 218 is Glu or Asp
  • AA 219 is Ser, Ala, or Gly
  • AA 219 is Ser, Ala, or Gly
  • AA 219 is Ser, Ala,
  • Y is - AA 223 - AA 224 - AA 225 - AA 226 - AA 227 - AA 228 - AA 229 -Y a , where Y a is OH, NH 2 , NHR 2 , NHR 2 R 3 , or OR 4 ; AA 223 is His, Phe, or Tyr; AA 224 is Gly, Ala, or Ser; AA 225 is Pro; AA 226 is He, Leu, Val; AA 227 is Lys or Arg; AA 228 is Asn or Gin; and AA 229 is Lys or Arg.
  • AA 210 is Arg; AA 21 !
  • X is X - AA 205 - AA 206 - AA 207 - AA 208 - AA 209 -; Y is - AA 223 - AA 224 - Y a , wherein X a is R !
  • Y a is OH, NH 2 , NHR 2 , NHR 2 R 3 , or OR 4 ;
  • AA 205 is Asp or Glu;
  • AA 206 is Trp, Tyr, or Phe;
  • AA 207 is Asp or Glu;
  • AA 208 is Val, Leu, He, Thr, Ser, or Ala;
  • AA 209 is He, Leu, or Val;
  • AA 223 is His, Tyr, or Phe; and
  • AA 224 is Gly, Ala, or Ser.
  • AA 205 is Asp;
  • AA 206 is Trp;
  • AA 207 is Asp;
  • AA 208 is Val;
  • AA 209 is He;
  • AA 210 is Arg;
  • AA 212 is Lys; AA 213 is Thr; AA 214 is Lys; AA 215 is Thr; AA 216 is Lys; AA 217 is He; AA 218 is Glu; AA 219 is Ser; AA 220 is Leu; AA 221 is Lys; AA 222 is Glu; AA 223 is His; and AA 224 is Gly.
  • X is H or a chain of amino acids of from 1 to 5 residues, preferably corresponding to Asp- Trp-Asp-Val-Ile-
  • Y is OH or a chain of amino acids of from 1 to 76 residues, preferably corresponding to -Arg-Asp-Lys-Thr-Lys-Thr-Lys-He-Glu- Ser-Leu-Lys-Glu-His-Glu-Pro-Ile-Lys-Asn-Lys-Met-Ser-Glu-Ser-Pro-Asn- Lys-Thr-Val-Ser-Glu-Glu-Lys-Ala-Lys-Gln-Tyr-Leu-Glu-Glu-Phe-His-Gln- Thr-Ala-Leu-Glu-His-Pro-Glu-Leu-Ser-Glu-Leu-Lys-Thr-Val-Thr-Gly-Thr- Asn-Pro-Val-P
  • R 1 , R 2 , or R 4 can be a polyethylene glycol moiety selected from the group consisting of: H 3 C(OCH 2 CH 2 ) cc OCH 2 C(0)-, H(OCH 2 CH 2 ) cc OCH 2 C(0)-, H 3 C(OCH 2 CH 2 ) cc OC(0)-, H(OCH 2 CH 2 ) cc OC(0)-, H 3 C(OCH 2 CH 2 ) cc NHC(0)-, H(OCH 2 CH 2 ) cc NHC(0)-, H 3 C(OCH 2 CH 2 ) cc NHC(S)-, H(OCH 2 CH 2 ) cc NHC(S)-, H 3 C(OCH 2 CH 2 ) cc C(0)-, H(OCH 2 CH 2 ) cc C(0)-, H(OCH 2 CH 2 ) cc C(0)-, H(OCH 2 CH 2 ) cc C(0)-, H(OCH 2 CH 2
  • the compounds of the invention By selectively preventing the catalytic domain of toxins or viral factors from translocating across endosomal membranes, the compounds of the invention, or derivatives or peptidomimetics thereof, can inhibit mammalian cell death caused by such toxins/factors. Therefore, these compounds can be used in the prophylaxis or treatment of diseases caused by toxin-producing bacteria or in the prophylaxis or treatment of adverse events that are caused by the direct exposure to mammals of toxins or toxin derivatives, such as, for example, fusion toxin-proteins. In another example, the compounds of the invention are useful for the prophylaxis or treatment of viral diseases by inhibiting the translocation across endosomal membrane of viral/retro viral transcription factors.
  • the invention features the use of any of the compounds of the invention in the manufacture of a medicament for inhibiting cell death in a mammal, preferably a human.
  • the compound inhibits the translocation of a viral or bacterial toxin from the lumen of an endosome to the cytosol of said cell.
  • the toxin is an AB toxin, such as, for example Diphtheria toxin, a Botulinum toxin, Anthrax toxin LF, and Anthrax toxin EF.
  • the compound inhibits the translocation of a viral or retroviral transcription factor, such as, for example, human immunodeficiency virus (HIV-1) reverse transcriptase or Tat.
  • HIV-1 human immunodeficiency virus
  • the invention features a compound having a nucleic acid sequence encoding any of the peptide sequences of the invention.
  • the peptide sequence is selected from the group consisting of: - Arg-Asp-Lys-Thr-Lys-Thr-Lys-Ile-Glu-Ser-Leu-Lys-Glu-His-Gly-Pro-Ile-Lys- Asn-Lys- ; -Asp-Trp- Asp- Val-Ile- Arg- Asp-Lys-Thr-Lys-Thr-Lys-Ile-Glu-Ser- Leu-Lys-Glu-His-Gly-; and -Arg-Asp-Lys-Thr-Lys-Thr-Lys-Ile-Glu-Ser-Leu- Lys-Glu-His-Gly-Pro-Ile-Lys- Asn-Lys-.
  • the peptide sequence is Arg- Asp-Lys-Thr-Lys-Thr-Lys-Ile-Glu-Ser-Leu-Lys-Glu-His-Glu- Pro-Ile-Lys-Asn-Lys-Met-Ser-Glu-Ser-Pro-Asn-Lys-Thr-Val-Ser-Glu-Glu- Lys-Ala-Lys-Gln-Tyr-Leu-Glu-Glu-Phe-His-Gln-Thr-Ala-Leu-Glu-His-Pro- Glu-Leu-Ser-Glu-Leu-Lys-Thr-Val-Thr-Gly-Thr-Asn-Pro-Val-Phe-Ala-Gly- Ala-Asn-Tyr-Ala-Ala-Trp- Ala-Val-Asn-Val-Ala-Gln- Val-Ile- Asp-Ser-Glu- Thr- Ala
  • the nucleic acid is operably linked to an inducible promoter.
  • inducible promoter systems include those where the expression of the peptide sequence can moderated by treating the transfected cell with an agent selected from the group consisting of: doxycycline; retinal; cyclosporin or analogs thereof; FK506; FK520; and rapamycin or analogs thereof.
  • the invention features pharmaceutical compositions of the peptides of the invention.
  • the pharmaceutical compositions can include agents or compounds that facilitate delivery of the peptides to therapeutic targets. Such delivery strategies are described in Therapeutic Protein and Peptide Formulation and Delivery (ACS Symposium Series, No 675) (1997), edited by Shahrokh, et al.
  • the invention features a method of identifying a compound that inhibits cell death in a mammal comprising the following steps: a) isolating endosomes, desirably early endosomes, from the cell, b) placing the endosomes in a cytosolic buffer, c) contacting the endosomes with a fusion protein-toxin, wherein the protein comprises a binding moiety for a component of the cell membrane of the cell and the toxin comprises a fragment of
  • Diphtheria toxin d) contacting the endosomes with a cytosolic translocation factor complex, e) contacting the endosomes with said compound, and f) measuring translocation of the toxin, where a decreased level of said translocation relative to that observed in the absence of the compound indicates that the compound inhibits said cell death.
  • the invention features a method of identifying a compound that promotes cell death in a mammal comprising the following steps: a) isolating endosomes, desirably early endosomes, from the cell, b) placing the endosomes in a cytosolic buffer, c) contacting the endosomes with a fusion protein-toxin, wherein the protein comprises a binding moiety for a component of the cell membrane of the cell and the toxin comprises a fragment of Diphtheria toxin, d) contacting the endosomes with a cytosolic translocation factor complex, e) contacting the endosomes with said compound, and f) measuring translocation of the toxin, where an increased level of said translocation relative to that observed in the absence of the compound indicates that the compound inhibits said cell death.
  • the fusion protein can be any protein or protein fragment that binds to a component of mammalian cellular membranes and is subsequently internalized.
  • the fusion protein is IL-2.
  • Other examples include monoclonal antibodies that bind to cellular membrane epitopes.
  • the fusion protein-toxin is DAB 389 IL-2.
  • cytosolic translocation factor comprises Hsp 90 and thioredoxin reductase. Assessing translocation can include measuring the ADP- ribosylation of elongation factor-2.
  • the invention features a composition that contains heat shock protein 90 (Hsp 90), or a protein that is substantially identical to Hsp 90, complexed to a cellular fraction, where the composition is formed by adding Hsp 90 to the cytosol of a mammalian cell, followed by isolating the cellular cytosolic fraction that has a molecular weight of between 100 kDa and 250 kDa and also facilitates the translocation of the fusion protein DAB 389 IL-2 from the interior to the exterior of endosomes.
  • the Hsp 90 that is added is a human recombinant protein.
  • thioredoxin reductase is also part of the composition.
  • the invention features a composition that contains thioredoxin reductase (TrR-1), or a protein that is substantially identical to TrR-1, complexed to a cellular fraction, where the composition is formed by adding TrR-1 to the cytosol of a mammalian cell, followed by isolating the cytosolic fraction that has a molecular weight of between 100 kDa and 250 kDa and also facilitates the translocation of the fusion protein DAB 389 IL-2 from the interior to the exterior of endosomes.
  • the TrR-1 that is added is a human recombinant protein.
  • Hsp 90 is also part of the composition.
  • the composition includes TrR-1 and Hsp 90, where both of these components are human recombinant proteins.
  • bovine recombinant when not referring to the amino acid cysteine, "C” stands for catalytic; “CTF” stands for cytosolic translocation factor; “DT” stands for diphtheria toxin; “EF-2” stands for Elongation Factor 2; “ESI” stands for electrospray ionization; “hr” stands for human recombinant; “Hsp” stands for heat shock protein; “MALDI” stands for matrix assisted laser desorption ionization; “MS” stands for mass spectrometry; when not referring to the amino acid threonine, “T” stands for transmembrane; “TrR-1” stands for: thioredoxin reductase; “v” stands for vesicular.
  • alkoxy or "alkyloxy,” as used interchangeably herein, represent an alkyl group attached to the parent molecular group through an oxygen atom.
  • exemplary unsubstituted alkoxy groups are of from 1 to 6 carbons
  • alkyl represents a monovalent group derived from a straight or branched chain saturated hydrocarbon of, unless otherwise specified, from 1 to 6 carbons and is exemplified by methyl, ethyl, n- and iso- propyl, n-, sec-, iso- and tert-butyl, neopentyl and the like and may be optionally substituted with one, two, three or, in the case of alkyl groups of two carbons or more, four substituents independently selected from the group consisting of: (1) alkoxy of one to six carbon atoms; (2) alkylsulf ⁇ nyl of one to six carbon atoms; (3) alkylsulfonyl of one to six carbon
  • aryl represents a mono- or bicyclic carbocyclic ring system having one or two aromatic rings and is exemplified by phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl and the like and may be optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of: (1) alkanoyl of one to six carbon atoms; (2) alkyl of one to six carbon atoms; (3) alkoxy of one to six carbon atoms; (4) alkoxyalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (5) alkylsulfinyl of one to six carbon atoms; (6) alkylsulfinylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms
  • arylalkoxy or “aralkoxy,” as used interchangeably herein, represent an arylalkyl group attached to the parent molecular group through an oxygen atom. Exemplary unsubstituted arylalkoxy groups are of from 7 to 16 carbons.
  • arylalkyl or “aralkyl,” as used interchangeably herein, represent an aryl group attached to the parent molecular group through an alkyl group. Exemplary unsubstituted arylalkyl groups are of from 7 to 16 carbons.
  • heteroaryl represents that subset of heterocycles, as defined herein, which are aromatic: i.e., they contain 4n+2 pi electrons within the mono- or multicyclic ring system. Exemplary unsubstituted heteroaryl groups are of from 1 to 9 carbons.
  • heterocycle or “heterocyclyl,” as used interchangeably herein represent a 5-, 6- or 7-membered ring, unless otherwise specified, containing one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur.
  • the 5-membered ring has zero to two double bonds and the 6- and 7-membered rings have zero to three double bonds.
  • heterocycle also includes bicyclic, tricyclic and tetracyclic groups in which any of the above heterocyclic rings is fused to one or two rings independently selected from the group consisting of an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring and another monocyclic heterocyclic ring such as indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl and the like.
  • Heterocyclics include pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl, morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, furyl, thienyl, thiazolidin
  • F is selected from the group consisting of -CH 2 -, -CH 2 0- and -0-
  • G is selected from the group consisting of -C(O)- and -(C(R')(R")) V -, wherein R' and R" are independently selected from the group consisting of hydrogen or alkyl of one to four carbon atoms, and v is one to three and includes groups such as 1,3-benzodioxolyl, 1,4-benzodioxanyl and the like.
  • any of the heterocycle groups mentioned herein may be optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of: (1) alkanoyl of one to six carbon atoms; (2) alkyl of one to six carbon atoms; (3) alkoxy of one to six carbon atoms; (4) alkoxyalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (5) alkylsulfinyl of one to six carbon atoms; (6) alkylsulfinylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (7) alkylsulfonyl of one to six carbon atoms; (8) alkylsulfonylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (9) aryl; (10) arylalkyl, wherein the alkyl group is of one to six carbon atoms
  • heterocyclyloxy or " (heterocycle) oxy,” as used interchangeably herein, represents a heterocycle group, as defined herein, attached to the parent molecular group through an oxygen atom.
  • exemplary unsubstituted heterocyclyloxy groups are of from 1 to 9 carbons.
  • amino acid residue represents a - N(R A )C(R B )(R c )C(0)- linkage, wherein R A is selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, as defined herein; and R and R are independently selected from the group consisting of: (a) hydrogen, (b) optionally substituted alkyl, (c) optionally substituted cycloalkyl, (d) optionally substituted aryl, (e) optionally substituted arylalkyl, (f) optionally substituted heterocyclyl, and (g) optionally substituted heterocyclylalkyl, each of which is as defined herein.
  • R is H and R corresponds to those side chains of natural amino acids found in nature, or their antipodal configurations.
  • exemplary natural amino acids include alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, aspartamine, ornithine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine, each of which, except glycine, as their D- or L-form.
  • Non-naturally occuring (i.e., unnatural) amino acid residues in their D- or L-form such as, for example, homophenylalanine, phenylglycine, cyclohexylglycine, cyclohexylalanine, cyclopentyl alanine, cyclobutylalanine, cyclopropylalanine, cyclohexylglycine, norvaline, norleucine, ornithine, thiazoylalanine (2-, 4- and 5- substituted), pyridylalanine (2-, 3- and 4-isomers), naphthalalanine (1- and 2-isomers) and the like.
  • non-naturally occuring amino acid residues in their D- or L-form such as, for example, homophenylalanine, phenylglycine, cyclohexylglycine, cyclohexylalanine, cyclopentyl alanine, cyclobut
  • cytosolic buffer any buffering system into which endosomes can be placed where they remain intact and viable. In one example; 3% sucrose in 100 mM HEPES-KOH pH 7.9, 1.4 mM KCl, 30 mM MgCl 2 , 2 mM EDTA, and 5 mM DTT constitutes a cytosolic buffer.
  • cytosolic translocation factor complex is a group of component proteins that includes Hsp 90 and TrR-1, with the complex also having the ability to facilitate the translocation of the catalytic domain of diphtheria toxin from the interior to the exterior of an endosome.
  • a “pharmaceutically acceptable excipient” is meant a carrier that is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered.
  • One exemplary pharmaceutically acceptable excipient is physiological saline.
  • Other physiologically acceptable excipients and their formulations are known to one skilled in the art and described, for example, in “Remington: The Science and Practice of Pharmacy” (20th ed., ed. A.R. Gennaro AR., 2000, Lippincott Williams & Wilkins).
  • operably linked is meant that a nucleic acid molecule and one or more regulatory sequences (e.g., a promoter) are connected in such a way as to permit expression and/or secretion of the product (i.e., a polypeptide) of the nucleic acid molecule when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.
  • polypeptide or “peptide” is meant any chain of from 2 to 100 natural or unnatural amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally- occurring or non-naturally occurring polypeptide or peptide, as is described herein.
  • Naturally occurring amino acids are any one of the following, alanine (A or Ala), cysteine (C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H, or His), isoleucine (I or He), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), ornithine (O or Om), proline (P or Pro), hydroxyproline (Hyp), glutamine (Q or Gin), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), or tyrosine (Y or Tyr).
  • a or Ala alanine
  • cysteine C or Cys
  • aspartic acid D or Asp
  • E or Glu glut
  • substantially identical is meant a protein, polypeptide, or nucleic acid exhibiting at least 75%, but preferably 85%, more preferably 90%o, most preferably 95%, or even 99% identity to a reference amino acid or nucleic acid sequence.
  • the length of comparison sequences will generally be at least 10 amino acids, and preferably at least 20 amino acids.
  • the length of comparison sequences will generally be at least 30 nucleotides, preferably at least 60 nucleotides, and more preferably at least 120 nucleotides.
  • Figure 1 shows the partial purification of cytosolic proteins required to mediate DT C domain translocation from the lumen of early endosomes in vitro.
  • A Early endosomes from human T cells (HUT102/6TG), preloaded with DAB 389 IL-2, were incubated for 30 min. at 37° with 2 mM ATP (A) and/or 4 ⁇ g of HUT102/6TG crude cytosol (C).
  • C* denotes heat inactivation of cytosol prior to incubation with endosomes.
  • Both the supernatant and the pellet fractions were assayed for ADP-ribosyltransferase activity, and the autoradiographic signals were measured by densitometry.
  • Figure 2 shows the in vitro acidification of early endosomes requires ATP and does not require any cytosolic protein factors.
  • A Fluorescence emission of 1 ng/ml SNARF-1 70 kDa dextran conjugate standards at pH 7.5 and 4.5 was measured at an excitation wavelength of 534 nM and an emission wavelength of 645 nM.
  • B Purified early endosomes pre-loaded with the pH sensitive SNARF-1 70 kDa Dextran Conjugate were incubated in translocation assay buffer for 20 min. at 37°C with 2 mM ATP and/or 0.1 ⁇ g/ ⁇ L of MONO-Q purified cytosol. In each instance, assays were performed in triplicate and fluorescence was monitored using a Perkin-Elmer 650S Fluorescence Detector.
  • FIG. 3 shows the partial purification of CTFs results in the increase of translocation in vitro specific activity.
  • A Translocation in vitro specific activity of CTFs increases following each stage of purification. Reactions were performed as previously described, and only the ADP-ribosyltransferase activity of the supernatant fluid fractions is shown.
  • CE crude extract
  • DEAE DEAE-Sepharose anion exchange chromatography (150-190 M NaCl fractions)
  • S200 Sephacryl 200 sizing chromatography (250-100 kDa fractions)
  • MQ Mono-Q anion exchange chromatography (27.3 mS fractions).
  • B Colloidal Coomassie stained 10% SDS-PAGE protein band profiles following MONO-Q anion exchange chromatography. Partially purified CTF complex fractions from both T cells and yeast cells were eluted at a conductance of 27.3 mS.
  • Figure 4 shows the identification of CTF(s) using mass spectrometry.
  • FIG. 2B, 5B Representative total ion chromatogram from an online capillary liquid chromatography mass spectrometric analysis of the 'in-geP tryptic digest following immunoprecipitation of the 84 kDa band (Fig. 2B, 5B) from human partially purified CTFs using rabbit polyclonal anti-Hsp 90 antibodies.
  • B Mass spectrum from LC-MS elution at time 19.5 to 20.5 min, as indicated by the shaded region in (A). Peaks are labeled with the m/z value, the charge state, the corresponding amino acid segment, and specification of the Hsp 90 isoform.
  • FIG. 1 Colloidal Coomassie stained 10% SDS- PAGE protein band profiles of immunoprecipitated human Hsp 90. Arrow indicates Hsp 90 as identified by ESI LC-MS/MS analysis. Figure 6 shows that Hsp 90 is essential for mediating DAB 389 IL-2 C domain translocation from the lumen of early endosomes to the external milieu.
  • TrR-1 is a component of the CTF complex.
  • Y partially purified CTF complexes were depeleted of TrR-1 by immunoprecipitation with anti-human TrR-1 antibodies or affinity purification using 2',5' ADP-Sepharose. TrR-1 depleted CTF complexes were then assayed for translocation activity in vitro under reducing conditions.
  • TrR-1 Either partially purified CTF complex or brTrR-1 was added back to TrR-1 depleted CTF complexes as indicated, and translocation activity in vitro was assayed as previously described.
  • B Colloidal Coomassie stained 10%) SDS-P AGE protein band profiles of 2 ',5' ADP-sepharose affinity purified yeast TrR-1. Arrow indicates yeast TrR-1 as identified by ESI LC-MS/MS analysis.
  • Figure 8 shows that TrR-1 function is essential for mediating DAB 389 IL- 2 C domain translocation from the lumen of early endosomes to the external milieu under non-reducing conditions.
  • Partially purified CTF complex both human and yeast, were assayed for translocation activity in vitro under non- reducing conditions using translocation buffer containing 10 ⁇ M NADPH without DTT.
  • the TrR-1 stereospecific inhibitor ct-s-13-retinoic acid and the inactive tr ⁇ s-13-retinoic acid isomer were pre-incubated with partially purified CTF complex as indicated for 15 min at room temperature prior to assaying for translocation activity in vitro under non-reducing conditions.
  • Excess brHsp 90 was added to cl3RA treated CTF complexes as indicated and translocation activity in vitro was assayed under non-reducing conditions.
  • Figure 9 shows a Blast analysis (BLAST NCBI, Vector NTI version 8.0) of Anthrax toxin-EF, Anthrax toxin-LF, Botulinum A, Botulinum-Cl, Botulinum D, Diphtheria toxin, and HIV reverse transcriptase isolated from 6 different patients.
  • Figure 10 shows (A) The entry motif of diphtheria toxin, anthrax lethal factor, anthrax edema factor and boutlinum neurotoxin serotype A that was identified by BLAST analysis of the diphtheria toxin transmembrane helix Tl.
  • Figure 11 shows an autoradiographic analysis of diphtheria toxin catalytic domain translocation and release from purified early endosomes in the presence and absence of a peptide corresponding to DT amino acid residues 205-224.
  • the translocation assay (TA) was performed in the presence of ATP and partially purified cytosolic translocation factor complex from HUT 102 6TG cells. Control translocation is performed in the absence of DT205-224 peptide.
  • the ADP-ribosyltransferase reactions were performed on supernatant fluid and lysed pellet fractions following ultracentrifugation. Under the TA conditions used, approximately 50% of the diphtheria toxin catalytic domain is specifically translocated from the lumen of the early endosome to the external milieu.
  • FIG. 12 shows a plasmid that incorporates DT 210-299. This sequence was cloned into the pTRACER expression vector and placed under control of the CMV promoter to form plasmid pTl . Plasma pTl was transfected into HUT 102 cells and stable transfectants were isolated using zeocine selection.
  • FIG. 13A shows the sensitivity of HUT 102 (closed circles) and HUT102-T1 cells (closed squares) to the fusion toxin DAB389IL-2 in a dose response study.
  • Figure 13B shows the sensitivity of HUT 102 (closed circles) and HUT102-T1 cells (closed squares) to Pseudomonas exotoxin A, a bacterial protein toxin known to enter the cell at the level of the trans-golgi (rather than from an acidified early endosome).
  • Figure 14 shows the amino acid and corresponding nucleic acid sequences of DT 210-299.
  • Hsp 90 or TrR-1 Following depletion of either Hsp 90 or TrR-1 from partially purified human T cell and yeast CTF-complexes, we were not successful in reconstituting in vitro translocation of the C-domain by the addition of hrHsp 90 or brTrR-1, either alone or in combination.
  • Hsp 90 chaperonin and TrR-1 are components of a complex(es) that is(are) necessary for facilitating C-domain translocation across the early endosomal membrane.
  • Hsp 90 is ubiquitously expressed and is known to be a component of several multi-molecular chaparonin complexes which are highly conserved in eukaryotes (Chang et al, J. Biol Chem. 269:24983-8, 1994).
  • the interaction of Hsp 90 with other co-chaparonins and the formation of discrete complexes is known to mediate Hsp 90 substrate recognition (Caplan et al, Trends Cell. Biol 9:262-8, 1999).
  • Hsp 90 does not usually directly bind nor refold nascent polypeptides, it is known to refold a growing list of newly synthesized proteins including membrane associated protein kinases (Bijlmakers and Marsh, Hsp 90 is essential for the synthesis and subsequent membrane association, but not the maintenance, of the Src-kinase p56(lck). Mol. Biol. Cell 11 : 1585-95, 2000). In addition to its refolding activity, Hsp 90 complexes are also known to regulate the trafficking of membrane associated proteins through interactions with cytoskeleton motors (Pratt et al, Cell. Signal. 11:839-51, 1999).
  • the CTF complex is capable of refolding thermally denatured diphtheria toxin fragment A in vitro, and refolding requires the ATPase activity of Hsp 90.
  • Hsp 90 ATPase activity by either geldanamycin or radicicol alone does not inhibit translocation of ADP-ribosyltransferase activity across the early endosomal membrane. As such, it would appear that refolding of denatured C-domain into an active conformation and translocation are mutually exclusive events.
  • TrR- 1 activity is required for at least the cytosolic release of the DAB 389 lL-2 C-domain from purified early endosomes, we cannot conclude whether or not TrR-1 is directly involved in the reduction of the interchain disulfide bond. Since we have identified thioredoxin peroxidase in CTF-complexes purified from yeast, it is possible that TrR-1 functions indirectly through a cascade of reductases ⁇ e.g., thioredoxin, Moskaug et al, J. Biol.
  • anthrax lethal toxin and edema factor as well as the botulinum neurotoxins must pass through an acidic early endosomal compartment in order to deliver their respective catalytic domain into the cytosol of targeted cells.
  • cyclophilin is required for the cytosolic entry of HIV (Braaten et al, J. Virol. 70:4220-7, 1996), the vacuolar import of fructose- 1,6-bisphophatase (Brown et al, J. Biol. Chem. 276(51):48017-26, 2001), and the activation of peroxiredoxins (Lee et al, J. Biol. Chem.
  • HIV-1 Tat protein a strong trans-activator that enables productive transcription from the HIV-1 long terminal repeat and is required for HIV replication, enters T-cells essentially like diphtheria toxin, using clathrin-mediated endocytosis before low-pH induced and Hsp90-assisted endosomal translocation.
  • Example 1 Partial purification of human T cell and yeast cytosolic factors required for the in vitro translocation of ADP-ribosyltransferase activity across the membrane of early endosomes
  • translocation active fractions 150mM - 190mM NaCl
  • the translocation active fractions 250 - 100 kDa were pooled and further fractionated by MonoQ high performance liquid chromatography under conditions free of reducing agents.
  • the translocation active fraction was found to elute from the MonoQ column at 27.3 mS.
  • CTF complex activity from human T cell and yeast cell extracts were increased by 650-fold and 800-fold, respectively.
  • Further analysis of the MonoQ pooled fractions by SDS-polyacrylamide gel electrophoresis and colloidal Commassie staining revealed multiple protein bands ranging in apparent molecular weight from ca. 12 - 100 kDa (Fig. 3B).
  • Example 2 Identification of individual components of the CTF complex Tryptic peptides from "in gel” digestion of individual protein bands resolved by SDS-polyacrylamide gel electrophoresis were subjected to analysis by mass spectroscopy using matrix-assisted laser desorption/ionization-time-of- flight (MALDI-TOF) and nano-electrospray ionization quadrupole orthogonal-
  • MALDI-TOF matrix-assisted laser desorption/ionization-time-of- flight
  • TOF ESI-QoTOF
  • Peptide maps and tandem mass spectrometry sequence data allowed for the unequivocal identification of Hsp 90 (alpha and beta) and TrR-1 in the partially purified CTF complex mixture from human T cells (Fig. 4; Table 1).
  • the corresponding yeast homologs, Hsp 82 and TrR-1, as well as thioredoxin peroxidase were identified in the partially purified CTF complex from yeast cells (Table 1).
  • the cumulative peptide coverage for each protein identified through mass spectrometry sequencing was between 65 - 85% of the total protein (Table 2). Ions unassigned in the LC-MS/MS spectra were indicative of truncation, sequence variation, and/or post-translational modification.
  • Table 1 Summary of the data obtained for each of the CTF components identified in this study: MALDI, ESI-MS/MS, LC-MS/MS, western blot (WB), in vitro translocation assay (TA), mammalian cell cytotoxicity assay (CA)
  • Table 2 Summary of information dependent acquisition tandem MS sequence data from tryptic digests of human Hsp 90 (alpha and beta) and yeast TrR-1.
  • TrR-1 is essential, but not sufficient for DT C-domain translocation in vitro
  • TrR-1 was also identified by mass spectrometry sequence analysis of CTF complexes from human T cell and yeast extracts, we have used both immunoprecipitation and specific inhibitors to demonstrate a functional role of TrR-1 in the translocation and/or release of the C-domain from early endosomes. As shown in Figure 7 A, immunoprecipitation of TrR-1 from human CTF-complexes and 2',5'-ADP-sepharose affinity chromatographic depletion of yeast TrR-1 from CTF complex mixtures abolished C-domain translocation in vitro.
  • TrR-1 is a component of a complex and that another factor(s) essential for translocation were co-depleted with TrR-1 (Fig. 7B).
  • TrR-1 activity plays an essential role in the translocation and/or release of the C-domain from early endosomes.
  • Example 6 Partial Purification of the DT C-domain CTF complex
  • the requirements for DT C-domain translocation across the early endosomal membrane and release into the external milieu were monitored using an in vitro translocation assay modified from Umata et al, J. Biol. Chem. 265:21940-5, 1990 and Lemichez et al, supra.
  • the early endosomal compartment of HUT 102 6TG cells was pre-loaded with DAB 389 lL-2 in the presence of bafilomycin Al .
  • Early endosomes were purified by sucrose density gradient centrifugation, and then incubated in the presence of ATP and cytosolic extracts from either HUT 102 6TG cells or yeast.
  • IC 50 3 x 10 "n M vs. 4 x 10 " M).
  • Figure IB the ADP-ribosyltransferase activity as measured by densitometry of the combined [ P]-labeled EF2 from each paired pellet and supernatant fluid fraction is plotted as percent ADP-ribosyltransferase activity in the supernatant fluid.
  • translocation of the C-domain is linear for up to 45 min, at which time ca. 80% of the total activity is found in the supernatant fluid fraction.
  • diphtheria toxin Like diphtheria toxin, these other toxins require binding to their specific cell surface receptor, receptor mediated endocytosis, passage through an acidic early endosomal vesicle compartment, and unfolding of their respective catalytic domain prior to translocation and delivery to the cytosol (Wesche et al, supra; Simpson et al, Journal of Pharmacology & Experimental Therapeutics. 269:256-62, 1994). Since diphtheria toxin C-domain translocation across the endosomal vesicle membrane is mediated by a CTF complex, we hypothesize the presence of an amino acid sequence motif that interacts with component(s) of either CTF complex and/or endosomal vesicle-associated factors in the translocation process.
  • FIG. 10 shows the entry motif that was found for diphtheria toxin, anthrax lethal factor and edema factor, and botulinum toxin serotype A. While not shown in this figure, the entry motif is also found in all other six serotypes of botulinum toxin.
  • Panel A we have indicated the relative position of the entry motif on the structure of diphtheria toxin, anthrax lethal factor, anthrax edema factor, and botulinum toxin serotype A.
  • this motif is positioned on the surface of the protein, is an amphipathic alpha helix, and located in a region of the toxin consistent with a potential function in the translocation process.
  • Panel B of Figure 10 shows the motif in an N- to C- terminal orientation directly through the alpha-helix; whereas, Panel C shows the motif in a side view. Taken together, these views of the entry motif reveal a striking conservation of structure and distribution of charge and hydrophobicity.
  • DAB( ⁇ 204-263) 389 IL-2 was found to insert into planar lipid bilayers and form a channel identical to that of wild type fusion protein toxin.
  • This non-toxic mutant is >10,000-times less active than the wild type. Further, it is devoid of the first three helices of the transmembrane domain and requires 50 - 100-times more protein than the wild type fusion protein toxin to make channels (vanderSpek JC et al, J. Biol. Chem. 90:8524-8528, 1993).
  • DAB 389 (L221E)IL-2 carries a mutation in a highly conserved region of the entry motif.
  • DAB 389 (L221E)IL-2 binds to the high affinity IL-2 receptor, forms channels in purified early endosomes, and that its C-domain fails to be translocated in vitro and remains in the pellet fraction in the CTF assay system described above.
  • the entry motif in diphtheria toxin is positioned in transmembrane helix 1 is composed of amino acids 205 - 225.
  • the amino acid sequence of this region is as follows: Asp 205 -T ⁇ -Asp-Val-Ile-Arg-Asp-Lys-Thr-Lys-Thr-Lys- Ile-Glu-Ser-Leu-Lys-Glu-His-Gly-Pro 225 .
  • a peptide corresponding to the sequence of amino acids 205 to 224 was synthesized and examined for its ability to inhibit DAB 389 lL-2 intoxication in the same cytotoxicity assay used to evaluate geldanamycin, radicicol, and retinoic acid (see above).
  • the results, shown in Figure 11, indicate that the translocation of diphtheria toxin catalytic domain was inhibited.
  • X is H, a nitrogen protecting group, R 1 -C(0)-, or a chain of amino acids of from 1 to 5 residues substituted at the N-terminus with R ! -C(0)-, a nitrogen protecting group, or H;
  • Y is OH, NH 2 , NHR 2 , NHR 2 R 3 , OR 4 , or a chain of amino acids of from 1 to 76 residues substituted at the C-terminus with OH, NH 2 , NHR , NHR R , OR , or a carboxyl protecting group, where R is a - 6 alkyl, C 6 or C 10 aryl, .
  • R and R is, independently, H, a C ⁇ - 6 alkyl, C 6 or do aryl, - 9 heterocyclyl, C _ 16 aralkyl, C 2 - 15 heterocyclylalkyl, or a polyethylene glycol moiety
  • R 4 is H, C ⁇ - 6 alkyl, C 6 or C 10 aryl, - 9 heterocyclyl, - 6 alkoxy, C - ⁇ 6 aralkyl, C 2 - 15 heterocyclylalkyl, a carboxyl protecting group or a polyethylene glycol moiety
  • -AA is Arg or Lys
  • AA is Asp or Glu
  • AA is Lys or Arg
  • AA 213 is Thr
  • Modifications of Compounds of the Invention It is also possible to modify the structure of a compound of the invention for such purposes as enhancing therapeutic or prophylactic efficacy, or stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo). Such modified peptides, when designed to retain at least one activity of the naturally- occurring form of the protein, are considered functional equivalents of CTF.
  • modified peptides can be produced, for instance, by amino acid substitution, deletion, or addition.
  • a compound of the invention that inhibits translocation from the endosome to the cytosol of a cell e.g., a peptidyl inhibitor of the invention
  • an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the ability of the peptide to serve as an inhibitor.
  • Conservative replacements or substitutions are those that take place within a family of amino acids that are related in their side chains, and apply to those that result from genetically encoding or those that are synthetically produced.
  • Amino acids can be divided into four families: (1) acidic residues, such as aspartatic acid or glutamic acid; (2) basic residues, such as lysine, arginine, or histidine; (3) nonpolar residues, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan; and (4) uncharged polar residues, such as glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.
  • acidic residues such as aspartatic acid or glutamic acid
  • basic residues such as lysine, arginine, or histidine
  • nonpolar residues such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan
  • amino acid repertoire can be grouped as (1) acidic residues, such as aspartate, glutamate; (2) basic residues, such as lysine, arginine histidine, (3) aliphatic residues, such as glycine, alanine, valine, leucine, isoleucine, serine, threonine, with serine and threonine optionally be grouped separately as aliphatic- hydroxyl; (4) aromatic residues, such as phenylalanine, tyrosine, tryptophan; (5) amide residues, such as asparagine, glutamine; and (6) sulfur-containing ' residues, such as cysteine and methionine (see, for example, Biochemistry, 2nd ed., Ed.
  • amino acid replacement can be based on steric criteria, e.g. isosteric replacements, without regard for polarity or charge of amino acid sidechains.
  • one or more amino acid residues in a compound of the invention can be replaced with another amino acid residue from the same family.
  • mutations can be introduced randomly along all or part of a nucleic acid encoding a compound of the invention, such as by saturation mutagenesis, and the resultant mutants can be screened for their ability to inhibit translocation, by methods described herein.
  • the peptide can be expressed by any recombinant technology known in the art, and the activity of the peptide can be determined.
  • the compounds of the present invention include analogs that contain moieties that improve pharmacodynamic properties, such as, for example, those that increase in vivo half-life; or that improve physical properties, such as, for example, increased resistance to in vivo degradation or increased cell- membrane permeability.
  • polymer vehicles may be used to modify the compounds of the present invention.
  • Various means for attaching chemical moieties useful as vehicles are currently available, see e.g., Patent Cooperation Treaty ("PCT”) International Publication No.
  • WO 96/11953 entitled “N- Terminally Chemically Modified Protein Compositions and Methods”.
  • a preferred polymer vehicle is polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the PEG group may be of any convenient molecular weight and may be linear or branched.
  • the average molecular weight of the PEG will preferably range from about 2 kiloDalton ("kDa") to about 100 kDa, more preferably from about 5 kDa to about 50 kDa.
  • the PEG groups will generally be attached to the compounds of the invention via acylation or reductive alkylation through a reactive group on the PEG moiety (e.g., an aldehyde, amino, isothiocyanate, or an activated carboxylic acid) to a reactive group on the inventive compound (e.g., an amino, or activated carboxyl group).
  • a reactive group on the PEG moiety e.g., an aldehyde, amino, isothiocyanate, or an activated carboxylic acid
  • a reactive group on the inventive compound e.g., an amino, or activated carboxyl group.
  • a useful strategy for the PEGylation of synthetic peptides consists of combining, through forming a conjugate linkage in solution, a peptide and a
  • the peptides can be prepared by solid phase synthesis, as described herein. Through selective deprotection strategies, the peptides are
  • precursors can be purified and fully characterized prior to reacting with the
  • the PEG moiety contains functionality reactive towards functional groups contained on biomolecules (e.g. proteins, aminoglycosylglycans), making this moiety a heterobifunctional crosslinker.
  • the reactive functionality on the PEG moiety is a maleimide, vinyl carbonyl, vinyl sulfonyl group, or alpha-halocarbonyl, and is reacted with a biomolecule containing a free thiol.
  • a maleimide vinyl carbonyl, vinyl sulfonyl group, or alpha-halocarbonyl
  • Such reactions are extremely facile and can be performed a low reactant concentrations, such as are found in in vitro experiments or in vivo.
  • Other bifunctional agents are known to be useful for cross-linking the peptides or their functional derivatives to a water-insoluble support matrix or to other macromolecular vehicles.
  • cross-linking agents include, for example, l,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N- hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N- maleimido-l,8-octane.
  • Derivatizing agents such as methyl-3-[(p- azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.
  • alkyl linkers such as -NH-(CH 2 ) 5 C(0)-. These alkyl linkers may further be substituted by any non-sterically hindering group such as d- 6 alkyl, C 2 - acyl, halogen (e.g., Cl, Br), CN, NH , aryl, heterocyclyl, etc.
  • Other linkers include those made up of amino acids linked together by amide bonds.
  • the linker is made up of from 1 to 20 amino acids linked by amide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those in the art.
  • the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine.
  • a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine.
  • preferred linkers are polyglycines (particularly (Gly , (Gly) 5 ), poly(Gly-Ala), and polyalanines.
  • linkers are: (Gly) 3 Lys(Gly) 4 ; (Gly) 3 AsnGlySer(Gly) 2 ; (Gly) 3 Cys(Gly) 4 ; and GlyProAsnGlyGly.
  • the peptide linker is designed to be cleaved in vivo at a specific dipeptide amide bond by proteolytic enzymes.
  • Polysaccharide polymers are another type of water soluble polymer which may be used for modification of the compounds of the invention.
  • Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by ⁇ l-6 linkages.
  • the dextran itself is available in many molecular weight ranges, and is readily available in molecular weights from about 1 kD to about 70 kD.
  • Dextran is a suitable water soluble polymer for use in the present invention as a vehicle by itself or in combination with another vehicle (see, for example, WO 96/11953 and WO 96/05309).
  • the use of dextran conjugated to therapeutic or diagnostic immunoglobulins has been reported; see, for example, European Patent Publication No. 0 315 456.
  • Dextran of about 1 kD to about 20 kD is preferred when dextran is used as a vehicle in accordance with the present invention.
  • oligosaccharide groups may conveniently be attached to sites that are known to be glycosylation sites in proteins.
  • oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides are attached to asparagine (Asn) residues when they are part of the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline.
  • X is preferably one of the 19 naturally occurring amino acids other than proline.
  • the structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different.
  • sialic acid is usually the terminal residue of both N- linked and O-linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycosylated compound.
  • site(s) may be incorporated in the linker of the compounds of this invention and are preferably glycosylated by a cell during recombinant production of the polypeptide compounds (e.g., in mammalian cells such as CHO, BHK, COS). However, such sites may further be glycosylated by synthetic or semi-synthetic procedures known in the art.
  • a peptide of the invention can be modified by the replacement of one or more peptidyl (-C(O)NR-) linkages (bonds) by a non- peptidyl linkage.
  • exemplary non-peptidyl linkages are -CH 2 -carbamate (-CH 2 - OC(O)NR-), phosphonate, -CH 2 -sulfonamide (-CH 2 -S(0) 2 NR-), urea (- NHC(O)NH-), -CH 2 -secondary amine, and alkylated amide [-C(0)NR A - wherein R A is alkyl).
  • one or more individual amino acid residues can be modified.
  • Various derivatizing agents are known to react specifically with selected sidechains or terminal residues. For example, lysinyl residues and amino terminal residues may be reacted with succinic or other carboxylic acid anhydrides, which reverse the charge of the lysinyl residues.
  • Suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
  • imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues may be modified by reaction with any one or combination of several conventional reagents, including phenylglyoxal, 2,3- butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginyl residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
  • tyrosyl residues Specific modification of tyrosyl residues has been performed, with examples including introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3 -nitro derivatives, respectively.
  • Compounds of the present invention may be changed at the DNA level as well.
  • the DNA sequence of any portion of the compound may be changed to codons more compatible with the chosen host cell. Codons may be substituted to eliminate restriction sites or to include silent restriction sites, which may aid in processing of the DNA in the selected host cell.
  • the vehicle, linker and peptide DNA sequences may be modified to include any of the foregoing sequence changes.
  • the present invention provides a method of inhibiting cell death in a mammal by administering to the cell a nucleic acid sequence that encodes a peptide that prevents the translocation of the catalytic domain of a toxin from the lumen of endosomes to the cytosol of a mammalian cell.
  • a nucleic acid sequence that encodes a peptide that prevents the translocation of the catalytic domain of a toxin from the lumen of endosomes to the cytosol of a mammalian cell.
  • Examples are peptides that include the amino acid sequences of SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO.7, and SEQ ID NO.7.
  • the present invention provides a method of inhibiting cell death in a mammal by administering to the cell a nucleic acid sequence that encodes a peptide that prevents the translocation of a viral or retroviral transcription factor from the lumen of endosomes to the cytosol of a mammalian cell.
  • a nucleic acid sequence that encodes a peptide that prevents the translocation of a viral or retroviral transcription factor from the lumen of endosomes to the cytosol of a mammalian cell.
  • Examples are peptides that include the of amino acid sequences of SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO.7, and SEQ ID NO.7.
  • the nucleic acid sequences of the present invention or portions thereof can be inserted into a vector used to propagate the sequences in a cell.
  • Such vectors are introduced into cells (e.g., prokaryotic or eukaryotic), and the cells are propagated to produce multiple copies of the vector.
  • a useful type of vector is an expression vector. Coding regions of the nucleic acid sequences of the present invention or fragments thereof can be inserted into an expression vector under conditions appropriate for expression of the sequences.
  • Such vectors are introduced into cells (e.g., prokaryotic or eukaryotic cells) under conditions appropriate for expression.
  • the cell is eukaryotic (e.g., mammalian, avian, insect, or yeast).
  • the cell is human.
  • the invention thus provides nucleic acid constructs which encode the various peptide compounds, or fragments thereof, of the invention, various DNA vectors containing those constructs for use in transducing prokaryotic and eukaryotic cells, cells transduced with the nucleic acids, fusion proteins encoded by the above nucleic acids, and target gene constructs.
  • Each of the nucleic acids of this invention may further contain an expression control sequence operably linked to the coding sequence and may be provided within a DNA vector, e.g., for use in transducing prokaryotic or eukaryotic cells.
  • Some or all of the nucleic acids of a given composition, including any optional nucleic acids may be present within a single vector or may be apportioned between two or more vectors.
  • the vector or vectors are viral vectors useful for producing recombinant viruses containing one or more of the nucleic acids.
  • the recombinant nucleic acids may be provided as inserts within one or more recombinant viruses which may be used, for example, to transduce cells in vitro or cells present within an organism, including a human or non-human mammalian subject.
  • nucleic acids encoding peptides or peptidyl fragments of the present invention may be present within a single recombinant virus or within a set of recombinant viruses, each of which containing one or more of the set of recombinant nucleic acids.
  • Viruses useful for such embodiments include any virus useful for gene transfer, including adenoviruses, adeno-associated viruses (AAV), retroviruses, hybrid adenovirus-AAV, herpes viruses, lenti viruses, etc.
  • the recombinant nucleic acid containing the target gene is present in a first virus and one or more or the recombinant nucleic acids encoding the transcription regulatory protein(s) are present in one or more additional viruses.
  • a recombinant nucleic add encoding a fusion protein containing a bundling domain and a transcription activation domain, and optionally, a ligand binding domain may be provided in the same recombinant virus as the target gene construct, or alternatively, on a third virus.
  • non- viral approaches naked DNA, liposomes or other lipid compositions, etc. may be used to deliver nucleic acids of this invention to cells in a recipient organism.
  • a plasmid was created (see Figure 12 and SEQ ID NO. 1) in which the segment of the diphtheria toxin structural gene encoding amino acids 210-299, which contains the entry motif which is the basis for the compounds of the invention, was cloned into the expression vector pTRACER (obtained from Invitrogen) and placed under control to the CMV promoter to form plasmid: pTl .
  • pTRACER obtained from Invitrogen
  • pTl was subsequently used to transfect HUT102 cells.
  • a stable transfectant clone was purified and designated HUT102-T1.
  • HUT 102 cells (closed circles) were sensitive to DAB389IL-2 with an IC 50 of about 2 x 10 "10 M.
  • HUT102-T1 (closed squares) were completely resistant to the action of DAB389IL-2 to concentrations greater than 10 "8 M.
  • HUT 102 cells transfected with an empty vector (pTRACER) were as sensitive to DAB389IL-2 as HUT 102 cells devoid of the vector (IC 50 of about 1 x 10 "10 M).
  • knock out of the diphtheria toxin 210-299 amino acid region in HUT102-T1 cells also resulted in the loss of resistance to DAB389IL-
  • HUT102 cells (closed circles) and HUT102-T1 cells (closed squares) to Pseudomonas exotoxin A, a bacterial protein toxin known to enter the cell at the level of the trans-golgi rather than from an acidified early endosome.
  • both cell lines were equally sensitive to the action of exotoxin A.
  • DAB389IL-2 and Pseudomonas exotoxin A catalyze the ADP-ribosylation of elongation factor 2 by precisely the same mechanism.
  • the invention also provides methods for rendering a cell capable of regulated expression of a target gene which involves introducing into the cell one or more of the nucleic acids of this invention to yield engineered cells which can express the appropriate fusion protein(s) of this invention to regulate transcription of a target gene.
  • the recombinant nucleic acid(s) may be introduced in viral or other form into cells maintained in vitro or into cells present within an organism.
  • the resultant engineered cells and their progeny containing one or more of these recombinant nucleic acids or nucleic acid compositions of this invention may be used in a variety of important applications, including human gene therapy, analogous veterinary applications, the creation of cellular or animal models (including transgenic applications) and assay applications.
  • Such cells are useful, for example, in methods involving the addition of a ligand, preferably a cell permeant ligand, to the cells (or administration of the ligand to an organism containing the cells) to regulate expression of a target gene.
  • the coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention.
  • the cells are human and a nucleic acid of the present invention is operably linked to an inducible promoter.
  • inducible promoters are known to those skilled in the art. For example, the tetracycline-inducible system of Gossen and Bujard (Proc. Natl. Acad. Sci. USA 89:5547-5551, 1992; U.S. Pat. No.
  • coli tetracycline-resistance operon
  • HSV herpes simplex virus
  • CMV minimal cytomegalovirus
  • the tetR portion of the transactivator binds the tetO sequences of the promoter and the VP16 portion facilitates transcription.
  • tetracycline binds the tetR portion of tTA, which in turn prevents binding of the tetR portion to the tetO sequence(s) of the promoter, thus inhibiting transcription. Since even low concentrations of tetracycline are sufficient to block tTA function, and since most mammalian cells can tolerate tetracycline, this system provides a tightly regulated on/off switch for gene expression that can be controlled by varying the tetracycline concentration to which the cells are exposed.
  • ligand binding domain/ligand pairs include retinol binding protein or variants thereof and retinol or derivatives thereof; cyclophilin or variants thereof and cyclosporin or analogs thereof; FKBP or variants thereof and FK506, FK520, rapamycin, analogs thereof or synthetic FKBP ligands.
  • the present invention also provides methods of inhibiting cell death in a mammal, preferably a human, by administering to the cell a compound of the invention, or analog thereof, which prevents the translocation of the catalytic domain of a toxin from the lumen of endosomes to the cytosol of the cell.
  • the toxin is an AB toxin, such as, for example Diphtheria toxin, one of the seven serotypes of Botulinum toxin, Anthrax toxin LF, or Anthrax toxin EF.
  • the compound inhibits the translocation of a viral or retroviral transcription factor, such as, for example, human immunodeficiency virus (HIV-1) reverse transcriptase or Tat.
  • a viral or retroviral transcription factor such as, for example, human immunodeficiency virus (HIV-1) reverse transcriptase or Tat.
  • HIV-1 human immunodeficiency virus
  • Compounds of the invention include peptide sequences that contain the entry motif consensus sequence.
  • Compounds of the invention also include peptidyl compounds that are further modified to improve their pharmacological properties, as described in detail herein.
  • the inventions also features compounds that include nucleic acid sequences that encode a peptide that contains the entry motif peptide sequence.
  • a method of identifying a compound that inhibits cell death in a mammal includes the following steps: a) isolating endosomes from said cell; b) placing said endosomes in a cytosolic buffer; c)contacting said endosomes with a fusion protein-toxin, wherein said protein comprises a binding moiety for a component of the cell membrane of said cell and said toxin comprises a fragment of Diphtheria toxin; d)contacting said endosomes with a cytosolic translocation factor complex; e) contacting said endosomes with said compound; and e)measuring translocation of said toxin, wherein an decreased level of said translocation relative to that observed in the absence of said compound indicates that said compound inhibits said cell death.
  • a method of identifying a compound that inhibits cell death in a mammal includes the following steps: contacting a mammalian cell or cell population with a fusion protein-toxin, where the protein has a binding moiety for a component of mammalian cellular membranes and where the toxin contains a fragment of Diphtheria toxin that includes the catalytic domain; introducing a cytosolic translocation factor complex (e.g., one that includes a compound of the invention) to the cytosol of the cell(s); contacting the cell(s) with a test compound; and measuring cell death relative to a control cell or cell population which has been similarly treated with fusion protein toxin and a cytosolic translocation factor complex, but not treated with the test compound.
  • a cytosolic translocation factor complex e.g., one that includes a compound of the invention
  • measuring cell death includes a FACS analysis.
  • Introduction of a test compound and/or the CTF complex can be accomplished by treating the cell or cell population with the compound and waiting for passive diffusion through the cell membrane to the cytosol. If necessary, aids to passive transport (e.g., agents that increase cell permeability) can be used.
  • ChariotTM reagent One method for introducing proteins or peptides into the cells of a mammalian cell culture is the ChariotTM reagent (Morris et al., Nature Biotechnology 19:1173-1176, 2001; available from Active Motif, Carlsbad, CA. This reagent quickly and efficiently delivers biologically active proteins, peptides and antibodies directly into cultured mammalian cells at an efficiency of 60-95%. Less than two hours after delivery, live cells can be assayed to determine the effects of the introduced materials, without the need for fixing. In addition to the introduction of the compounds of the invention into the cultured cells, the use of this reagent also aids in the cellular uptake of the compound to be screened, as well as reporter construct.
  • the Chariot reagent can be used in the presence or absence of serum and is independent of the endosomal pathway, which can modify macromolecules during internalization. Additionally, the use of this method for introducing a protein or peptide bypasses the transcription-translation process, which reduces the time required to complete the assay from overnight to less than two hours.
  • HUT1026TG cells (ATCC TIB 1620), were maintained in RPMI 1640 (Bio-Whittaker) supplemented with 10% fetal bovine serum (Hyclone Labs), 2 mM glutamine (Bio-Whittaker), 50 IU/ml penicillin and 50 ⁇ g/ml streptomycin (Bio-Whittaker) at 37°C in 5% C0 2 .
  • Yeast strain NLY22 " (gift from Dr. Kevin Jarrel, Modular Genetics) was maintained in YPD media (Difco) and on YPD agar plates at 30°C. Purification ofEF-2 EF-2 was partially purified using a procedure by Chung et al, supra.
  • EF-2 was further purified by DEAE-Sepharose (Retrac IBF) anion exchange chromatography. EF-2 was eluted with a linear gradient, 0-200 mM NaCl, in 50 mM Tris-HCl pH 8.0, 50 mM Mg(OAc) 2 , 0.1 M KCl, 4 mM CaCl 2 , 5 mM 2-ME and 1 ⁇ g phenylmethylsulfonyl fluoride (PMSF) (Sigma) per ml. Fractions containing EF-2 were identified as above.
  • EF-2 was approximately 80% homogeneous as resolved by 7% SDS-PAGE and stained with colloidal Coomassie (Invitrogen). Protein concentration was determined by Bradford Assay according to standard protocols using Coomassie Protein Assay Reagent (Pierce).
  • HUT102/6TG CTF Complex Crude cytosolic extract was isolated from HUT 102/6TG cells according to the protocol modified from Bomsel et al, Cell 62:719-31, 1990. Briefly, cells were washed three times with cold PBS containing 5 mg/ml BSA, once with cold PBS alone, and twice with cold cytosol buffer (CB; 3% sucrose in 100 mM HEPES-KOH pH 7.9, 1.4 mM KC1 flesh 30 mM MgCl 2 , 2 mM EDTA, 5 mM DTT).
  • CB cold cytosol buffer
  • Cells were lysed by 20 passages through a 25 G needle in CB containing protease inhibitors: 10 ⁇ g/ml aprotinin (Sigma), 1 ⁇ g/ml pepstatin (Sigma), 1 ⁇ g/ml antipain (Sigma), and 1 ⁇ m PMSF (Sigma).
  • the lysate was centrifuged at 1,000 x g for 15 min at 4°C.
  • the post-nuclear supernatant was then centrifuged at 170,000 x g for 1 hr at 4°C.
  • cytosol dialysis buffer CDB; 1% sucrose in 20 mM TRIS-HC1 pH 8.0, 2mM EDTA, 2mM 2-ME
  • Crude cytosol was fractionated according to standard chromatographic protocols. Briefly, crude extract was loaded onto an in-house packed DEAE- Sepharose (Retrac IBF) XK 26 column (Amersham Pharmacia) for anion exchange chromatography.
  • a peristaltic FPLC pump P-l (Amersham Pharmacia) and Single Path Monitor UV-1 (Amersham Pharmacia) were used during chromatography.
  • the column was pre- equilibrated with buffer B3: 50 mM Tris-HCl pH 8.0, 1 mM EDTA, 5 mM 2-mercaptoethanol, and 1 ⁇ g PMSF per ml, and 'loaded' sample was washed using the same buffer.
  • CTFs were eluted with a linear gradient, 0-400 mM NaCl, in buffer B3 at a flow rate of 5 ml/min. Fractions containing CTFs were identified using an in vitro TA and in vitro RA in series (see below).
  • CTFs were identified as previously described, and correlated with elution of 100 to 250 kDa sized proteins, but contained proteins as small as 20 to 25 kDa when visualized by 7%- 12% SDS-PAGE and stained with colloidal Coomassie. Partially purified CTFs were further purified by anion exchange chromatography using a MONO Q HR 5/5 column (Amersham Pharmacia) on a Biosys2000 HPLC (Beckman).
  • the column was pre-equilibrated with buffer B4: 50 mM Tris-HCl pH 8.0 and 1 mM EDTA. Sample loads of 2 ml were washed using buffer B4 and CTFs were eluted using serial hyperbolic step gradients, 0 to 1.0 M NaCl in buffer B4 at a flow rate 2 ml/min. CTFs were identified as previously described and eluted at a conductance of 27.3 mS. Translocation in vitro competent fractions were pooled, dialyzed against 50 mM Tris HCl pH 7.4, 1% sucrose overnight at 4°C, and then concentrated using YM-10 Microcon Centrifugal Filters (Amicon) according to manufacturer's directions. Protein concentration was determined as previously described. Controls indicated that the purified CTF complex had no intrinsic ADP-ribosyltransferase activity.
  • Yeast crude cytosolic extract was isolated using the same procedure described above for HUT 102/6TG cells except NLY22 " cells were lysed by vortexing cells with 212-300 micron glass beads (Sigma). Cell lysis was monitored by decrease in exclusion of Trypan Blue Dye (Gibco BRL). Controls indicated that the purified CTF complex had no intrinsic -ADP- ribosyltransferase activity
  • TA Translocation of the C domain was carried out using protocol modified by Lemichez et al, supra: 25 ⁇ l reaction mixtures containing 4 ⁇ l early endosomes in translocation buffer (TB; 50 mM Tris-HCl pH 7.4, 25 mM EDTA).
  • TB contained 20 mM DTT.
  • TB contained 10 ⁇ M NADPH (Alexis). ATP and cytosol were added to 2 mM and 5.0 to 0.09 ⁇ g/ ⁇ l as indicated, respectively.
  • Translocation mixtures were incubated at 37°C for 30 min., and the supernatant fluid and pellet were separated by ultra-centrifugation at 180,000 x g at 4°C for 20 min. The pellet fraction was resuspended in 25 ⁇ l TB containing 0.2% Triton x-100 (Sigma) and both the lysed pellet and supernatant fluid were boiled for 5 min.
  • geldanamycin Alomone Labs
  • radicicol Sigma
  • cis- 13 -retinoic acid Sigma
  • trans- 13 -retinoic acid Sigma
  • hrHsp 90 StressGen
  • brTrR-1 American Diagnostica Inc.
  • hrTrx American Diagnostica Inc.
  • Crosslinked antibody was pelleted by centrifugation at 1,000 x g for 5 min at 4°C, and the pellet was washed 2x with 10 c.v. 0.2 M ethanolamine (Sigma) for 30 min. at 25 C, and 2x with PBS for 30 min. at 25 C.
  • Immunoprecipitations using the cross-linked antibody agarose conjugates were performed according to standard protocols. Briefly, 200 ⁇ l of MONO-Q partially purified CTFs (approximately 0.1 ⁇ g/ ⁇ l) in 50 mM Tris HCl, 1% sucrose, containing 1% NP-40 and 25 mM NaCl was incubated with 20 ⁇ l of antibody-agarose conjugate on a rocker overnight at 4°C.
  • IPs were collected by centrifugation at 1,000 x g for 5 min at 4°C, and supernatant fluid was evaluated in the in vitro TA.
  • Pellet was washed 3x with 100 ⁇ l cold 50 mM Tris HCl, 1 mM EDTA containing 1% NP-40 and 50 mM NaCl, and resuspended in 50 ⁇ l lx SDS-PAGE loading buffer and boiled for 5 min.
  • Antibody-agarose beads were pelleted by centrifugation at 1,000 x g for 5 min at 25 C and the supernatant was analyzed by 10% SDS-PAGE, stained with colloidal Coomassie, and selected bands were evaluated by MS as described below.
  • Yeast TrR-1 was affinity purified using 2 ',5' ADP-Sepharose agarose (Amersham Biosciences) using a protocol modified from Hunt et al, Eur. J. Biochem. 131:303-11, 1983. Briefly, 20 ⁇ g of 2',5' ADP-Sepharose agarose was washed 2x with 200 ⁇ l 50 mM Tris HCl, 1 mM EDTA for 20 min.
  • MONO-Q partially purified CTFs 200 ⁇ l of approximately 0.1 ⁇ g/ ⁇ l
  • Tris HCl 50 mM Tris HCl, 1 mM EDTA, 1% Sucrose, 25 mM NaCl
  • Affinity purified TrR-1 was collected by centrifugation at 1,000 x g for 5 min at 4°C. The supernatant fluid was assayed for translocation activity in vitro.
  • the pellet was washed 2x in 100 ⁇ l 50 mM Tris HCl pH 7.5, 1 mM EDTA, 1% sucrose, and then resuspended in 50 ⁇ l 50 mM Tris HCl pH 7.5, 1 mM EDTA, 1% sucrose, containing 20 ⁇ M NADPH and incubated for 2 hours at 25 C.
  • the supernatant fluid was collected following centrifugation at 1,000 x g for 5 min at 4°C and the supernatant fluid was analyzed by 10% SDS-PAGE, stained with colloidal Coomassie, and selected bands were evaluated by MS as described below.
  • Ammonium bicarbonate 50 mM was added when necessary to keep the gel pieces wet. Digestions were incubated for 6-8 hours at 37°C. Peptides were extracted from the gel pieces using 100 ⁇ l 20 mM ammonium bicarbonate for 20 min, followed by 2x 200 ⁇ l 1% TFA in 50% ACN for 20 min, and finally lx 100 ⁇ l 100% ACN for 10 min. Supernatant fluids were pooled and dried in a SpeedVac. The pellets were resuspended in 0.1% TFA and desalted using ZipTipc ⁇ 8 pipette tips (Millipore) according to manufacturer's directions.
  • Capillary HPLC of tryptic peptides HPLC was performed using an LC Packings (Dionex Corp.) capillary LC system composed of a Famous autosampler, a Switchos microcolumn switching unit and an Ultimate pump. Sample loads of 5 ⁇ L were preconcentrated and desalted online with a "small molecule" C 18 CapTrap (Michrome Bioresources) using a solution of 5% FA, 0.1% TFA at a flow rate of 50 ⁇ L per min. for 4 min.
  • Capillary HPLC columns were prepared in house as follows: 300 ⁇ m ID x 15 cm fused silica capillaries were pressure bomb- packed (Mass Evolution, Inc) at 2000 PSI with Magic C 18 -3 ⁇ m-200 A pore reversed phase packing material (Michrome Bioresources) using 2-propanol as a carrier solvent. Columns were washed with 10% acetic acid, followed by methanol, then the HPLC mobile phase prior to use at a flow rate of 2 ⁇ L per min. Elution was by linear gradient; 95% A (5% ACN, 0.1% formic acid) to 55% B (85% ACN, 10% 2-propanol, 0.1% formic acid) over 50 min. followed by 60 min. of column regeneration.
  • MALDI and ESI Mass Spectrometry (MS), tandem MS, and LC-MS/MS MALDI MS were acquired in positive polarity on a Bruker Reflex IV mass spectrometer with delayed extraction in the reflectron mode using a UV nitrogen laser. A laser power of 28-45% was used and 50 to 100 laser shots were summed for each spectrum. The matrix used was 2,5-dihydroxybenzoic acid (Sigma). Data was analyzed using BioanalystTM (Applied Biosystems Inc.) reconstruction algorithms. For initial screening and searches, acquired mass values were compared to theoretical protein digests using the Mascot search engine (Matrix Science Ltd). Reported scores, based on a probability of match, were statistically significant for each protein identified in Table 1. ESI MS and MS/MS were performed using an Applied Biosystems
  • MS and MS/MS were acquired in the positive polarity mode over the range of m/z 320-1800 (MS) and m/z 100-1800 (MS/MS) with resolution > 1 :9,000 (fwhm) and better than 50 ppm mass accuracy (external calibration).
  • MS/MS QSTARi Pulsar ESI quadrupole/orthogonal acceleration time-of-flight mass spectrometer
  • MS and MS/MS were acquired in the positive polarity mode over the range of m/z 320-1800 (MS) and m/z 100-1800 (MS/MS) with resolution > 1 :9,000 (fwhm) and better than 50 ppm mass accuracy (external calibration).
  • a Protana source was employed using uncoated glass nanospray tips pulled in house to 1 ⁇ m ID using a capillary puller (Sutter Instrument Co.) ESI was initiated at ⁇ 1200 V via a Pt wire inserted into the glass tip.
  • Tandem mass spectra were acquired using Ar as the collision gas and sufficient collision energy to obtain complete sequence information of the precursor.
  • Pulsed ion enhancement of product ions was used for MS/MS of low S/N precursors.
  • the LC was coupled to the mass spectrometer using 50 ⁇ m ID distal coated nanospray tips pulled to 15 ⁇ m ID, 75 ⁇ m OD at the tip (New Objectives Inc.).
  • ESI was carried out at 4500 V.
  • Information dependent acquisition (IDA) was used to obtain MS/MS spectra of peaks during elution from the LC system. MS peaks which exceeded a threshold of 10 counts/s were subjected to MS/MS using preset collision energies proportional to the m/z value of the precursor (ca. 18-60 V, lab frame). Pulsed ion enhancement was used for all LC MS/MS spectra.
  • Cytotoxicity assays for the fusion protein toxins were performed essentially as described by vanderSpek et al, J. Biol. Chem. 269: 21455- 21459, 1994. Cytotoxicity assays to evaluate the affects of geldanamycin, radicicol, and retinoic acid upon DAB 389 IL-2 intoxication were modified as follows: Cells were seeded at 5 x 10 4 cells per well and pre-incubated with inhibitors geldanamycin, radicicol, cis- 13 -retinoic acid, for 30 min. at 37°C, 5% C0 2 and subsequently incubated with varying concentrations of DAB 389 IL- 2 and inhibitor for 15 min.
  • reaction products are isolated and purified by conventional methods, typically by solvent extraction into a compatible solvent, or by using a washing protocol for resin bound intermediates.
  • the products may be further purified by column chromatography or other appropriate methods, including medium pressure or high pressure liquid chromatography.
  • compounds of the invention can be conveniently prepared using solid phase synthesis methodology (Merrifield, J. Am. Chem. Soc. 85:2149, 1964; Houghten, Proc. Natl. Acad. Sci. USA, 82:5132, 1985).
  • Solid phase synthesis begins at the carboxy terminus of the compound by attaching a protected amino acid, or other carboxylic acid-containing compound, to an inert solid support.
  • the inert solid support can be any macromolecule capable of serving as an anchor for the C-terminus of the initial amino acid.
  • the macromolecular support is a cross-linked polymeric resin (e.g. a polyamide or polystyrene resin).
  • the C- terminal amino acid is coupled to a polystyrene resin to form a benzyl ester.
  • Particularly useful benzyl-type resins such as trityl resin, chlorotrityl resin, and Wang resin, are those in which the linkage of the carboxy group (or carboxamide) to the resing is acid-lable.
  • a macromolecular support is selected such that the peptide anchor link is stable under the conditions used to deprotect the ⁇ -amino group of the blocked amino acids in peptide synthesis.
  • a base-labile ⁇ -protecting group is used, then it is desirable to use an acid- labile link between the peptide and the solid support.
  • an acid- labile ether resin is effective for base-labile Fmoc-amino acid peptide synthesis.
  • a peptide anchor link and ⁇ -protecting group that are differentially labile to acidolysis can be used.
  • an aminomethyl resin such as the phenylacetamidomethyl (Pam) resin works well in conjunction with Boc-amino acid peptide synthesis.
  • the ⁇ - amino protecting group of the initial amino acid is removed with, for example, trifluoroacetic acid (TFA) in methylene chloride and neutralizing in, for example, triethylamine (TEA).
  • TFA trifluoroacetic acid
  • TAA triethylamine
  • the next . ⁇ -amino and side chain protected amino acid in the synthesis is added.
  • the remaining ⁇ -amino protected and, if necessary, side chain protected amino acids are then coupled sequentially in the desired order by condensation to obtain an intermediate compound connected to the solid support.
  • some amino acids may be coupled to one another to form a fragment of the desired peptide followed by addition of the peptide fragment to the growing solid phase peptide chain.
  • the condensation reaction between two amino acids, or an amino acid and a peptide, or a peptide and a peptide an be carried out according to the usual condensation methods such as the axide method, mixed acid anhydride method, DCC (N,N'-dicyclohexylcarbodiimide) or DIG (N,N' ⁇ diisopropylcarbodiimide) methods, active ester method, p-nitrophenyl ester method, BOP (benzotriazole-l-yl-oxy-tris[dimethylamino] phosphonium hexafluorophosphate)method, N-hydroxysuccinicacid imido ester method, O- benzotriazolyl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) or
  • Suitable protecting groups for ⁇ -amino and side chain amino groups are exemplified by benzyloxycarbonyl (abbreviated Z), isonicotinyloxycarbonyl (iNOC), o-chlorobenzyloxycarbonyl [Z(2C1)], p-nitrobezyloxycarbonyl [Z(N0 2 )], p-methoxybenzyloxycarbonyl [Z(OMe)], t-butoxycarbonyl (Boc), t- amyloxycarbonyl(Aoc), isobornyloxycarbonyl, adamantyloxycarbonyl, 2-(4- biphenyl)-2-propyloxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl(Fmoc), methylsulfonyethoxycarbonyl (Msc), trifluoroacetyl, phthalyl, formyl, 2- nitrophenylsulfenyl (NP
  • Protective groups for the carboxy functional group are exemplified by benzyl ester, (OBz), cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb), t-butyl ester (OtBu), 4-pyridylmethyl ester (OPic), and the like. It is often desirable that amino acids such as arginine, cysteine, and serine possessing a functional group other than amino and carboxy groups be protected by a suitable protecting group.
  • the guanidino group may be protected with nitro, p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl, p- methoxybenzenesulfonyl, 4-methoxy-2, 6-dimethylbenzenesulfonyl (Nds), 1,3,5-trimethylphenysulfonyl (Mts), and 2,3,6-trimethyl-4- methoxyphenylsulfonyl (Mtr), and the like.
  • the thiol group can be protected with p-methoxybenzyl, trityl, and the like.
  • the compounds of the invention are synthesized with the help of blocking groups that protect the side chain amide bond- forming substituents of the N-terminal and C-terminal flanking residues.
  • the protecting group or groups used for the side chain amide bond-forming substituents of the N-terminal and C-terminal flanking residues can be the same or different than the protecting group or groups used to block the side chain functional groups of other residues in the peptide.
  • the protecting group or groups used to block the side chain amide bond- forming substituents is (are) differentially removable with respect to the protecting groups used for other side chain functional groups, i.e.
  • the side chain amide bond-forming substituents can be deprotected without deprotecting the other side chain functional groups, in addition to being differentially removable with respect to the ⁇ -amino protecting group used in peptide synthesis.
  • the side chain amide bond- forming substituents of the flanking residues are orthogonally protected with respect to each other such that the side chain amide bond- forming substituent of one flanking residue can be deprotected without deprotecting the side chain amide bond-forming substituent of the other flanking residue.
  • Suitable protecting groups for use in orthogonally protecting the side chain amide bond- forming substituents of the flanking residues with respect to other functional groups and/or with respect to each other include pairs of differentially removable carboxy protective groups, such as a reduction-labile carboxy protective group, e.g. allyl or benzyl esters, paired with a base-labile carboxy protective group, e.g. fluorenylmethylesters, methyl or other primary alkyl esters.
  • a reduction-labile carboxy protective group e.g. allyl or benzyl esters
  • a base-labile carboxy protective group e.g. fluorenylmethylesters, methyl or other primary alkyl esters.
  • Fluorenylmethyl, methyl or other primary alkyl groups or other base-labile carboxy protective groups can be removed from their corresponding esters to yield a free carboxy group (without deprotecting allyl or benzyl esters or other reduction-labile esters) by saponification of the esters with a suitable base such as piperidine and sodium hydroxide in a suitable solvent such as dimethylacetamide, or methanol and water, for a period of 10 to 120 minutes, and preferably 20 minutes, 0 °C to 50°C.
  • the allyl or benzyl or other reduction-labile esters can be removed when desired by reduction in the presence of a suitable transition metal catalyst, such as Pd(PPh 3 ) 4 , Pd(PPh 3 ) 2 Cl 2 , Pd(OAc) 2 or Pd on carbon with a source of hydrogen, e.g. H 2 gas, in a suitable solvent such as dimethylacetamide, dimethylformamide, N- methylpyrrolidinoneor methanol for a period of 10 to 500 minutes, and preferably 100 minutes, at 0 °C to 50°C.
  • a suitable transition metal catalyst such as Pd(PPh 3 ) 4 , Pd(PPh 3 ) 2 Cl 2 , Pd(OAc) 2 or Pd on carbon with a source of hydrogen, e.g. H 2 gas
  • a suitable solvent such as dimethylacetamide, dimethylformamide, N- methylpyrrolidinoneor methanol for a period of 10 to 500 minutes, and preferably 100
  • reduction- labile groups When such reduction- labile groups are used to provide orthogonal protection in combination with base-labile protecting groups, it is preferable to either (1) utilize a synthetic scheme that calls for the removal of the base-labile protecting groups before the removal of the reduction-labile protecting groups or (2) restore the Pd catalyst with a nucleophile that does not deprotect the base-labile protecting groups.
  • the carboxy substituents of the flanking residues can be orthogonally protected with respect the other functional groups and/or with respect to each other by using an acid-labile protecting group, such as a tertiary alkyl ester, e.g. t-butyl ester, in combination with a reduction-labile protecting group, such as the allyl or benzyl esters described above.
  • the tertiary alkyl or other acid-labile ester group can be removed by acidolysis, e.g. with trifluoroacetic acid in methylene chloride, and the allyl or benzyl or other reduction-labile esters can be removed by reduction in the presence of a transition metal catalyst as described above.
  • the carboxy substituents of the flanking residues can be orthogonally protected with respect to other functional groups and/or with respect to each other by using a fluoride ion-labile protecting group, such as 2-(trimethylsilyl)ethyl and silyl esters, in combination with a reduction- labile protecting group, such as the allyl or benzyl esters described above, or in combination with a base-labile protecting group, such as the fluorenylmethyl, methyl or other primary alkyl esters described above, without deprotecing the reduction-labile or base-labile esters.
  • a fluoride ion-labile protecting group such as 2-(trimethylsilyl)ethyl and silyl esters
  • a reduction- labile protecting group such as the allyl or benzyl esters described above
  • a base-labile protecting group such as the fluorenylmethyl, methyl or other primary alkyl esters described above
  • the 2-(trimethylsilyl)ethyl, silyl or other fluoride-labile ester group can be removed by reaction with a suitable fluoride ion source, such as tetrabutylammonium fluoride in the presence of a suitable solvent, such as dimethylacetamide(DMA), dimethylformamide (DMF), tetrahydrofuran (THF), or acetonitrile.
  • a suitable fluoride ion source such as tetrabutylammonium fluoride
  • a suitable solvent such as dimethylacetamide(DMA), dimethylformamide (DMF), tetrahydrofuran (THF), or acetonitrile.
  • Suitable protecting groups for use in orthogonally protecting the side chain amide bond- forming substituents of the flanking residues with respect to other functional groups and/or with respect to each other also include pairs of differentially removable amino protective groups, such as an allyloxycarbonyl or other reduction-labile amino protective group paired with a t-butoxycarbonyl (Boc) or other acid-labile amino protective group, and a reduction-labile amino protective group paired with a fluorenylmethoxycarbonyl (Fmoc) or other base- labile amino protective group.
  • pairs of differentially removable amino protective groups such as an allyloxycarbonyl or other reduction-labile amino protective group paired with a t-butoxycarbonyl (Boc) or other acid-labile amino protective group, and a reduction-labile amino protective group paired with a fluorenylmethoxycarbonyl (Fmoc) or other base- labile amino protective group.
  • An allyloxycarbonyl (or other reduction-labile blocking group) protected amino group can be deprotected by reduction using a transition metal catalyst as in the procedure for removing reduction-labile carboxy protective groups described above, without deprotecting a Boc or Fmoc protected amino group.
  • an acid-labile amino protective group and a base-labile amino protective group can be removed by acidolysis and base saponif ⁇ cation, respectively, without removing a reduction-labile amino protective group.
  • the amino substituents of the flanking residues can be orthogonally protected with respect to other functional groups and/or with respect to each other by using a fluoride-labile protecting group, such as 2-trimethylsilylethylcarbamate (Teoc), in combination with a reduction-labile protecting group, such as allyloxylcarbonyl, or in combination with a base- labile protecting group, such as fluorenylmethoxycarbonyl, as described above.
  • a fluoride-labile protecting group such as 2-trimethylsilylethylcarbamate (Teoc)
  • Teoc 2-trimethylsilylethylcarbamate
  • a reduction-labile protecting group such as allyloxylcarbonyl
  • a base- labile protecting group such as fluorenylmethoxycarbonyl
  • the Teoc or other fluoride-labile group can be removed by reaction a with a suitable fluoride ion source, such as tetrabutylammonium fluoride, as in the procedures for removal of fluoride-labile carboxy protective groups described above, without deprotecting an allyloxycarbonyl or fluorenylmethoxycarbonyl protected amino group.
  • a reduction-labile amino protective group and a base-labile amino protective group can be removed by reduction and base saponification, respectively, without removing a fluoride-labile amino protective group.
  • the carboxy substituent and the amino substituent can be orthogonally protected with respect to each other by using a reduction-labile protecting group to block one substituent, e.g. allyl ester or allyloxycarbonyl, and a fluoride-labile, acid-labile or base-labile protecting group to block other substituent, e.g. silyl ester, t-butyl ester, fluorenylmethyl ester, Teoc, Boc, or Fmoc.
  • a reduction-labile protecting group to block one substituent
  • a fluoride-labile, acid-labile or base-labile protecting group to block other substituent, e.g. silyl ester, t-butyl ester, fluorenylmethyl ester, Teoc, Boc, or Fmoc.
  • a reduction-labile protecting group is used to block the side chain amide bond-forming substituent of one flanking residue and the protecting group for the side chain amide bond- forming substituent of the other flanking residue is selected such that it provides orthogonal protection with respect to both the reduction-labile protecting group and the ⁇ -amino protecting group used in the synthesis.
  • orthogonal protection of the side chain amide bond-forming substituents would be provided by a reduction-labile protecting group and an acid-labile protecting group.
  • orthogonal protection of the side chain amide bond-forming substituents would be provided by a reduction-labile protecting group and a base-labile protecting group.
  • the side chain amide bond- forming substituents of the flanking residues are orthogonally protected with respect to each other, with respect to ⁇ -amino protecting group used in the synthesis, and with respect to the protecting groups used to block other side chain functional groups in the peptide chain.
  • the side chain amide bond- forming substituents of the flanking residues are orthogonally protected with respect to each other, and with respect to ⁇ -amino protecting group, but only one of the side chain amide bond- forming substituents is orthogonally protected with respect to the protecting groups used to block other side chain functional groups.
  • the amide bond-forming reaction will be specific to the desired side chain amide bond- forming substituent, and will reduce the production of unwanted difunctional linker derivatives.
  • cyclization will require the deprotection of the side chain amide bond- forming substituent of the other flanking residue, and may cause concomitant deprotection of other side chain functional groups, unwanted derivatives are less likely to form given that the peptide chains are anchored to a solid support and that the linker length will regioselectively favor a amide bond- forming reaction between the unbound functional group of the linker and the side chain amide bond-forming substituent of the other flanking residue. If further peptide chain synthesis is desired after cyclization, any side chain functional groups on other amino acid residues left unprotected by the cyclization reactions can be reprotected before chain synthesis is resumed.
  • blocked amino acids described above can be obtained from commercial sources such as Novabiochem (San Diego, Calif), Bachem Calif. (Torrence, Calif.) or Peninsula Labs (Belmont, Calif).
  • functionalized or protected amino acids, including unnatural amino acids can be prepared by methods known in the art.
  • the compounds of the invention can be prepared by, or in conjunction with, solution phase peptide synthesis, for example, the solution phase peptide synthesis methods described in Principles of Peptide Synthesis, 2d ed, M. Bodanszky, Springer- Verlag (1993) or in The Practice of Peptide Synthesis, 2d ed, M. Bodanszky and A. Bodanszky, Springer- Verlag (1994).
  • solution phase peptide synthesis methods can be easily modified to incorporate the desired flanking residues, with or without orthogonally-protected side chain amide bond-forming substituents, into the compound of interest, using procedures similar to those used in the solid phase synthesis methods described herein.

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Abstract

In vitro delivery of the diphtheria toxin (DT) catalytic (C) domain from the lumen of purified early endosomes to the external milieu requires the addition of both ATP and a cytosolic translocation factor (CTF) complex. The results presented here demonstrate that both Hsp 90 and TrR-1 activity plays an essential role in the cytosolic release of the C-domain and is mediated by a consensus peptide sequence found on several bacterial toxins and in HIV-1 reverse transcriptase. The invention features methods for inhibiting cell death that include the administration of compounds based on this consensus sequence that inhibit the translocation of the catalytic domain of toxins or transcription factors. Also featured are methods for identifying compounds that inhibit cell death, and methods for identifying compounds that promote cell death by blocking or accelerating, respectively, the rate of toxin/factor endosomal translocation.

Description

NOVEL COMPOSITIONS AND METHODS FOR PROMOTING, INHIBITING, AND DETECTING PROTEIN ENTRY INTO CELLS
This invention was made with Government Support under Contract Number CA60934 awarded by the National Institutes of Health. The U.S. Government has certain rights in the invention.
BACKGROUND OF THE INVENTION This invention features methods and compositions that relate to the cytosolic translocation factor complex (CTL) responsible for the translocation of the catalytic domain of diphtheria toxin from the lumen of endosomes to the cytosol.
Diphtheria toxin (DT) (58 kDa) is a typical single chain AB toxin composed of three functional domains: the amino terminal catalytic (C) domain corresponds to fragment A (21 kDa), and the transmembrane (T) and carboxy terminal receptor binding (R) domains comprise fragment B (37 kDa) of the toxin (Choe et al., Nature 357: 216-22, 1992). A disulfϊde bond between
Cysl86 and Cys201 subtends a protease sensitive loop and connects fragment A with fragment B. Furin mediated cleavage within this loop and retention of the disulfϊde bond have been shown to be pre-requisites for intoxication of eukaryotic cells (Tsuneoka et al., J iol. Chem. 268:26461-5, 1993; Ariansen et al., Biochem. 32:83-90, 1993). Substitution of the native R domain with human interleukin-2 (IL-2) has resulted in the formation of a fusion protein toxin, DAB389IL-2, whose cytotoxic action is specifically targeted only to cells expressing the high affinity IL-2 receptors (Bacha et al, J. Exp. Med. 167:612- 622, 1988; Waters et al, Eur. J. Immunol. 20:785-91, 1990; Ratts and vanderSpek, Diphtheria Toxin: Structure Function and its Clinical Applications. In Chimeric Toxins, H. Lorberboum-Galski, P. Lazarovici, editors. Taylor and Francis, London, New York. p. 14-36, 2002). Since DAB3s9IL-2 binds with greater affinity to its receptor compared to native DT, this fusion protein toxin has proven to be an effective and novel probe for studying internalization of the C-domain by target cells (Williams et al, J. Biol Chem. 265:11885-9, 1990). While much is known about the mechanisms of receptor binding and receptor mediated endocytosis of native DT and the DT-related fusion proteins, little is known about the precise molecular mechanisms of C-domain translocation across the endosomal membrane and its release into the cytosol. Unfolding of the DT C domain was first postulated as a pre-requisite for translocation by Kagen et al., (1981) and Donovan et al, Proc. Nail. Acad. Sci. USA 78:172-6, 1981. The necessity for complete denaturation of the DT C domain prior to translocation was then indirectly demonstrated by Wiedlocha et al, EMBO J. 11 :4835-42, 1992 and by Falsnes et al. (1994). At present there are two conflicting hypotheses for translocation of denatured DT C-domain across the early endosomal membrane. Studies using artificial lipid bilayers suggest that the DT T-domain itself exhibits chaperonin-like properties and is solely sufficient to promote C-domain delivery across the bilayer (Ren et al, Science 284:955-7, 1999; Oh et al, Proc. Natl Acad. Sci. USA 96:8467-70, 1999). In contrast, studies using partially purified early endosomes that were pre-loaded with toxin suggest that C-domain translocation across the vesicle membrane is dependent upon ATP and the presence of cytosolic components which include β-COP (Lemichez et al, Mol. Microbio. 23:445-57, 1997).
Since protease digestion patterns of DT inserted into planar lipid bilayers differ from those of DT inserted into the plasma membrane (Moskaug et al, Biochem. J. 291 :473-7, 1993; Cabiaux et al, Mol. Microbiol 11:43-50, 1994), it seems likely that interaction(s) between the toxin and proteins associated with the endosomal membrane {e.g., receptor) influence the orientation and/or stoichiometry of insertion of the T-domain and translocation of the C-domain. In addition, Ren et al, supra and Hammond et al, Biochem. 41 :3243-53, 2002, have shown that although the DT T-domain has chaperonin-like properties, it has a significantly greater affinity for other molten globule-like polypeptides compared to its own C-domain. Anthrax toxin is a binary complex that may be assembled from three distinct protein chains: protective antigen (PA), lethal factor (LF), and edema factor (EF) (Mourez et al, Trends in Microbiology 10: 287-293, 2002). Protective antigen (p83) binds to a universal cell surface receptor and a 20 kDa fragment is removed by digestion with the endoprotease furin (MoUoy et al, J. Biol Chem.; 267: 16396-16402, 1992). The remaining 63 kDa fragment of protective antigen remains on the cell surface and spontaneously oligomerizes into a heptamer. The heptameric complex is then capable of binding either lethal factor or edema factor and facilitating their delivery into the cytosol. While a detailed understanding of this process is not understood, the overall route of entry appears to closely follow that of diphtheria toxin. Protective antigen with bound lethal factor or edema factor is then internalized by receptor mediated endocytosis into an early endosomal compartment. Acidification of this compartment is necessary to induce a conformational change in protective antigen allowing the formation of a cation selective channel in the membrane. In fact, Wesche et al, Biochem. 37:15737-46, 1998, have shown that acid- induced translocation of lethal factor resembles that of diphtheria toxin C- domain. Moreover, the translocation of lethal factor has been suggested to involve complete unfolding in order to allow passage through the channel formed by protective antigen, and then refolding into a catalytically active conformation in the cytosol (Wesche et al, supra). In contrast, edema factor has been reported to remain associated with the vesicle compartment (Guidi- Rontani et al, Cell Microbiol. 2: 259-264, 2000). SUMMARY OF THE INVENTION We hypothesize that there is a common mechanism of catalytic domain entry for bacterial toxins such as, for example, diphtheria, anthrax lethal factor, anthrax lethal edema factor, and the seven serotypes of botulinum toxin, as well as viral transcription factors, such as, for example, HIV-1 reverse transcriptase and Tat, and that that process requires both a cytosolic translocation factor (CTF) complex and components of the outer surface of endocytic vesicles. Described herein are compounds that include a consensus peptide sequence (the entry motif) held in common by these toxins/factors. Accordingly, the invention features a compound of formula I: X - AA210- AA211 - AA212- AA213 - AA214- AA215- AA 16- AA217- AA218- AA219- AA220- AA221 - AA222-Y (I), where X is H or a chain of amino acids of from 1 to 5 residues substituted at the N- terminus with R1-C(0)-, a nitrogen protecting group, or H; Y is OH, NH2, NHR2, NHR2R3, OR4, or a chain of amino acids of from 1 to 76 residues substituted at the C-terminus with OH, NH2, NHR2, NHR2R3, OR4, where R1 is a Ci-6 alkyl, C6 or C10 aryl, -9 heterocyclyl, -6 alkoxy, C7-16 aralkyl, C2-15 heterocyclylalkyl, C7-16 aralkoxy, C2-ι5 heterocyclyloxy, or a polyethylene glycol moiety; each of R and R is, independently, H, a -6 alkyl, C6 or C10 aryl, -9 heterocyclyl, C7.16 aralkyl, C2 5 heterocyclylalkyl, or a polyethylene glycol moiety; R4 is H, -6 alkyl, C6 or C10 aryl, -9 heterocyclyl, C .6 alkoxy, C -16 aralkyl, C2-15 heterocyclylalkyl, a carboxyl protecting group, or a 'j i π i l 01 polyethylene glycol moiety; AA is Arg or Lys; AA is Asp or Glu; AA is Lys or Arg; AA213 is Thr, Ser, Ala, Gly, Val, Asn, or Gin; AA214 is Lys or Arg; AA215 is Thr, Ser, Ala, Gly, Val, Asn, or Gin; AA216 is Lys or Arg; AA217 is lie, Leu, or Val; AA218 is Glu or Asp; AA219 is Ser, Ala, or Gly; AA220 is Leu, He, or Val; AA221 is Lys or Arg; and AA222 is Glu or Asp. In one embodiment, Y is - AA223 - AA224 - AA225 - AA226 - AA227 - AA228- AA229-Ya, where Ya is OH, NH2, NHR2, NHR2R3, or OR4; AA223 is His, Phe, or Tyr; AA224is Gly, Ala, or Ser; AA225 is Pro; AA226 is He, Leu, Val; AA227 is Lys or Arg; AA228 is Asn or Gin; and AA229 is Lys or Arg. Preferably, AA210 is Arg; AA21 ! is Asp; AA212 is Lys; AA213 is Thr; AA214 is Lys; AA215 is Thr; AA216 is Lys; AA217is He; AA218 is Glu; AA219is Ser; AA220 is Leu; AA221 is Lys; AA222 is Glu; AA223 is His; AA224is Gly; AA225 is Pro; AA226is He; AA227 is Lys; AA228 is Asn; and AA229 is Lys. In another embodiment, X is X - AA205 - AA206 - AA207 - AA208 - AA209 -; Y is - AA223 - AA224- Ya, wherein Xa is R!-C(0)- or H, Ya is OH, NH2, NHR2, NHR2R3, or OR4; AA205 is Asp or Glu; AA206 is Trp, Tyr, or Phe; AA207is Asp or Glu; AA208 is Val, Leu, He, Thr, Ser, or Ala; AA209is He, Leu, or Val; AA223 is His, Tyr, or Phe; and AA224 is Gly, Ala, or Ser. Preferably, AA205 is Asp; AA206 is Trp; AA207 is Asp; AA208 is Val; AA209 is He; AA210 is Arg; AA21 ! is Asp; AA212 is Lys; AA213 is Thr; AA214 is Lys; AA215 is Thr; AA216 is Lys; AA217 is He; AA218 is Glu; AA219 is Ser; AA220 is Leu; AA221 is Lys; AA222 is Glu; AA223 is His; and AA224is Gly. Other examples include those compounds of formula I where X is H or a chain of amino acids of from 1 to 5 residues, preferably corresponding to Asp- Trp-Asp-Val-Ile-, and Y is OH or a chain of amino acids of from 1 to 76 residues, preferably corresponding to -Arg-Asp-Lys-Thr-Lys-Thr-Lys-He-Glu- Ser-Leu-Lys-Glu-His-Glu-Pro-Ile-Lys-Asn-Lys-Met-Ser-Glu-Ser-Pro-Asn- Lys-Thr-Val-Ser-Glu-Glu-Lys-Ala-Lys-Gln-Tyr-Leu-Glu-Glu-Phe-His-Gln- Thr-Ala-Leu-Glu-His-Pro-Glu-Leu-Ser-Glu-Leu-Lys-Thr-Val-Thr-Gly-Thr- Asn-Pro-Val-Phe-Ala-Gly-Ala-Asn-Tyr-Ala-Ala-Trp-Ala-Val-Asn-Val-Ala- Gln-Val-Ile-Asp-Ser-Glu-Thr-Ala-Asp-Asn-Leu-Glu-Lys. For any of the compounds of the inventions, R1, R2, or R4 can be a polyethylene glycol moiety selected from the group consisting of: H3C(OCH2CH2)ccOCH2C(0)-, H(OCH2CH2)ccOCH2C(0)-, H3C(OCH2CH2)ccOC(0)-, H(OCH2CH2)ccOC(0)-, H3C(OCH2CH2)ccNHC(0)-, H(OCH2CH2)ccNHC(0)-, H3C(OCH2CH2)ccNHC(S)-, H(OCH2CH2)ccNHC(S)-, H3C(OCH2CH2)ccC(0)-, H(OCH2CH2)ccC(0)-, H3C(OCH2CH2)ccNHCH2C(0)-, H(OCH2CH2)ccNHCH2C(0)-, H3C(OCH2CH2)ccOC(0)C(CH3)2-, and H(OCH2CH2)ccOC(0)C(CH3)2-, where cc is a range of numbers that results in an average molecular weight of the polyethylene glycol moiety of between 1,000-40,000, preferably 20,000 or 40,000, or a polyethylene glycol moiety selected from the group consisting of: maleimide- (CH2)bbC(0)NHCH2CH2(OCH2CH2)aaOCH2C(0)-, maleimide- (CH2)bbC(0)NHCH2CH2(OCH2CH2)aaNHCH2C(0)-, maleimide-
(CH2)bbC(0)NHCH2CH2(OCH2CH2)aaNHC(S)-, maleimide-(CH2)b NHC(S), maleimide-(CH2)bbC(0)-, or maleimide-(CH2) b-, where aa is 1-10 and bb is 1- 4. For those compounds that contain a polyethylene glycol chain that includes a maleimide functional moiety, the compound can be further reacted with a monoclonal antibody, or fragment thereof, to form a covalent bond between a sulfur atom of the antibody and the maleimide moiety of the compound. By selectively preventing the catalytic domain of toxins or viral factors from translocating across endosomal membranes, the compounds of the invention, or derivatives or peptidomimetics thereof, can inhibit mammalian cell death caused by such toxins/factors. Therefore, these compounds can be used in the prophylaxis or treatment of diseases caused by toxin-producing bacteria or in the prophylaxis or treatment of adverse events that are caused by the direct exposure to mammals of toxins or toxin derivatives, such as, for example, fusion toxin-proteins. In another example, the compounds of the invention are useful for the prophylaxis or treatment of viral diseases by inhibiting the translocation across endosomal membrane of viral/retro viral transcription factors. Accordingly, in another aspect, the invention features the use of any of the compounds of the invention in the manufacture of a medicament for inhibiting cell death in a mammal, preferably a human. In one embodiment, the compound inhibits the translocation of a viral or bacterial toxin from the lumen of an endosome to the cytosol of said cell. In one example the toxin is an AB toxin, such as, for example Diphtheria toxin, a Botulinum toxin, Anthrax toxin LF, and Anthrax toxin EF. In another embodiment, the compound inhibits the translocation of a viral or retroviral transcription factor, such as, for example, human immunodeficiency virus (HIV-1) reverse transcriptase or Tat. In another aspect, the invention features a compound having a nucleic acid sequence encoding any of the peptide sequences of the invention. In one embodiment, the peptide sequence is selected from the group consisting of: - Arg-Asp-Lys-Thr-Lys-Thr-Lys-Ile-Glu-Ser-Leu-Lys-Glu-His-Gly-Pro-Ile-Lys- Asn-Lys- ; -Asp-Trp- Asp- Val-Ile- Arg- Asp-Lys-Thr-Lys-Thr-Lys-Ile-Glu-Ser- Leu-Lys-Glu-His-Gly-; and -Arg-Asp-Lys-Thr-Lys-Thr-Lys-Ile-Glu-Ser-Leu- Lys-Glu-His-Gly-Pro-Ile-Lys- Asn-Lys-. In another embodiment, the peptide sequence is Arg- Asp-Lys-Thr-Lys-Thr-Lys-Ile-Glu-Ser-Leu-Lys-Glu-His-Glu- Pro-Ile-Lys-Asn-Lys-Met-Ser-Glu-Ser-Pro-Asn-Lys-Thr-Val-Ser-Glu-Glu- Lys-Ala-Lys-Gln-Tyr-Leu-Glu-Glu-Phe-His-Gln-Thr-Ala-Leu-Glu-His-Pro- Glu-Leu-Ser-Glu-Leu-Lys-Thr-Val-Thr-Gly-Thr-Asn-Pro-Val-Phe-Ala-Gly- Ala-Asn-Tyr-Ala-Ala-Trp- Ala-Val-Asn-Val-Ala-Gln- Val-Ile- Asp-Ser-Glu- Thr- Ala- Asp- Asn-Leu-Glu-Lys- .
In another embodiment the nucleic acid is operably linked to an inducible promoter. Examples of inducible promoter systems include those where the expression of the peptide sequence can moderated by treating the transfected cell with an agent selected from the group consisting of: doxycycline; retinal; cyclosporin or analogs thereof; FK506; FK520; and rapamycin or analogs thereof. In another aspect, the invention features pharmaceutical compositions of the peptides of the invention. In one embodiment, the pharmaceutical compositions can include agents or compounds that facilitate delivery of the peptides to therapeutic targets. Such delivery strategies are described in Therapeutic Protein and Peptide Formulation and Delivery (ACS Symposium Series, No 675) (1997), edited by Shahrokh, et al. and in Formulation and Delivery of Proteins and Peptides (ACS Symposium Series, No 567) (1994), edited by Cleland and Langer, both of which are hereby incorporated by reference. In another aspect, the invention features a method of identifying a compound that inhibits cell death in a mammal comprising the following steps: a) isolating endosomes, desirably early endosomes, from the cell, b) placing the endosomes in a cytosolic buffer, c) contacting the endosomes with a fusion protein-toxin, wherein the protein comprises a binding moiety for a component of the cell membrane of the cell and the toxin comprises a fragment of
Diphtheria toxin, d) contacting the endosomes with a cytosolic translocation factor complex, e) contacting the endosomes with said compound, and f) measuring translocation of the toxin, where a decreased level of said translocation relative to that observed in the absence of the compound indicates that the compound inhibits said cell death.
In another aspect, the invention features a method of identifying a compound that promotes cell death in a mammal comprising the following steps: a) isolating endosomes, desirably early endosomes, from the cell, b) placing the endosomes in a cytosolic buffer, c) contacting the endosomes with a fusion protein-toxin, wherein the protein comprises a binding moiety for a component of the cell membrane of the cell and the toxin comprises a fragment of Diphtheria toxin, d) contacting the endosomes with a cytosolic translocation factor complex, e) contacting the endosomes with said compound, and f) measuring translocation of the toxin, where an increased level of said translocation relative to that observed in the absence of the compound indicates that the compound inhibits said cell death. The fusion protein can be any protein or protein fragment that binds to a component of mammalian cellular membranes and is subsequently internalized. In a desirable embodiment, the fusion protein is IL-2. Other examples include monoclonal antibodies that bind to cellular membrane epitopes. In a most desirable embodiment, the fusion protein-toxin is DAB389IL-2. In another embodiment, cytosolic translocation factor comprises Hsp 90 and thioredoxin reductase. Assessing translocation can include measuring the ADP- ribosylation of elongation factor-2. In another aspect, the invention features a composition that contains heat shock protein 90 (Hsp 90), or a protein that is substantially identical to Hsp 90, complexed to a cellular fraction, where the composition is formed by adding Hsp 90 to the cytosol of a mammalian cell, followed by isolating the cellular cytosolic fraction that has a molecular weight of between 100 kDa and 250 kDa and also facilitates the translocation of the fusion protein DAB389IL-2 from the interior to the exterior of endosomes. In one embodiment, the Hsp 90 that is added is a human recombinant protein. In another embodiment thioredoxin reductase is also part of the composition. In yet another aspect, the invention features a composition that contains thioredoxin reductase (TrR-1), or a protein that is substantially identical to TrR-1, complexed to a cellular fraction, where the composition is formed by adding TrR-1 to the cytosol of a mammalian cell, followed by isolating the cytosolic fraction that has a molecular weight of between 100 kDa and 250 kDa and also facilitates the translocation of the fusion protein DAB389IL-2 from the interior to the exterior of endosomes. In one embodiment, the TrR-1 that is added is a human recombinant protein. In another embodiment Hsp 90 is also part of the composition. In yet another embodiment, the composition includes TrR-1 and Hsp 90, where both of these components are human recombinant proteins.
Abbreviations and Definitions
The following abbreviations are used throughout the application: "br" stands for bovine recombinant; when not referring to the amino acid cysteine, "C" stands for catalytic; "CTF" stands for cytosolic translocation factor; "DT" stands for diphtheria toxin; "EF-2" stands for Elongation Factor 2; "ESI" stands for electrospray ionization; "hr" stands for human recombinant; "Hsp" stands for heat shock protein; "MALDI" stands for matrix assisted laser desorption ionization; "MS" stands for mass spectrometry; when not referring to the amino acid threonine, "T" stands for transmembrane; "TrR-1" stands for: thioredoxin reductase; "v" stands for vesicular.
The terms "alkoxy" or "alkyloxy," as used interchangeably herein, represent an alkyl group attached to the parent molecular group through an oxygen atom. Exemplary unsubstituted alkoxy groups are of from 1 to 6 carbons The term "alkyl," as used herein, represents a monovalent group derived from a straight or branched chain saturated hydrocarbon of, unless otherwise specified, from 1 to 6 carbons and is exemplified by methyl, ethyl, n- and iso- propyl, n-, sec-, iso- and tert-butyl, neopentyl and the like and may be optionally substituted with one, two, three or, in the case of alkyl groups of two carbons or more, four substituents independently selected from the group consisting of: (1) alkoxy of one to six carbon atoms; (2) alkylsulfϊnyl of one to six carbon atoms; (3) alkylsulfonyl of one to six carbon atoms; (4) amino; (5) aryl; (6) arylalkoxy; (7) aryloyl; (8) azido; (9) carboxaldehyde; (10) cycloalkyl of three to eight carbon atoms; (11) halo; (12) heterocyclyl; (13) (heterocycle)oxy; (14) (heterocycle) oyl; (15) hydroxyl; (16) N-protected amino; (17) nitro; (18) oxo; (19) spiroalkyl of three to eight carbon atoms; (20) thioalkoxy of one to six carbon atoms; (21) thiol; (22) -C02R , wherein R is selected from the group consisting of (a) alkyl, (b) aryl and (c) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (23) -C(0)NR R , wherein RB and Rc are independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (24) -S02RD, wherein RD is selected from the group consisting of (a) alkyl, (b) aryl and (c) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (25) -S02NRBRF, wherein RE and RF are independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, wherein the alkylene group is of one to six carbon atoms; and (26) -NR R , wherein R and R are independently selected from the group consisting of (a) hydrogen; (b) an N-protecting group; (c) alkyl of one to six carbon atoms; (d) alkenyl of two to six carbon atoms; (e) alkynyl of two to six carbon atoms; (f) aryl; (g) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (h) cycloalkyl of three to eight carbon atoms and (i) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms, and the alkylene group is of one to ten carbon atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group. The term "aryl," as used herein, represents a mono- or bicyclic carbocyclic ring system having one or two aromatic rings and is exemplified by phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl and the like and may be optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of: (1) alkanoyl of one to six carbon atoms; (2) alkyl of one to six carbon atoms; (3) alkoxy of one to six carbon atoms; (4) alkoxyalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (5) alkylsulfinyl of one to six carbon atoms; (6) alkylsulfinylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (7) alkylsulfonyl of one to six carbon atoms; (8) alkylsulfonylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (9) aryl; (10) arylalkyl, wherein the alkyl group is of one to six carbon atoms; (11) amino; (12) aminoalkyl of one to six carbon atoms; (13) aryl; (14) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (15) aryloyl; (16) azido; (17) azidoalkyl of one to six carbon atoms; (18) carboxaldehyde; (19) (carboxaldehyde)alkyl, wherein the alkylene group is of one to six carbon atoms; (20) cycloalkyl of three to eight carbon atoms; (21) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms and the alkylene group is of one to ten carbon atoms; (22) halo; (23) haloalkyl of one to six carbon atoms; (24) heterocyclyl; (25) (heterocyclyl)oxy; (26) (heterocyclyl)oyl; (27) hydroxy; (28) hydroxyalkyl of one to six carbon atoms; (29) nitro; (30) nitroalkyl of one to six carbon atoms; (31) N-protected amino; (32) N-protected aminoalkyl, wherein the alkylene group is of one to six carbon atoms; (33) oxo; (34) thioalkoxy of one to six carbon atoms; (35) thioalkoxyalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (36) - (CH2)qC02RA, wherein q is zero to four and RA is selected from the group consisting of (a) alkyl, (b) aryl and (c) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (37) -(CH2)qCONR R , wherein R and R are independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (38) -(CH2)qS02RI), wherein RD is selected from the group consisting of (a) alkyl, (b) aryl and (c) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (39) -(CH2)qS02NRBRF, , wherein RE and RF are independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (40) - (CH2)qNRGRH, wherein RG and RH are independently selected from the group consisting of (a) hydrogen; (b) an N-protecting group; (c) alkyl of one to six carbon atoms; (d) alkenyl of two to six carbon atoms; (e) alkynyl of two to six carbon atoms; (f) aryl; (g) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (h) cycloalkyl of three to eight carbon atoms and (i) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms, and the alkylene group is of one to ten carbon atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group; (41) oxo; (42) thiol; (43) perfluoroalkyl; (44) perfluoroalkoxy; (45) aryloxy; (46) cycloalkoxy; (47) cycloalkylalkoxy; and (48) arylalkoxy. The terms "arylalkoxy" or "aralkoxy," as used interchangeably herein, represent an arylalkyl group attached to the parent molecular group through an oxygen atom. Exemplary unsubstituted arylalkoxy groups are of from 7 to 16 carbons. The terms "arylalkyl" or "aralkyl," as used interchangeably herein, represent an aryl group attached to the parent molecular group through an alkyl group. Exemplary unsubstituted arylalkyl groups are of from 7 to 16 carbons. The term "heteroaryl," as used herein, represents that subset of heterocycles, as defined herein, which are aromatic: i.e., they contain 4n+2 pi electrons within the mono- or multicyclic ring system. Exemplary unsubstituted heteroaryl groups are of from 1 to 9 carbons. The terms "heterocycle" or "heterocyclyl," as used interchangeably herein represent a 5-, 6- or 7-membered ring, unless otherwise specified, containing one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur. The 5-membered ring has zero to two double bonds and the 6- and 7-membered rings have zero to three double bonds. The term "heterocycle" also includes bicyclic, tricyclic and tetracyclic groups in which any of the above heterocyclic rings is fused to one or two rings independently selected from the group consisting of an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring and another monocyclic heterocyclic ring such as indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl and the like. Heterocyclics include pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl, morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl, isoindazoyl, triazolyl, tetrazolyl, oxadiazolyl, uricyl, thiadiazolyl, pyrimidyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroinidolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl, benzothienyl and the like. Heterocyclic groups also include compounds of the formula
Figure imgf000015_0001
F is selected from the group consisting of -CH2-, -CH20- and -0-, and G is selected from the group consisting of -C(O)- and -(C(R')(R"))V-, wherein R' and R" are independently selected from the group consisting of hydrogen or alkyl of one to four carbon atoms, and v is one to three and includes groups such as 1,3-benzodioxolyl, 1,4-benzodioxanyl and the like. Any of the heterocycle groups mentioned herein may be optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of: (1) alkanoyl of one to six carbon atoms; (2) alkyl of one to six carbon atoms; (3) alkoxy of one to six carbon atoms; (4) alkoxyalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (5) alkylsulfinyl of one to six carbon atoms; (6) alkylsulfinylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (7) alkylsulfonyl of one to six carbon atoms; (8) alkylsulfonylalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (9) aryl; (10) arylalkyl, wherein the alkyl group is of one to six carbon atoms; (11) amino; (12) aminoalkyl of one to six carbon atoms; (13) aryl; (14) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (15) aryloyl; (16) azido; (17) azidoalkyl of one to six carbon atoms; (18) carboxaldehyde; (19) (carboxaldehyde)alkyl, wherein the alkylene group is of one to six carbon atoms; (20) cycloalkyl of three to eight carbon atoms; (21) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms and the alkylene group is of one to ten carbon atoms; (22) halo; (23) haloalkyl of one to six carbon atoms; (24) heterocycle; (25) (heterocycle)oxy; (26) (heterocycle)oyl; (27) hydroxy; (28) hydroxyalkyl of one to six carbon atoms; (29) nitro; (30) nitroalkyl of one to six carbon atoms; (31) N-protected amino; (32) N-protected aminoalkyl, wherein the alkylene group is of one to six carbon atoms; (33) oxo; (34) thioalkoxy of one to six carbon atoms; (35) thioalkoxyalkyl, wherein the alkyl and alkylene groups are independently of one to six carbon atoms; (36) - (CH2)qC02RA, wherein q is zero to four and RA is selected from the group consisting of (a) alkyl, (b) aryl and (c) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (37) -(CH2)qCONR R , wherein R and R are independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (38) -(CH2)qS02RD, wherein RD is selected from the group consisting of (a) alkyl, (b) aryl and (c) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (39) -(CH2)qS02NRBRF, , wherein RB and RF are independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (40) - (CH2)qNR R , wherein R and R are independently selected from the group consisting of (a) hydrogen; (b) an N-protecting group; (c) alkyl of one to six carbon atoms; (d) alkenyl of two to six carbon atoms; (e) alkynyl of two to six carbon atoms; (f) aryl; (g) arylalkyl, wherein the alkylene group is of one to six carbon atoms; (h) cycloalkyl of three to eight carbon atoms and (i) cycloalkylalkyl, wherein the cycloalkyl group is of three to eight carbon atoms, and the alkylene group is of one to ten carbon atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group; (41) oxo; (42) thiol; (43) perfluoroalkyl; (44) perfluoroalkoxy; (45) aryloxy; (46) cycloalkoxy; (47) cycloalkylalkoxy; and (48) arylalkoxy. The terms "heterocyclyloxy" or " (heterocycle) oxy," as used interchangeably herein, represents a heterocycle group, as defined herein, attached to the parent molecular group through an oxygen atom. Exemplary unsubstituted heterocyclyloxy groups are of from 1 to 9 carbons. The term " amino acid residue," as used herein, represents a - N(RA)C(RB)(Rc)C(0)- linkage, wherein RA is selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, as defined herein; and R and R are independently selected from the group consisting of: (a) hydrogen, (b) optionally substituted alkyl, (c) optionally substituted cycloalkyl, (d) optionally substituted aryl, (e) optionally substituted arylalkyl, (f) optionally substituted heterocyclyl, and (g) optionally substituted heterocyclylalkyl, each of which is as defined herein. For natural amino acids, R is H and R corresponds to those side chains of natural amino acids found in nature, or their antipodal configurations. Exemplary natural amino acids include alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, aspartamine, ornithine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine, each of which, except glycine, as their D- or L-form. As used herein, for the most part, the names of naturally-occuring amino acids and aminoacyl residues used herein follow the naming conventions suggested by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature as set out in Nomenclature of α-
Amino Acids (Recommendations, 1974), Biochemistry, 14 (2), (1975).
Compounds of the present invention include non-naturally occuring (i.e., unnatural) amino acid residues in their D- or L-form such as, for example, homophenylalanine, phenylglycine, cyclohexylglycine, cyclohexylalanine, cyclopentyl alanine, cyclobutylalanine, cyclopropylalanine, cyclohexylglycine, norvaline, norleucine, ornithine, thiazoylalanine (2-, 4- and 5- substituted), pyridylalanine (2-, 3- and 4-isomers), naphthalalanine (1- and 2-isomers) and the like. Stereochemistry is as designated by convention, where a bold bond indicates that the substituent is oriented toward the viewer (away from the page) and a dashed bond indicates that the substituent is oriented away from the viewer (into the page). If no stereochemical designation is made, it is to be assumed that the structure definition includes both stereochemical possibilities. What is meant by "cytosolic buffer" is any buffering system into which endosomes can be placed where they remain intact and viable. In one example; 3% sucrose in 100 mM HEPES-KOH pH 7.9, 1.4 mM KCl, 30 mM MgCl2, 2 mM EDTA, and 5 mM DTT constitutes a cytosolic buffer. What is meant by "cytosolic translocation factor complex" is a group of component proteins that includes Hsp 90 and TrR-1, with the complex also having the ability to facilitate the translocation of the catalytic domain of diphtheria toxin from the interior to the exterior of an endosome.
By a "pharmaceutically acceptable excipient" is meant a carrier that is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable excipient is physiological saline. Other physiologically acceptable excipients and their formulations are known to one skilled in the art and described, for example, in "Remington: The Science and Practice of Pharmacy" (20th ed., ed. A.R. Gennaro AR., 2000, Lippincott Williams & Wilkins). ( By "operably linked" is meant that a nucleic acid molecule and one or more regulatory sequences (e.g., a promoter) are connected in such a way as to permit expression and/or secretion of the product (i.e., a polypeptide) of the nucleic acid molecule when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences. By "polypeptide" or "peptide" is meant any chain of from 2 to 100 natural or unnatural amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally- occurring or non-naturally occurring polypeptide or peptide, as is described herein. Naturally occurring amino acids are any one of the following, alanine (A or Ala), cysteine (C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H, or His), isoleucine (I or He), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), ornithine (O or Om), proline (P or Pro), hydroxyproline (Hyp), glutamine (Q or Gin), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), or tyrosine (Y or Tyr). By "substantially identical" is meant a protein, polypeptide, or nucleic acid exhibiting at least 75%, but preferably 85%, more preferably 90%o, most preferably 95%, or even 99% identity to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 10 amino acids, and preferably at least 20 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 30 nucleotides, preferably at least 60 nucleotides, and more preferably at least 120 nucleotides.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the partial purification of cytosolic proteins required to mediate DT C domain translocation from the lumen of early endosomes in vitro. (A) Early endosomes from human T cells (HUT102/6TG), preloaded with DAB389IL-2, were incubated for 30 min. at 37° with 2 mM ATP (A) and/or 4 μg of HUT102/6TG crude cytosol (C). C* denotes heat inactivation of cytosol prior to incubation with endosomes. Endosomes were pelleted, and the supernatant fluid (S) was assayed for DT C domain ADP-ribosyltransferase activity by measuring the incorporation of [ 2P]-NAD+ into AD[32P]-ribosyl- EF2 following 7% SDS-PAGE and autoradiography. (B) Time course for translocation of ADP-ribosyltransferase activity from DA189(vsv-G)B3S9lL-2 across the early endosomal membrane. Translocations were performed as described above, except the reactions were incubated at different temperatures (0°, 15°, 37° C) for 15, 30, and 45 min. Both the supernatant and the pellet fractions were assayed for ADP-ribosyltransferase activity, and the autoradiographic signals were measured by densitometry. The sum of densitometry units from each pair of supernatant fluid and pellet fractions is plotted as the percentage of activity in the supernatant fractions at that time point. (n=3; error bar: standard deviation).
Figure 2 shows the in vitro acidification of early endosomes requires ATP and does not require any cytosolic protein factors. (A) Fluorescence emission of 1 ng/ml SNARF-1 70 kDa dextran conjugate standards at pH 7.5 and 4.5 was measured at an excitation wavelength of 534 nM and an emission wavelength of 645 nM. (B) Purified early endosomes pre-loaded with the pH sensitive SNARF-1 70 kDa Dextran Conjugate were incubated in translocation assay buffer for 20 min. at 37°C with 2 mM ATP and/or 0.1 μg/μL of MONO-Q purified cytosol. In each instance, assays were performed in triplicate and fluorescence was monitored using a Perkin-Elmer 650S Fluorescence Detector.
Figure 3 shows the partial purification of CTFs results in the increase of translocation in vitro specific activity. (A) Translocation in vitro specific activity of CTFs increases following each stage of purification. Reactions were performed as previously described, and only the ADP-ribosyltransferase activity of the supernatant fluid fractions is shown. CE: crude extract; DEAE: DEAE-Sepharose anion exchange chromatography (150-190 M NaCl fractions); S200: Sephacryl 200 sizing chromatography (250-100 kDa fractions); MQ: Mono-Q anion exchange chromatography (27.3 mS fractions). (B) Colloidal Coomassie stained 10% SDS-PAGE protein band profiles following MONO-Q anion exchange chromatography. Partially purified CTF complex fractions from both T cells and yeast cells were eluted at a conductance of 27.3 mS. Figure 4 shows the identification of CTF(s) using mass spectrometry.
(A) Representative total ion chromatogram from an online capillary liquid chromatography mass spectrometric analysis of the 'in-geP tryptic digest following immunoprecipitation of the 84 kDa band (Fig. 2B, 5B) from human partially purified CTFs using rabbit polyclonal anti-Hsp 90 antibodies. (B) Mass spectrum from LC-MS elution at time 19.5 to 20.5 min, as indicated by the shaded region in (A). Peaks are labeled with the m/z value, the charge state, the corresponding amino acid segment, and specification of the Hsp 90 isoform. (C) Tandem mass spectrum for m/z 575.984+ [redundant sequence from Hsp 90 alpha and beta; See inset on (B)]. Complementary a and b (N- terminal derived) as well as y (C-terminal derived) ions are labeled in the spectrum with m/z value and charge state. All observed a, b, and y ions are indicated in the peptide diagram. Data was analyzed using Bioanalyst™ (Applied Biosystems Inc.) reconstruction algorithms. For initial screening and searches, acquired mass values were compared to theoretical protein digests using the Mascot search engine (Matrix Science Ltd). Figure 5 shows that Hsp 90 is a component of the CTF complex. (A) Hut 102/6TG (H) and yeast NLY22" (Y) partially purified CTF complexes were co-incubated with 1 μg anti- Hsp 90 and 1 μg anti- Hsp 82 antibodies, respectively, for 10 min. at room temperature prior to standard analysis in the in vitro translocation assay as previously described. Analogously, CTF complexes were depleted of Hsp 90 or Hsp 82 by immunoprecipitation and assayed for translocation activity in vitro. Either partially purified CTF complex or human recombinant (hr)-Hsp 90 was added back to Hsp 90 depleted CTF complexes as indicated, and translocation activity in vitro was assayed as previously described. (B) Colloidal Coomassie stained 10% SDS- PAGE protein band profiles of immunoprecipitated human Hsp 90. Arrow indicates Hsp 90 as identified by ESI LC-MS/MS analysis. Figure 6 shows that Hsp 90 is essential for mediating DAB389IL-2 C domain translocation from the lumen of early endosomes to the external milieu. The Hsp 90 specific inhibitors, geldanamycin and radicicol, were pre-incubated with partially purified CTF complex as indicated for 15 min at room temperature prior to assaying for translocation activity in vitro. Excess partially purified HUT 102/6TG CTF complex and hr-Hsp 90 were added to geldanamycin/radicicol treated CTF complexes as indicated and assayed for translocation activity in vitro previously described. Figure 7 shows that TrR-1 is a component of the CTF complex. (A) Hut 102/6TG (H) and yeast NLY22" (Y) partially purified CTF complexes were depeleted of TrR-1 by immunoprecipitation with anti-human TrR-1 antibodies or affinity purification using 2',5' ADP-Sepharose. TrR-1 depleted CTF complexes were then assayed for translocation activity in vitro under reducing conditions. Either partially purified CTF complex or brTrR-1 was added back to TrR-1 depleted CTF complexes as indicated, and translocation activity in vitro was assayed as previously described. (B) Colloidal Coomassie stained 10%) SDS-P AGE protein band profiles of 2 ',5' ADP-sepharose affinity purified yeast TrR-1. Arrow indicates yeast TrR-1 as identified by ESI LC-MS/MS analysis. Figure 8 shows that TrR-1 function is essential for mediating DAB389IL- 2 C domain translocation from the lumen of early endosomes to the external milieu under non-reducing conditions. Partially purified CTF complex, both human and yeast, were assayed for translocation activity in vitro under non- reducing conditions using translocation buffer containing 10 μM NADPH without DTT. The TrR-1 stereospecific inhibitor ct-s-13-retinoic acid and the inactive tr< s-13-retinoic acid isomer were pre-incubated with partially purified CTF complex as indicated for 15 min at room temperature prior to assaying for translocation activity in vitro under non-reducing conditions. Excess brHsp 90 was added to cl3RA treated CTF complexes as indicated and translocation activity in vitro was assayed under non-reducing conditions. Figure 9 shows a Blast analysis (BLAST NCBI, Vector NTI version 8.0) of Anthrax toxin-EF, Anthrax toxin-LF, Botulinum A, Botulinum-Cl, Botulinum D, Diphtheria toxin, and HIV reverse transcriptase isolated from 6 different patients. Figure 10 shows (A) The entry motif of diphtheria toxin, anthrax lethal factor, anthrax edema factor and boutlinum neurotoxin serotype A that was identified by BLAST analysis of the diphtheria toxin transmembrane helix Tl. (B) top panel shows the ribbon diagram of the X-ray structures of diphtheria toxin [C = catalytic domain; T = transmembrane domain; R = receptor binding domain; arrow = furin cleavage site], anthrax lethal factor [circle = protective antigen binding domain; arrow = aa 263 site of fusion protein formation allowing heterologous peptide to be delivered into target cells]; anthrax edema factor [circle = protective antigen binding domain; and botulinum neurotoxin serotype A; middle panel shows the conservation of structure by an end view; bottom panel shows conservation of structure in a lateral view: Red = D & E; Blue = L &K; Turquoise = N, Q; Orange = S &T; Green = hydrophobic non- aromatic residues. Figure 11 shows an autoradiographic analysis of diphtheria toxin catalytic domain translocation and release from purified early endosomes in the presence and absence of a peptide corresponding to DT amino acid residues 205-224. The translocation assay (TA) was performed in the presence of ATP and partially purified cytosolic translocation factor complex from HUT 102 6TG cells. Control translocation is performed in the absence of DT205-224 peptide. The ADP-ribosyltransferase reactions were performed on supernatant fluid and lysed pellet fractions following ultracentrifugation. Under the TA conditions used, approximately 50% of the diphtheria toxin catalytic domain is specifically translocated from the lumen of the early endosome to the external milieu. In contrast, the addition of the DT205-224 peptide (final concentration = 50 μM) to the translocation assay mixture inhibits translocation of the catalytic domain. Figure 12 shows a plasmid that incorporates DT 210-299. This sequence was cloned into the pTRACER expression vector and placed under control of the CMV promoter to form plasmid pTl . Plasma pTl was transfected into HUT 102 cells and stable transfectants were isolated using zeocine selection. A stable transfectant clone was then purified and designated HUT102-T1 Figure 13A shows the sensitivity of HUT 102 (closed circles) and HUT102-T1 cells (closed squares) to the fusion toxin DAB389IL-2 in a dose response study. In a control experiment, Figure 13B shows the sensitivity of HUT 102 (closed circles) and HUT102-T1 cells (closed squares) to Pseudomonas exotoxin A, a bacterial protein toxin known to enter the cell at the level of the trans-golgi (rather than from an acidified early endosome). Figure 14 shows the amino acid and corresponding nucleic acid sequences of DT 210-299.
DETAILED DESCRIPTION In order to further define the requirements of C-domain translocation across the endosomal membrane, we have used an in vitro C-domain translocation assay essentially as described by Lemichez et al, supra. This assay employs purified early endosomes that have been pre-loaded with DAB389lL-2 and monitors the translocation of -ADP-ribosyltransferase activity from the endosomal lumen to the external milieu. We have used translocated ADP-ribosylatransferase activity to monitor the purification of cytosolic components that are required for this process. As described herein, we demonstrate that the in vitro translocation of the DAB389lL-2 ADP-ribosyltransferase activity across the membrane of early endosomes and its release into the external milieu requires a cytosolic translocation factor (CTF) complex. Using translocated ADP- ribosyltransferase activity as an assay, we have partially purified the CTF- complex from both human T cell and yeast extracts. Mass spectrometry sequencing of individual protein bands revealed by colloidal Commassie staining of SDS-polyacrylamide gels has allowed the identification of Hsp 90 and TrR-1 from human T cells, and the homologous Hsp 82 and TrR-1 from yeast extracts. A functional role for these proteins in the translocation and/or cytosolic release of -ADP-ribosyltransferase activity was established through immunoprecipitation and the use of specific inhibitors. Furthermore, the identification of CTF complex homologs in partially purified yeast extracts suggests that DT C-domain translocation may proceed by a fundamental mechanism of entry.
Following depletion of either Hsp 90 or TrR-1 from partially purified human T cell and yeast CTF-complexes, we were not successful in reconstituting in vitro translocation of the C-domain by the addition of hrHsp 90 or brTrR-1, either alone or in combination. These results suggest that Hsp 90 chaperonin and TrR-1 are components of a complex(es) that is(are) necessary for facilitating C-domain translocation across the early endosomal membrane. In marked contrast, we were able to reconstitute in vitro C-domain translocation activity in either geldanamycin / radicicol or -13-retinoic acid treated CTF complexes by the addition of recombinant proteins to the mixture. Taken together, these observations lead us to conclude that both the chaparonin Hsp 90 and TrR-1 are required for C-domain translocation, but are not in themselves sufficient. Hsp 90 is ubiquitously expressed and is known to be a component of several multi-molecular chaparonin complexes which are highly conserved in eukaryotes (Chang et al, J. Biol Chem. 269:24983-8, 1994). The interaction of Hsp 90 with other co-chaparonins and the formation of discrete complexes is known to mediate Hsp 90 substrate recognition (Caplan et al, Trends Cell. Biol 9:262-8, 1999). Although Hsp 90 does not usually directly bind nor refold nascent polypeptides, it is known to refold a growing list of newly synthesized proteins including membrane associated protein kinases (Bijlmakers and Marsh, Hsp 90 is essential for the synthesis and subsequent membrane association, but not the maintenance, of the Src-kinase p56(lck). Mol. Biol. Cell 11 : 1585-95, 2000). In addition to its refolding activity, Hsp 90 complexes are also known to regulate the trafficking of membrane associated proteins through interactions with cytoskeleton motors (Pratt et al, Cell. Signal. 11:839-51, 1999). The CTF complex is capable of refolding thermally denatured diphtheria toxin fragment A in vitro, and refolding requires the ATPase activity of Hsp 90. However, the inhibition of Hsp 90 ATPase activity by either geldanamycin or radicicol alone does not inhibit translocation of ADP-ribosyltransferase activity across the early endosomal membrane. As such, it would appear that refolding of denatured C-domain into an active conformation and translocation are mutually exclusive events. The synergistic effects of geldanamycin and radicicol on the inhibition of ADP-ribosyltransferase translocation are of interest, and are consistent with previous reports (Rosenhagen et al, Biol. Chem. 382:499-504, 2001). It is possible that when used in combination, these inhibitors result in either a disruption of Hsp 90 substrate recognition and/or the disruption of Hsp 90 - co-chaperone interactions thereby leading to an inhibition of C-domain translocation. The inability to reconstitute yeast CTF- complexes with mammalian factors supports the later hypothesis. Following furin-mediated nicking of the α-carbon backbone of either DT or DAB389IL-2, retention of the interchain disulfide bond between the C- and T-domains of the toxin presumably is essential for insertion and threading of the denatured C-domain into and through the nascent channel formed by the T-domain (vanderSpek et al, Protein Engn.; 7: 985-989, 1994). Moreover, post-translocation reduction of this disulfide bond is also required for the release of the C-domain into the cytosol since unreduced C-domain and membrane inserted T-domain are both targeted for proteolytic degradation (Moskaug et al, Biochem. J. 291:473-7, 1993; Madshus et al, J. Biol. Chem. 269:4648-52, 1994). Indeed, the pivotal role of this event is underscored by the observation that reduction of this interchain disulfide bond is the rate limiting step in the diphtherial intoxication of eukaryotic cells (Papini et al, J. Biol. Chem. 268:1567-74, 1993). Observations reported here confirm and extend these earlier findings, and strongly suggest that TrR-1 is a component of the CTF-complex required for the release of the C-domain from the early endosome. These observations also confirm and extend the earlier observations of Sandvig et al, Biochem. J. 194:821-7, 1981, who reported that retinoic acids inhibit the action of several AB toxins, including diphtheria toxin, on eukaryotic cells. Although the data reported here clearly demonstrates that TrR- 1 activity is required for at least the cytosolic release of the DAB389lL-2 C-domain from purified early endosomes, we cannot conclude whether or not TrR-1 is directly involved in the reduction of the interchain disulfide bond. Since we have identified thioredoxin peroxidase in CTF-complexes purified from yeast, it is possible that TrR-1 functions indirectly through a cascade of reductases {e.g., thioredoxin, Moskaug et al, J. Biol. Chem. 262:10339-45, 1987). It is widely accepted that anthrax lethal toxin and edema factor, as well as the botulinum neurotoxins must pass through an acidic early endosomal compartment in order to deliver their respective catalytic domain into the cytosol of targeted cells. The unfolding of the catalytic domains of anthrax lethal factor (Wesche et al, supra) and botulinum toxin serotype D (Bade et al, Naunyn Schmiedebergs Arch Pharmacol 385 Sup 2: R12, 2002), as well as the TrR-1 mediated reduction of the botulinum neurotoxins (Kistner and Habermann, Naunyn Schmiedebergs Arch. Pharmacol. 345:227-34, 1992; Bigalke and Shoer, Clostridial Neurotoxins in Bacterial Protein Toxins, K Aktories, I. Just, editors. Springer- Verlag, Berlin. 407-444, 2000), have been postulated to be a pre-requisites for their delivery to the cytosol. Accordingly, the findings reported here may have wider implications. Importantly, several protein complexes of similar composition have been described in protein- trapping proteomic analysis of yeast. For example, Ho et al, Nature 415:180- 3, 2002, has shown that cyclophilin trapped complexes from yeast contain Hsp 82, TrR-1, and Sec 27. Moreover, cyclophilin is required for the cytosolic entry of HIV (Braaten et al, J. Virol. 70:4220-7, 1996), the vacuolar import of fructose- 1,6-bisphophatase (Brown et al, J. Biol. Chem. 276(51):48017-26, 2001), and the activation of peroxiredoxins (Lee et al, J. Biol. Chem.
276:29826-32, 2001). Recently, Vendeville et al, {Mol Biol. Cell, epub prior to publication, March 12, 2004) reported that the HIV-1 Tat protein, a strong trans-activator that enables productive transcription from the HIV-1 long terminal repeat and is required for HIV replication, enters T-cells essentially like diphtheria toxin, using clathrin-mediated endocytosis before low-pH induced and Hsp90-assisted endosomal translocation. It should also be noted that trafficking mechanisms mediated by cyclophilin - Hsp 90 complexes are synergistically affected by geldanamycin and radicicol (Meyer et al, Cell Stress Chaperones 5:243-54, 2000). Taken in aggregate, observations reported here confirm and extend the hypothesis that multiple pathogens from diverse phylogenetic backgrounds, as well as many of their virulence determinants have convergently evolved to recruit host cell proteins {e.g., CTF-complexes) in order to facilitate their membrane translocation and release into the cytosol of eukaryotic cells. The following non-limiting examples are provided to further describe various aspects and embodiments of the present invention.
Example 1. Partial purification of human T cell and yeast cytosolic factors required for the in vitro translocation of ADP-ribosyltransferase activity across the membrane of early endosomes
Since C-domain translocation across the endosomal vesicle membrane requires the addition of cytosolic components to the reaction mixture, we used translocation of ADP-ribosyltransferase activity to monitor the partial purification of the active component(s) from both human T cell (HUT102/6TG) and yeast (NLY22" ) extracts. Following DEAE anion exchange chromatography translocation active fractions (150mM - 190mM NaCl) were pooled and applied to a Sephacryl 200 sizing column. The translocation active fractions (250 - 100 kDa) were pooled and further fractionated by MonoQ high performance liquid chromatography under conditions free of reducing agents. The translocation active fraction was found to elute from the MonoQ column at 27.3 mS. As shown in Figure 3 A, after fractionation on MonoQ, CTF complex activity from human T cell and yeast cell extracts were increased by 650-fold and 800-fold, respectively. Further analysis of the MonoQ pooled fractions by SDS-polyacrylamide gel electrophoresis and colloidal Commassie staining revealed multiple protein bands ranging in apparent molecular weight from ca. 12 - 100 kDa (Fig. 3B).
Example 2. Identification of individual components of the CTF complex Tryptic peptides from "in gel" digestion of individual protein bands resolved by SDS-polyacrylamide gel electrophoresis were subjected to analysis by mass spectroscopy using matrix-assisted laser desorption/ionization-time-of- flight (MALDI-TOF) and nano-electrospray ionization quadrupole orthogonal-
TOF (ESI-QoTOF) spectrometers. Peptide maps and tandem mass spectrometry sequence data allowed for the unequivocal identification of Hsp 90 (alpha and beta) and TrR-1 in the partially purified CTF complex mixture from human T cells (Fig. 4; Table 1). Importantly, the corresponding yeast homologs, Hsp 82 and TrR-1, as well as thioredoxin peroxidase were identified in the partially purified CTF complex from yeast cells (Table 1). The cumulative peptide coverage for each protein identified through mass spectrometry sequencing was between 65 - 85% of the total protein (Table 2). Ions unassigned in the LC-MS/MS spectra were indicative of truncation, sequence variation, and/or post-translational modification.
Table 1. Summary of the data obtained for each of the CTF components identified in this study: MALDI, ESI-MS/MS, LC-MS/MS, western blot (WB), in vitro translocation assay (TA), mammalian cell cytotoxicity assay (CA)
CTFs Data Obtained
Human T Cell (Hut 102/6TG) Hsp 90 alpha MALDI, ESI-MS MS, LC-MS/MS, WB, TA, CA Hsp 90 beta MALDI, ESI-MS/MS, LC-MS/MS, WB, TA, CA TrR-1 MALDI, ESI-MS/MS, WB, TA, CA
Yeast (NLY2T)
Hsp 82 MALDI, ESI-MS/MS, WB, TA, CA
TrR-1 MALDI, LC-MS/MS, TA, CA
Thioredoxin peroxidase (AHP1) MALDI, ESI-MS/MS
Table 2. Summary of information dependent acquisition tandem MS sequence data from tryptic digests of human Hsp 90 (alpha and beta) and yeast TrR-1.
Human Hsp 90 Alpha (30% coverage MS/MS only; 70% coverage MS + MS/MS) AA m/z (obs Mr(expt) Mrfcalc") Delta Sequence 47- 60 520.94 1559.81 1559.82 -0.01 ELISNSSDALDKIR 59- 74 452.99 1807.94 1807.93 0.01 IRYESLTDPSKLDSGK 59- 74 603.65 1807.93 1807.93 -0.00 IRYESLTDPSKLDSGK 61- 74 513.92 1538.74 1538.75 -0.00 YESLTDPSKLDSGK 61- 74 770.36 1538.71 1538.75 -0.04 YESLTDPSKLDSGK 7755--8877 553300..6633 11558888..8877 11558888..8877 --00..0000 ELHINLIPNKQDR 88- 100 675.36 1348.70 1348.73 -0.03 TLTIVDTGIGMTK 183 -201 576.06 2300.21 2300.20 0.00 GTKVILHLKEDQTEYLEER 186. -201 504.51 2014.01 2014.04 -0.03 VILHLKEDQTEYLEER 186- -201 672.35 2014.02 2014.04 -0.02 VILHLKEDQTEYLEER 118866-- -220011 11000077..9999 22001133..9966 22001144..0044 --00..0077 VILHLKEDQTEYLEER 284- -292 576.28 1150.54 1150.55 -0.01 YIDQEELNK 300 -314 917.36 1832.70 1832.77 -0.07 NPDDITNEEYGEFYK 315 ■ -327 509.92 1526.73 1526.74 -0.01 SLTNDWEDHLAVK 315' -327 764.35 1526.69 1526.74 -0.05 SLTNDWEDHLANK 332288- -333388 667744..8822 11334477..6633 11334477..6666 --00..0033 HFSNEGQLEFR 339 -345 408.26 814.50 814.51 -0.00 ALLFNPR 346 -355 422.22 1263.64 1263.64 0.00 RAPFDLFEΝR 347 -355 554.77 1107.52 1107.53 -0.01 APFDLFE R 387 -400 505.26 1512.76 1512.78 -0.02 GNNDSEDLPLΝISR 338877- -440000 775577..3366 11551122..7711 11551122..7788 --00..0077 GNNDSEDLPLΝISR 437 -443 474.73 947.44 947.44 0.00 FYEQFSK 465 -478 783.83 1565.65 1565.69 -0.04 YYTSASGDEMNSLK 465 -483 754.66 2260.94 2260.96 -0.02 YYTSASGDEMNSLKDYCTR 500 -510 618.30 1234.58 1234.59 -0.01 DQNAΝSAFNER 554400--556644 772299..6600 22991144..3377 22991144..5500 --00..1133 TLNSNTKEGLELPEDEEEKKKQEEK 547 -564 729.68 2186.03 2186.06 -0.03 EGLELPEDEEEKKKQEEK 574 -581 495.29 988.56 988.56 0.00 IMKDILEK 632 -647 479.50 1913.97 1914.03 -0.06 KHLEIΝPDHSΠETLR Human Hsp 90 Beta (30% coverage MS/MS only; 65% coverage MS + MS/MS) AA m/z fobs') Mrtexpt) Mrfcalc Delta Sequence 42-55 772.89 1543.77 1543.82 -0.05 ELISNASDALDKIR 54-69 452.99 1807.94 1807.93 0.01 IRYESLTDPSKLDSGK 54-69 603.65 1807.93 1807.93 -0.00 IRYESLTDPSKLDSGK 54-72 545.54 2178.14 2178.15 -0.01 IRYESLTDPSKLDSGKELK 56-69 513.92 1538.74 1538.75 -0.00 YESLTDPSKLDSGK 56-69 770.36 1538.71 1538.75 -0.04 YESLTDPSKLDSGK 56-72 637.33 1908.95 1908.97 -0.02 YESLTDPS LDSGKELK 70-82 522.29 1563.85 1563.86 -0.01 ELKIDIIPNPQER 83-95 675.36 1348.70 1348.73 -0.03 TLTLVDTGIGMTK 96-107 622.34 1242.66 1241.70 0.96 ADLINNLGTIAK (N→D) 178 - 196 576.06 2300.21 2300.20 0.00 GTKVILHLKEDQTEYLEER 181-196 504.51 2014.01 2014.04 -0.03 VILHLKEDQTEYLEER 181-196 672.35 2014.02 2014.04 -0.02 VILHLKEDQTEYLEER 181-196 1007.99 2013.96 2014.04 -0.07 VILHLKEDQTEYLEER 276-284 576.28 1150.54 1150.55 -0.01 YIDQEELNK 292-306 924.37 1846.73 1846.79 -0.06 NPDDITQEEYGEFYK 307-319 509.58 1525.72 1526.74 -1.01 SLTNDWEDHLAVK (D→N) 307-319 509.92 1526.73 1526.74 -0.01 SLTNDWEDHLAVK 307-319 764.35 1526.69 1526.74 -0.05 SLTNDWEDHLAVK 320-330 674.82 1347.63 1347.66 -0.03 HFSVEGQLEFR 331-337 415.27 828.52 828.52 -0.00 ALLFIPR 338-348 455.58 1363.73 1363.72 0.00 RAPFDLFENKK 379-392 505.26 1512.76 1512.78 -0.02 GWDSEDLPLNISR 379-392 757.36 1512.71 1512.78 -0.07 GWDSEDLPLNISR 429-435 446.22 890.42 890.42 0.01 FYEAFSK 457-475 731.64 2191.89 2191.93 -0.04 YHTSQSGDEMTSLSEYVSR 482-491 580.80 1159.58 1159.58 0.00 SIYYITGESK 492-502 625.30 1248.58 1248.61 -0.03 EQVANSAFVER 566-573 495.29 988.56 988.56 0.00 LMKEILDK 624-639 478.26 1909.00 1910.04 -1.04 KHLEINPDHPIVETLR(D->N) 625-639 594.98 1781.92 1781.94 -0.02 HLEINPDHPIVETLR 625-639 891.94 1781.87 1781.94 -0.07 HLEINPDHPIVETLR Yeast TrR-1 (57% coverage MS/MS only; 85% coverage MS + MS/MS) AA m/z fobs') Mrfexpt) Mrfcalc) Delta Sequence 1 - 24 627.60 2506.37 2506.35 0.02 -\-VHNKVTIIGSGPAAHTAAIYLAR Oxidation (M) 6 - 24 941.58 1881.14 1881.05 0.09 VTIIGSGPAAHTAAIYLAR 6 - 24 628.05 1881.12 1881.05 0.08 VTIIGSGPAAHTAAIYLAR 78 - 89 662.88 1323.75 1323.69 0.06 FGTEIITETVSK 90 - 98 510.82 1019.62 1019.57 0.05 VDLSSKPFK 99 - 124 921.80 2762.38 2762.36 0.01 LWTEFNEDAEPVTTDAIILATGASAK 125 - 137 564.30 1689.88 1689.79 0.08 RMHLPGEETYWQK Oxidation (M) 126 - 137 506.90 1517.68 1517.70 -0.02 MHLPGEETYWQK 154 - 175 1153.13 2304.24 2304.14 0.10 NKPLAVIGGGDSACEEAQFLTK 199 - 217 726.10 2175.27 2175.18 0.10 AEKNEKIEILYNTVALEAK 205 - 217 738.96 1475.90 1475.82 0.08 IEILYNTVALEAK 205 - 221 612.00 1832.98 1832.99 -0.01 IEILYNTVALEAKGDGK 205 - 221 917.54 1833.06 1832.99 0.07 IEILYNTVALEAKGDGK 233 - 244 651.36 1300.71 1300.65 0.06 KNEETDLPVSGL 233 - 255 622.85 2487.35 2487.26 0.09 KNEETDLPVSGLFYAIGHTPATK 245 - 255 402.57 1204.69 1204.62 0.07 FYAIGHTPATK 245 - 255 603.35 1204.69 1204.62 0.06 FYAIGHTPATK ι 271 - 293 756.40 2266.18 2266.11 0.06 TVPGSSLTSVPGFFAAGDVQDSK 296 - 313 890.01 1778.88 1778.93 -0.05 QAITSAGSGCMAALDAEK All peptide MS and MS/MS spectra were submitted to Mascot {32), and positive identifications were confirmed manually. Error tolerance was better than 50 ppm except for three peptides where either (N— »D) or (D-»N) conversion was observed.
Example 3. Hsp 90 is essential, but not sufficient for C-domain translocation in vitro
In order to determine whether Hsp 90 was a component of the CTF complex and to establish a functional role for this chaperonin in C-domain translocation, we conducted a series of experiments to examine the effects of using both polyclonal anti-Hsp 90 antibodies and specific inhibitors. As shown in Figure 5 A, either immunoprecipitation of Hsp 90 from the CTF complex or addition of anti-Hsp 90 to the CTF-complex prior to initiation of the translocation reaction resulted in marked loss of ADP-ribosyltransferase activity in the supernatant fluid fraction. There was no significant loss of ADP- ribosyltransferase activity in the pellet fractions, which argues against the possibility of any translocated but non-refolded pool of DT C domain. Since attempts to reconstitute the immuno-depleted CTF complex with human recombinant (hr) Hsp 90 failed to restore translocation activity, we conclude that additional, as yet unknown, protein(s) required for translocation were also removed by immunoprecipitation (Fig. 5B). Since geldanamycin and radicicol are well known inhibitors of Hsp 90, we next examined the effect of these two agents on C-domain translocation in vitro. These agents are known to bind to ATPase site of the chaperone and block ATP hydrolysis, thereby inhibiting refolding and release of substrate (Grenert et al, J. Biol. Chem. 272:23843-50, 1997; Schulte et al, Cell Stress Chaperones 3:100-8, 1998). Figure 6 shows that neither the addition of geldanamycin nor radicicol alone was capable of inhibiting C-domain translocation. However, when both inhibitors were used in combination C- domain translocation was inhibited. There are several reports demonstrating the synergistic inhibitory effects of geldanamycin and radicicol on Hsp 90, and inhibition is thought to result from either the disruption of substrate binding or the interaction with co-chaparonins (Rosenhagen et al, supra). This phenomenon appears to be Hsp 90 specific since the addition of hrHsp 90 to geldanamycin / radicicol treated human T cell CTF complexes restored C- domain translocation (Fig. 6). Interestingly, the addition of rhHsp 90 to the geldanamycin / radicicol treated CTF complex from yeast only partially restored C-domain translocation in vitro, suggesting that these agents disrupt a species specific Hsp 82 co-chaperone interaction necessary for reconstitution of translocation activity. Example 4. TrR-1 is essential, but not sufficient for DT C-domain translocation in vitro
Since TrR-1 was also identified by mass spectrometry sequence analysis of CTF complexes from human T cell and yeast extracts, we have used both immunoprecipitation and specific inhibitors to demonstrate a functional role of TrR-1 in the translocation and/or release of the C-domain from early endosomes. As shown in Figure 7 A, immunoprecipitation of TrR-1 from human CTF-complexes and 2',5'-ADP-sepharose affinity chromatographic depletion of yeast TrR-1 from CTF complex mixtures abolished C-domain translocation in vitro. Since there was no significant loss of -ADP- ribosyltransferase activity in the pellet fractions, we conclude that there is no pool of translocated but non-refolded DT C domain. Reconstitution experiments in which bovine recombinant (br) TrR-1 was added back to TrR-1 depleted CTF-complexes, from both T cell and yeast, failed to restore C- domain translocation. Since these experiments were performed under conditions known to reduce the interchain disulfide bond between the C- and T-domains, these results suggest that TrR-1 is a component of a complex and that another factor(s) essential for translocation were co-depleted with TrR-1 (Fig. 7B). We next examined the effect of the TrR-1 stereo-specific inhibitor cis-
13-retinoic acid on C-domain translocation (Schallreuter et al, Biochem. Biophys. Res. Commun. 160:573-9, 1989). The addition of cis- 13 -retinoic acid, but not trans- 13 -retinoic acid, to either human or yeast CTF complex mixtures resulted in the complete inhibition of C-domain translocation in vitro under non-reducing conditions (Fig. 8). Importantly, the addition of excess brTrR-1 to cis- 13 -retinoic treated complex restored C-domain translocation activity in vitro, suggesting that cis-13-retinoic acid inhibition is TrR-1 specific. Finally, when assayed under reducing conditions (20 mM DTT) cis- 13 -retinoic acid had no effect on C-domain translocation. Taken together, these results indicate that TrR-1 activity plays an essential role in the translocation and/or release of the C-domain from early endosomes.
Example 5. Geldanamycin and radicicol , and cis- 13 -retinoic acid protect HUT 102 6TG cells from the cytotoxic action ofDABs89lL-2 Since geldanamycin and radicicol were found to have a synergistic effect in blocking the in vitro translocation of ADP-ribosyltransferase from purified early endosomes, we examined the ability of these agents to protect intact cells from DAB389IL-2. A series of dose response experiments showed that neither the addition of 10 nM geldanamycin nor 10 nM radicicol alone confer protection against the fusion protein toxin. However, as seen in in vitro translocation assays, these agents in combination (10 nM each) were able to affect a two log shift in the DAB389IL-2 dose response curve (IC50 10"8M) compared to the untreated control (IC50 5 x lO"10M). In a similar fashion, cis-13-retinoic acid was found to affect a similar dose dependent shift in the DAB389IL-2 dose response curve for HUT 102 6TG cells, confirming and extending early observations made by Sandvig and Olsnes, supra.
Example 6. Partial Purification of the DT C-domain CTF complex The requirements for DT C-domain translocation across the early endosomal membrane and release into the external milieu were monitored using an in vitro translocation assay modified from Umata et al, J. Biol. Chem. 265:21940-5, 1990 and Lemichez et al, supra. The early endosomal compartment of HUT 102 6TG cells was pre-loaded with DAB389lL-2 in the presence of bafilomycin Al . Early endosomes were purified by sucrose density gradient centrifugation, and then incubated in the presence of ATP and cytosolic extracts from either HUT 102 6TG cells or yeast. Following incubation at 37°C, translocation of the C-domain across the endosomal membrane and release into the external medium was monitored by ADP- ribosyltransferase activity of both the pellet and supernatant fluid fractions following ultracentrifugation. The [32P]-adenosine diphosphate ribosylation (ADPR) of elongation factor 2 (EF2) was measured by autoradiography following SDS-polyacrylamide gel electrophoresis of reaction mixtures (Chung et al, Biochim. Biophys. Ada. 483:248-57, 1977). The limit of sensitivity of this assay is in the range of 10"14 - 10" M C-domain, a level well below that of detection by immunoblot. As shown in Figure 1A, upon dilution of bafilomycin Al and the addition of both ATP and cytosolic extracts to the reaction mixture, the C- domain is translocated across the endosomal membrane and released into the external medium. Moreover, pre-boiling the cytosolic extracts prior to their addition to the reaction mixture abolishes C-domain translocation. These results suggest that the C-domain translocation across the membrane of early endosomes requires cytosolic protein(s). The time course of C-domain translocation was examined using the epitope labeled fusion protein toxin DABI89(VSV-G)B389LL-2. The cytotoxic potency of the epitope tagged fusion toxin is almost identical to that of DAB389IL-2 (IC50 = 3 x 10"nM vs. 4 x 10" M). As shown in Figure IB, the ADP-ribosyltransferase activity as measured by densitometry of the combined [ P]-labeled EF2 from each paired pellet and supernatant fluid fraction is plotted as percent ADP-ribosyltransferase activity in the supernatant fluid. As can be seen, translocation of the C-domain is linear for up to 45 min, at which time ca. 80% of the total activity is found in the supernatant fluid fraction. As previously reported by Lemichez et al, supra, that while ADP-ribosyltransferase activity was translocated to the external medium, co-internalized horseradish peroxidase activity was found to remain in the pellet fraction throughout the incubation period. These results strongly suggest that C-domain translocation is specific, and not the result of spontaneous endosomal lysis during the incubation period. Finally, in the presence of added ATP and cytosolic extracts the translocation of the C- domain is dependent upon membrane fluidity and does not occur at temperatures below 15°C. In order to rule out the possibility that the crude T cell and yeast extracts contained an allosteric regulator(s) of v- ATPase activity rather than protein(s) that are required for C-domain translocation, early endosomes were charged with a 70-kDa dextran conjugated with the pH sensitive fluorescent dye, SNARF-1. As shown in Figure 2A, compared to pH 7.5, the fluorescence emission of 1 ng/ml SNARF-1 is decreased by ca. 4-fold at pH 4.5. As measured by the quenching of fluorescence emission of SNARF- 1 , in vitro acidification of the early endosomal lumen occurs upon dilution of bafilomycin Al and requires the addition of 2 mM ATP to the reaction mixture (Fig. 2B). Moreover, the time course for the acidification of early endosomes in vitro is virtually identical following the addition of either 2 mM ATP or 2 mM ATP plus partially purified T cell CTF complex. It is known that anthrax toxin lethal factor and edema factor, as well as the botulinum toxins (serotypes A-F) follow an analogous route of entry into the cell cytosol as diphtheria toxin. Like diphtheria toxin, these other toxins require binding to their specific cell surface receptor, receptor mediated endocytosis, passage through an acidic early endosomal vesicle compartment, and unfolding of their respective catalytic domain prior to translocation and delivery to the cytosol (Wesche et al, supra; Simpson et al, Journal of Pharmacology & Experimental Therapeutics. 269:256-62, 1994). Since diphtheria toxin C-domain translocation across the endosomal vesicle membrane is mediated by a CTF complex, we hypothesize the presence of an amino acid sequence motif that interacts with component(s) of either CTF complex and/or endosomal vesicle-associated factors in the translocation process. Given the common route of entry of diphtheria toxin, the anthrax toxins, and botulinum toxins, we further hypothesize that this motif would be a common feature among these toxins. In order to test the hypothesis of a common "entry motif might be present, BLAST (Basic Local Alignment Search Tool [Altschul et al., 1990]) analysis was used to compare diphtheria toxin to these other bacterial protein toxins. This analysis (see Figure 9) suggests that there is a conserved region between diphtheria toxin transmembrane helix Tl and each of these other toxins. A 12 amino acid region was further probed by position-specific-iterated (PSI)-BLAST (Karlin and Altschul, 1990) and AlignX (Vector NTI, version 6), and analysis indicated that this short conserved peptide motif is statistically significant. The consensus sequence is Glu-Lys-Xxx-Lys-Thr- Xxx-Xxx-Glu-Xxx-Leu-Lys- Glu, where Xxx is undefined. Figure 10 shows the entry motif that was found for diphtheria toxin, anthrax lethal factor and edema factor, and botulinum toxin serotype A. While not shown in this figure, the entry motif is also found in all other six serotypes of botulinum toxin. As shown in Panel A, we have indicated the relative position of the entry motif on the structure of diphtheria toxin, anthrax lethal factor, anthrax edema factor, and botulinum toxin serotype A. For each toxin, this motif is positioned on the surface of the protein, is an amphipathic alpha helix, and located in a region of the toxin consistent with a potential function in the translocation process. Panel B of Figure 10 shows the motif in an N- to C- terminal orientation directly through the alpha-helix; whereas, Panel C shows the motif in a side view. Taken together, these views of the entry motif reveal a striking conservation of structure and distribution of charge and hydrophobicity. This analysis provides the first indication that diphtheria toxin, anthrax lethal factor and edema factor toxins, and the botulinum toxins can share a common mechanism of entry. There are several lines of evidence that add further support to the hypothesis that the entry motif that we have identified is essential for the productive translocation of at least the C-domains of diphtheria toxin and anthrax lethal factor toxin. In the case of diphtheria toxin, several years ago, we isolated and described a new class of non-toxic mutants of DAB389IL-2. These mutants were found to bind the high affinity IL-2 receptor on the surface of HUT102/6TG cells with affinities comparable to the wild type fusion protein toxin, to retain full -ADP-ribosyltransferase activity, and to form channels in planar lipid bilayers (vanderSpek JC et al, J. Biol Chem. 90:8524-8528, 1993; vanderSpek et al, J. Biol. Chem. 269:21455-21459, 1994). While most of the mutations that were characterized carried small in- frame deletions within the transmembrane domain. One deletion mutant, DAB(Δ204-263)389IL-2, was found to insert into planar lipid bilayers and form a channel identical to that of wild type fusion protein toxin. This non-toxic mutant is >10,000-times less active than the wild type. Further, it is devoid of the first three helices of the transmembrane domain and requires 50 - 100-times more protein than the wild type fusion protein toxin to make channels (vanderSpek JC et al, J. Biol. Chem. 90:8524-8528, 1993). This study clear demonstrates a role for the first three helices in the transmembrane domain (Tl - T3), and most likely that role is two fold: orientation of the T-domain for efficient channel formation and treading the C-domain through the channel. In contrast, the point mutant DAB389(L221E)IL-2 carries a mutation in a highly conserved region of the entry motif. We have shown above that DAB389(L221E)IL-2 binds to the high affinity IL-2 receptor, forms channels in purified early endosomes, and that its C-domain fails to be translocated in vitro and remains in the pellet fraction in the CTF assay system described above. We therefore conclude that both the entry motif on the toxin and the CTF complex are necessary for productive delivery of the C-domain to the cytosol of target cells. In the case of anthrax lethal factor toxin, the entry motif is located between amino acid residues 27 - 39 in the mature protein, a region N- terminal to the PA binding domain (Figure 10). Additional support for the entry motif hypothesis comes from the analysis of anthrax lethal factor by N-terminal deletion mutagenesis. Arora and Leppla, J. Biol. Chem. 268:3334-41, 1993, have demonstrated that the deletion of amino acids 1 - 40 in lethal factor resulted in a complete loss of toxicity for macrophages. More recently, Lacy and Collier, Curr. Top. Microbiol. Immunol. 271 :61-85, 2002, have shown that deletion of the N-terminal 27 amino acids has no effect on binding or translocation of lethal factor to macrophages. The entry motif in diphtheria toxin is positioned in transmembrane helix 1 is composed of amino acids 205 - 225. The amino acid sequence of this region is as follows: Asp205-Tφ-Asp-Val-Ile-Arg-Asp-Lys-Thr-Lys-Thr-Lys- Ile-Glu-Ser-Leu-Lys-Glu-His-Gly-Pro225. A peptide corresponding to the sequence of amino acids 205 to 224 was synthesized and examined for its ability to inhibit DAB389lL-2 intoxication in the same cytotoxicity assay used to evaluate geldanamycin, radicicol, and retinoic acid (see above). The results, shown in Figure 11, indicate that the translocation of diphtheria toxin catalytic domain was inhibited. Compounds of the invention designed to take advantage of this phenomenon include compounds of formula I: X - AA210- AA211 - AA212- AA213- AA214- AA215- AA216- AA217- AA218 - AA219 - AA220 - AA221 - AA222 -Y (I),
where X is H, a nitrogen protecting group, R1-C(0)-, or a chain of amino acids of from 1 to 5 residues substituted at the N-terminus with R!-C(0)-, a nitrogen protecting group, or H; Y is OH, NH2, NHR2, NHR2R3, OR4, or a chain of amino acids of from 1 to 76 residues substituted at the C-terminus with OH, NH2, NHR , NHR R , OR , or a carboxyl protecting group, where R is a -6 alkyl, C6 or C10 aryl, .9 heterocyclyl, -6 alkoxy, C7-16 aralkyl, C2-ι5 heterocyclylalkyl, C7-16 aralkoxy, C2-15 heterocyclyloxy, or a polyethylene glycol moiety; each of R and R is, independently, H, a Cι-6 alkyl, C6 or do aryl, -9 heterocyclyl, C _16 aralkyl, C2-15 heterocyclylalkyl, or a polyethylene glycol moiety, and R4 is H, Cι-6 alkyl, C6 or C10 aryl, -9 heterocyclyl, -6 alkoxy, C -ι6 aralkyl, C2-15 heterocyclylalkyl, a carboxyl protecting group or a polyethylene glycol moiety; -AA is Arg or Lys; AA is Asp or Glu; AA is Lys or Arg; AA213 is Thr, Ser, Ala, Gly, Val, Asn, or Gin; AA214 is Lys or Arg; AA215 is Thr, Ser, Ala, Gly, Val, Asn, or Gin; AA216 is Lys or Arg; AA217 is lie, Leu, or Val; AA218 is Glu or Asp; AA219 is Ser, Ala, or Gly; AA220 is Leu, He, or Val; AA221 is Lys or Arg; and AA222 is Glu or Asp.
Modifications of Compounds of the Invention It is also possible to modify the structure of a compound of the invention for such purposes as enhancing therapeutic or prophylactic efficacy, or stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo). Such modified peptides, when designed to retain at least one activity of the naturally- occurring form of the protein, are considered functional equivalents of CTF.
Such modified peptides can be produced, for instance, by amino acid substitution, deletion, or addition.
For example, in a compound of the invention that inhibits translocation from the endosome to the cytosol of a cell (e.g., a peptidyl inhibitor of the invention), it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the ability of the peptide to serve as an inhibitor. Conservative replacements or substitutions are those that take place within a family of amino acids that are related in their side chains, and apply to those that result from genetically encoding or those that are synthetically produced. Amino acids can be divided into four families: (1) acidic residues, such as aspartatic acid or glutamic acid; (2) basic residues, such as lysine, arginine, or histidine; (3) nonpolar residues, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan; and (4) uncharged polar residues, such as glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In similar fashion, the amino acid repertoire can be grouped as (1) acidic residues, such as aspartate, glutamate; (2) basic residues, such as lysine, arginine histidine, (3) aliphatic residues, such as glycine, alanine, valine, leucine, isoleucine, serine, threonine, with serine and threonine optionally be grouped separately as aliphatic- hydroxyl; (4) aromatic residues, such as phenylalanine, tyrosine, tryptophan; (5) amide residues, such as asparagine, glutamine; and (6) sulfur-containing ' residues, such as cysteine and methionine (see, for example, Biochemistry, 2nd ed., Ed. by L. Stryer, W H Freeman and Co.: 1981). Alternatively, amino acid replacement can be based on steric criteria, e.g. isosteric replacements, without regard for polarity or charge of amino acid sidechains. Thus, one or more amino acid residues in a compound of the invention, can be replaced with another amino acid residue from the same family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a nucleic acid encoding a compound of the invention, such as by saturation mutagenesis, and the resultant mutants can be screened for their ability to inhibit translocation, by methods described herein. Following mutagenesis of the nucleic acid encoding the CTF peptide, the peptide can be expressed by any recombinant technology known in the art, and the activity of the peptide can be determined. The compounds of the present invention include analogs that contain moieties that improve pharmacodynamic properties, such as, for example, those that increase in vivo half-life; or that improve physical properties, such as, for example, increased resistance to in vivo degradation or increased cell- membrane permeability. In one example, polymer vehicles may be used to modify the compounds of the present invention. Various means for attaching chemical moieties useful as vehicles are currently available, see e.g., Patent Cooperation Treaty ("PCT") International Publication No. WO 96/11953, entitled "N- Terminally Chemically Modified Protein Compositions and Methods". This PCT publication discloses, among other things, the selective attachment of water soluble polymers to the N-terminus of proteins. A preferred polymer vehicle is polyethylene glycol (PEG). The PEG group may be of any convenient molecular weight and may be linear or branched. The average molecular weight of the PEG will preferably range from about 2 kiloDalton ("kDa") to about 100 kDa, more preferably from about 5 kDa to about 50 kDa. The PEG groups will generally be attached to the compounds of the invention via acylation or reductive alkylation through a reactive group on the PEG moiety (e.g., an aldehyde, amino, isothiocyanate, or an activated carboxylic acid) to a reactive group on the inventive compound (e.g., an amino, or activated carboxyl group). A useful strategy for the PEGylation of synthetic peptides consists of combining, through forming a conjugate linkage in solution, a peptide and a
PEG moiety, each bearing a special functionality that is mutually reactive toward the other. The peptides can be prepared by solid phase synthesis, as described herein. Through selective deprotection strategies, the peptides are
"preactivated" with an appropriate functional group at a specific site. The precursors can be purified and fully characterized prior to reacting with the
PEG moiety. Ligation of the peptide with PEG usually takes place in aqueous phase and can be easily monitored by reverse phase analytical HPLC. In a desirable embodiment, the PEG moiety contains functionality reactive towards functional groups contained on biomolecules (e.g. proteins, aminoglycosylglycans), making this moiety a heterobifunctional crosslinker.
Preferably, the reactive functionality on the PEG moiety is a maleimide, vinyl carbonyl, vinyl sulfonyl group, or alpha-halocarbonyl, and is reacted with a biomolecule containing a free thiol. Such reactions are extremely facile and can be performed a low reactant concentrations, such as are found in in vitro experiments or in vivo. Other bifunctional agents are known to be useful for cross-linking the peptides or their functional derivatives to a water-insoluble support matrix or to other macromolecular vehicles. Commonly used cross-linking agents include, for example, l,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N- hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N- maleimido-l,8-octane. Derivatizing agents such as methyl-3-[(p- azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Also included are alkyl linkers such as -NH-(CH2)5C(0)-. These alkyl linkers may further be substituted by any non-sterically hindering group such as d-6 alkyl, C2- acyl, halogen (e.g., Cl, Br), CN, NH , aryl, heterocyclyl, etc. Other linkers include those made up of amino acids linked together by amide bonds. In one example, the linker is made up of from 1 to 20 amino acids linked by amide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those in the art. In a more preferred embodiment, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. Even more preferably, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Thus, preferred linkers are polyglycines (particularly (Gly , (Gly)5), poly(Gly-Ala), and polyalanines. Other specific examples of linkers are: (Gly)3Lys(Gly)4; (Gly)3AsnGlySer(Gly)2; (Gly)3Cys(Gly)4; and GlyProAsnGlyGly. In some examples, the peptide linker is designed to be cleaved in vivo at a specific dipeptide amide bond by proteolytic enzymes. Polysaccharide polymers are another type of water soluble polymer which may be used for modification of the compounds of the invention. Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by αl-6 linkages. The dextran itself is available in many molecular weight ranges, and is readily available in molecular weights from about 1 kD to about 70 kD. Dextran is a suitable water soluble polymer for use in the present invention as a vehicle by itself or in combination with another vehicle (see, for example, WO 96/11953 and WO 96/05309). The use of dextran conjugated to therapeutic or diagnostic immunoglobulins has been reported; see, for example, European Patent Publication No. 0 315 456. Dextran of about 1 kD to about 20 kD is preferred when dextran is used as a vehicle in accordance with the present invention. Other carbohydrate (oligosaccharide) groups may conveniently be attached to sites that are known to be glycosylation sites in proteins. Generally, 0-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides are attached to asparagine (Asn) residues when they are part of the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids other than proline. The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar that is commonly found on both is N-acetylneuraminic acid (referred to as sialic acid). Sialic acid is usually the terminal residue of both N- linked and O-linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycosylated compound. Such site(s) may be incorporated in the linker of the compounds of this invention and are preferably glycosylated by a cell during recombinant production of the polypeptide compounds (e.g., in mammalian cells such as CHO, BHK, COS). However, such sites may further be glycosylated by synthetic or semi-synthetic procedures known in the art. In other examples, a peptide of the invention can be modified by the replacement of one or more peptidyl (-C(O)NR-) linkages (bonds) by a non- peptidyl linkage. Exemplary non-peptidyl linkages are -CH2-carbamate (-CH2- OC(O)NR-), phosphonate, -CH2-sulfonamide (-CH2-S(0)2NR-), urea (- NHC(O)NH-), -CH2-secondary amine, and alkylated amide [-C(0)NRA- wherein RA is alkyl).
In other examples, one or more individual amino acid residues can be modified. Various derivatizing agents are known to react specifically with selected sidechains or terminal residues. For example, lysinyl residues and amino terminal residues may be reacted with succinic or other carboxylic acid anhydrides, which reverse the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
Arginyl residues may be modified by reaction with any one or combination of several conventional reagents, including phenylglyoxal, 2,3- butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginyl residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group. Specific modification of tyrosyl residues has been performed, with examples including introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3 -nitro derivatives, respectively. Carboxyl sidechain groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R-N=C=N-R') such as 1-cyclohexyl- 3-(2-morpholinyl-(4-ethyl) carbodiimide or l-ethyl-3-(4-azonia-4,4- dimethylpentyl) carbodiimide, followed by reaction with an amine to form an amide. Compounds of the present invention may be changed at the DNA level as well. The DNA sequence of any portion of the compound may be changed to codons more compatible with the chosen host cell. Codons may be substituted to eliminate restriction sites or to include silent restriction sites, which may aid in processing of the DNA in the selected host cell. The vehicle, linker and peptide DNA sequences may be modified to include any of the foregoing sequence changes.
Compounds Containing Nucleic Acids Encoding Peptide Sequences of the Invention The present invention provides a method of inhibiting cell death in a mammal by administering to the cell a nucleic acid sequence that encodes a peptide that prevents the translocation of the catalytic domain of a toxin from the lumen of endosomes to the cytosol of a mammalian cell. Examples are peptides that include the amino acid sequences of SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO.7, and SEQ ID NO.7. In another aspect, the present invention provides a method of inhibiting cell death in a mammal by administering to the cell a nucleic acid sequence that encodes a peptide that prevents the translocation of a viral or retroviral transcription factor from the lumen of endosomes to the cytosol of a mammalian cell. Examples are peptides that include the of amino acid sequences of SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO.7, and SEQ ID NO.7. The nucleic acid sequences of the present invention or portions thereof can be inserted into a vector used to propagate the sequences in a cell. Such vectors are introduced into cells (e.g., prokaryotic or eukaryotic), and the cells are propagated to produce multiple copies of the vector. A useful type of vector is an expression vector. Coding regions of the nucleic acid sequences of the present invention or fragments thereof can be inserted into an expression vector under conditions appropriate for expression of the sequences. Such vectors, are introduced into cells (e.g., prokaryotic or eukaryotic cells) under conditions appropriate for expression. In one embodiment, the cell is eukaryotic (e.g., mammalian, avian, insect, or yeast). In a preferred embodiment, the cell is human. The invention thus provides nucleic acid constructs which encode the various peptide compounds, or fragments thereof, of the invention, various DNA vectors containing those constructs for use in transducing prokaryotic and eukaryotic cells, cells transduced with the nucleic acids, fusion proteins encoded by the above nucleic acids, and target gene constructs. Each of the nucleic acids of this invention may further contain an expression control sequence operably linked to the coding sequence and may be provided within a DNA vector, e.g., for use in transducing prokaryotic or eukaryotic cells. Some or all of the nucleic acids of a given composition, including any optional nucleic acids, may be present within a single vector or may be apportioned between two or more vectors. In certain embodiments, the vector or vectors are viral vectors useful for producing recombinant viruses containing one or more of the nucleic acids. The recombinant nucleic acids may be provided as inserts within one or more recombinant viruses which may be used, for example, to transduce cells in vitro or cells present within an organism, including a human or non-human mammalian subject. For example, nucleic acids encoding peptides or peptidyl fragments of the present invention may be present within a single recombinant virus or within a set of recombinant viruses, each of which containing one or more of the set of recombinant nucleic acids. Viruses useful for such embodiments include any virus useful for gene transfer, including adenoviruses, adeno-associated viruses (AAV), retroviruses, hybrid adenovirus-AAV, herpes viruses, lenti viruses, etc. In specific embodiments, the recombinant nucleic acid containing the target gene is present in a first virus and one or more or the recombinant nucleic acids encoding the transcription regulatory protein(s) are present in one or more additional viruses. In such multiviral embodiments, a recombinant nucleic add encoding a fusion protein containing a bundling domain and a transcription activation domain, and optionally, a ligand binding domain, may be provided in the same recombinant virus as the target gene construct, or alternatively, on a third virus. It should be appreciated that non- viral approaches (naked DNA, liposomes or other lipid compositions, etc.) may be used to deliver nucleic acids of this invention to cells in a recipient organism.
In one example, a plasmid was created (see Figure 12 and SEQ ID NO. 1) in which the segment of the diphtheria toxin structural gene encoding amino acids 210-299, which contains the entry motif which is the basis for the compounds of the invention, was cloned into the expression vector pTRACER (obtained from Invitrogen) and placed under control to the CMV promoter to form plasmid: pTl . pTl was subsequently used to transfect HUT102 cells. A stable transfectant clone was purified and designated HUT102-T1.
The relative sensitivity of both HUT- 102 and HUT102-T1 cells to the fusion protein toxin DAB389IL-2 was examined by dose response analysis. As shown in Fig. 13 A, HUT 102 cells (closed circles) were sensitive to DAB389IL-2 with an IC50 of about 2 x 10"10M. In contrast, HUT102-T1 (closed squares) were completely resistant to the action of DAB389IL-2 to concentrations greater than 10"8M. HUT 102 cells transfected with an empty vector (pTRACER) were as sensitive to DAB389IL-2 as HUT 102 cells devoid of the vector (IC50 of about 1 x 10"10M). In addition, knock out of the diphtheria toxin 210-299 amino acid region in HUT102-T1 cells also resulted in the loss of resistance to DAB389IL-
2. In another experiment, shown in Figure B, the sensitivity of both
HUT102 cells (closed circles) and HUT102-T1 cells (closed squares) to Pseudomonas exotoxin A, a bacterial protein toxin known to enter the cell at the level of the trans-golgi rather than from an acidified early endosome. In this instance, both cell lines were equally sensitive to the action of exotoxin A. It is noteworthy that both DAB389IL-2 and Pseudomonas exotoxin A catalyze the ADP-ribosylation of elongation factor 2 by precisely the same mechanism. These experiments show that cells transfected with a nucleic acid vector for a peptide sequence of the invention, which includes the entry motif, are resistant to the action of the diphtheria toxin related DAB3891L-2. Since a toxin that enters the cell by another route of entry was not effected by the presence of this peptide sequence, we conclude that expression of the peptide in the cytosol results in a competitive inhibition of diphtheria toxin catalytic domain entry into the cytosol. The invention also provides methods for rendering a cell capable of regulated expression of a target gene which involves introducing into the cell one or more of the nucleic acids of this invention to yield engineered cells which can express the appropriate fusion protein(s) of this invention to regulate transcription of a target gene. The recombinant nucleic acid(s) may be introduced in viral or other form into cells maintained in vitro or into cells present within an organism. The resultant engineered cells and their progeny containing one or more of these recombinant nucleic acids or nucleic acid compositions of this invention may be used in a variety of important applications, including human gene therapy, analogous veterinary applications, the creation of cellular or animal models (including transgenic applications) and assay applications. Such cells are useful, for example, in methods involving the addition of a ligand, preferably a cell permeant ligand, to the cells (or administration of the ligand to an organism containing the cells) to regulate expression of a target gene. Particularly important animal models include rodent (especially mouse and rat) and non-human primate models. The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. In certain gene therapy applications, the cells are human and a nucleic acid of the present invention is operably linked to an inducible promoter. Such inducible promoters are known to those skilled in the art. For example, the tetracycline-inducible system of Gossen and Bujard (Proc. Natl. Acad. Sci. USA 89:5547-5551, 1992; U.S. Pat. No. 5,464,758), has been used to regulate inducible expression of several genes (Furth et al. Proc. Natl. Acad. Sci. USA 91:9302-9306, 1994; Howe et al. J. Biol. Chem. 270:14168-14174, 1995; Resnitzky et al. Mol. Cell. Biol. 14:1669-1679, 1994; Shockett et al. Proc. Natl. Acad. Sci. USA 92:6522-6526, 1995). This system uses a chimeric transcription factor, termed tTA, which is composed of the repressor of Escherichia coli (E. coli) tetracycline-resistance operon (tetR) and the activation domain (carboxyl terminal domain) of virion protein 16 (VP16) of herpes simplex virus (HSV) (Triezebberg et al. Genes Dev. 2:718-729, 1988). The gene of interest is placed downstream of a minimal cytomegalovirus (CMV) 1 A promoter, derived from the immediate early CMV genes, which is linked to multiple copies of tetO, the binding site for the tetracycline repressor tetR. In the absence of tetracycline, the tetR portion of the transactivator binds the tetO sequences of the promoter and the VP16 portion facilitates transcription. When tetracycline is present, tetracycline binds the tetR portion of tTA, which in turn prevents binding of the tetR portion to the tetO sequence(s) of the promoter, thus inhibiting transcription. Since even low concentrations of tetracycline are sufficient to block tTA function, and since most mammalian cells can tolerate tetracycline, this system provides a tightly regulated on/off switch for gene expression that can be controlled by varying the tetracycline concentration to which the cells are exposed. This work has been extended by Yee et al, U.S. Patent No. 6,432,705, who describe an inducible promoter activated by a multi-chimeric transactivator that is particularly in the expression of retro viral vectors. A variety of other regulatable expression systems have been described involving allostery-based switches triggered by tetracycline, RU486 or ecdysone, as well as dimerization-based switches triggered by dimerizing agents such as rapamycin, coumermycin, dimers of FK506, synthetic FKBP- binders and/or CsA, or analogs thereof (see, for example, Clackson, Current Opinion in Chemical Biology, 1:210-218, 1997) U.S. Patent No. 6,566,073 describes methods for producing target proteins in vivo using fusion proteins containing conditional retention domains. Illustrative examples of ligand binding domain/ligand pairs include retinol binding protein or variants thereof and retinol or derivatives thereof; cyclophilin or variants thereof and cyclosporin or analogs thereof; FKBP or variants thereof and FK506, FK520, rapamycin, analogs thereof or synthetic FKBP ligands.
Methods of Inhibiting Cell Death by Administration of Peptides of the Invention The present invention also provides methods of inhibiting cell death in a mammal, preferably a human, by administering to the cell a compound of the invention, or analog thereof, which prevents the translocation of the catalytic domain of a toxin from the lumen of endosomes to the cytosol of the cell. In one example, the toxin is an AB toxin, such as, for example Diphtheria toxin, one of the seven serotypes of Botulinum toxin, Anthrax toxin LF, or Anthrax toxin EF. In another embodiment, the compound inhibits the translocation of a viral or retroviral transcription factor, such as, for example, human immunodeficiency virus (HIV-1) reverse transcriptase or Tat. Compounds of the invention include peptide sequences that contain the entry motif consensus sequence. Compounds of the invention also include peptidyl compounds that are further modified to improve their pharmacological properties, as described in detail herein. The inventions also features compounds that include nucleic acid sequences that encode a peptide that contains the entry motif peptide sequence.
Methods of Screening Biologically Active Compounds
In one example, a method of identifying a compound that inhibits cell death in a mammal includes the following steps: a) isolating endosomes from said cell; b) placing said endosomes in a cytosolic buffer; c)contacting said endosomes with a fusion protein-toxin, wherein said protein comprises a binding moiety for a component of the cell membrane of said cell and said toxin comprises a fragment of Diphtheria toxin; d)contacting said endosomes with a cytosolic translocation factor complex; e) contacting said endosomes with said compound; and e)measuring translocation of said toxin, wherein an decreased level of said translocation relative to that observed in the absence of said compound indicates that said compound inhibits said cell death. In another example, a method of identifying a compound that inhibits cell death in a mammal includes the following steps: contacting a mammalian cell or cell population with a fusion protein-toxin, where the protein has a binding moiety for a component of mammalian cellular membranes and where the toxin contains a fragment of Diphtheria toxin that includes the catalytic domain; introducing a cytosolic translocation factor complex (e.g., one that includes a compound of the invention) to the cytosol of the cell(s); contacting the cell(s) with a test compound; and measuring cell death relative to a control cell or cell population which has been similarly treated with fusion protein toxin and a cytosolic translocation factor complex, but not treated with the test compound. A decreased rate of cell death in a cell population treated with the test compound indicates that the compound may interfere with translocation of the toxin. This result can be subsequently confirmed in the more sensitive endosome test described above. In one example, measuring cell death includes a FACS analysis. Introduction of a test compound and/or the CTF complex can be accomplished by treating the cell or cell population with the compound and waiting for passive diffusion through the cell membrane to the cytosol. If necessary, aids to passive transport (e.g., agents that increase cell permeability) can be used. One method for introducing proteins or peptides into the cells of a mammalian cell culture is the Chariot™ reagent (Morris et al., Nature Biotechnology 19:1173-1176, 2001; available from Active Motif, Carlsbad, CA. This reagent quickly and efficiently delivers biologically active proteins, peptides and antibodies directly into cultured mammalian cells at an efficiency of 60-95%. Less than two hours after delivery, live cells can be assayed to determine the effects of the introduced materials, without the need for fixing. In addition to the introduction of the compounds of the invention into the cultured cells, the use of this reagent also aids in the cellular uptake of the compound to be screened, as well as reporter construct. The Chariot reagent can be used in the presence or absence of serum and is independent of the endosomal pathway, which can modify macromolecules during internalization. Additionally, the use of this method for introducing a protein or peptide bypasses the transcription-translation process, which reduces the time required to complete the assay from overnight to less than two hours.
Materials and Method
Cell Culture. HUT1026TG cells (ATCC TIB 1620), were maintained in RPMI 1640 (Bio-Whittaker) supplemented with 10% fetal bovine serum (Hyclone Labs), 2 mM glutamine (Bio-Whittaker), 50 IU/ml penicillin and 50 μg/ml streptomycin (Bio-Whittaker) at 37°C in 5% C02. Yeast strain NLY22" (gift from Dr. Kevin Jarrel, Modular Genetics) was maintained in YPD media (Difco) and on YPD agar plates at 30°C. Purification ofEF-2 EF-2 was partially purified using a procedure by Chung et al, supra. Following purification, fractions containing EF-2 were identified by ADP- ribosyltransferase using DAB389IL-2 (see in vitro RA). EF-2 was further purified by DEAE-Sepharose (Reactifs IBF) anion exchange chromatography. EF-2 was eluted with a linear gradient, 0-200 mM NaCl, in 50 mM Tris-HCl pH 8.0, 50 mM Mg(OAc)2, 0.1 M KCl, 4 mM CaCl2, 5 mM 2-ME and 1 μg phenylmethylsulfonyl fluoride (PMSF) (Sigma) per ml. Fractions containing EF-2 were identified as above. Aliquots were adjusted to a final concentration of 2 mM DTT, 5% glycerol, and stored at -70°C. Purified EF-2 was approximately 80% homogeneous as resolved by 7% SDS-PAGE and stained with colloidal Coomassie (Invitrogen). Protein concentration was determined by Bradford Assay according to standard protocols using Coomassie Protein Assay Reagent (Pierce).
Purification of Early Endosomes
Early endosomes were isolated from HUT102/6TG cells according to a protocol by Dubrez et al. (1986). The early endosomal compartment was loaded with DAB389IL-2(1 μM), DA189(Vsv-G)B389lL-2(l μM), SNARF1 -dextran (70 kD) conjugate (Molecular Probes) (8mg/ml), and/or horse radish peroxidase (Sigma) (5mg/ml) by using bafilomycin Al (Sigma) (1 μM) primed cells.
Purification ofHUT102/6TG CTF Complex Crude cytosolic extract was isolated from HUT 102/6TG cells according to the protocol modified from Bomsel et al, Cell 62:719-31, 1990. Briefly, cells were washed three times with cold PBS containing 5 mg/ml BSA, once with cold PBS alone, and twice with cold cytosol buffer (CB; 3% sucrose in 100 mM HEPES-KOH pH 7.9, 1.4 mM KC1„ 30 mM MgCl2, 2 mM EDTA, 5 mM DTT). Cells were lysed by 20 passages through a 25 G needle in CB containing protease inhibitors: 10 μg/ml aprotinin (Sigma), 1 μg/ml pepstatin (Sigma), 1 μg/ml antipain (Sigma), and 1 μm PMSF (Sigma). The lysate was centrifuged at 1,000 x g for 15 min at 4°C. The post-nuclear supernatant was then centrifuged at 170,000 x g for 1 hr at 4°C. The supernatant fraction was dialyzed overnight at 4°C against cytosol dialysis buffer (CDB; 1% sucrose in 20 mM TRIS-HC1 pH 8.0, 2mM EDTA, 2mM 2-ME) containing protease inhibitors as previously described. Crude cytosol was fractionated according to standard chromatographic protocols. Briefly, crude extract was loaded onto an in-house packed DEAE- Sepharose (Reactifs IBF) XK 26 column (Amersham Pharmacia) for anion exchange chromatography. A peristaltic FPLC pump P-l (Amersham Pharmacia) and Single Path Monitor UV-1 (Amersham Pharmacia) were used during chromatography. The column was pre- equilibrated with buffer B3: 50 mM Tris-HCl pH 8.0, 1 mM EDTA, 5 mM 2-mercaptoethanol, and 1 μg PMSF per ml, and 'loaded' sample was washed using the same buffer. CTFs were eluted with a linear gradient, 0-400 mM NaCl, in buffer B3 at a flow rate of 5 ml/min. Fractions containing CTFs were identified using an in vitro TA and in vitro RA in series (see below). Fractions containing in vitro translocation activity eluted between 150 to 190 mM NaCl and were pooled, and concentrated using YM-10 Centriplus Centrifugal Filters (Amicon) according to manufacturer's directions. Protein concentration was determined as previously described. CTFs were next fractionated by size exclusion chromatography using
Sephacryl S200 (Amersham Pharmacia) XK 26 column (Amersham Pharmacia) equilibrated with buffer B3. A single Path Monitor UV-1 (Amersham Pharmacia) was used to monitor chromatography. Sample loads of 5 ml were isocratically eluted in buffer B3. Flow rate was gravitationally 2005/014798
determined at approximately 2 ml per min. Resolution of the mobile phase was monitored by 7 - 12% SDS-PAGE electrophoresis and staining with colloidal Coomassie. CTFs were identified as previously described, and correlated with elution of 100 to 250 kDa sized proteins, but contained proteins as small as 20 to 25 kDa when visualized by 7%- 12% SDS-PAGE and stained with colloidal Coomassie. Partially purified CTFs were further purified by anion exchange chromatography using a MONO Q HR 5/5 column (Amersham Pharmacia) on a Biosys2000 HPLC (Beckman). The column was pre-equilibrated with buffer B4: 50 mM Tris-HCl pH 8.0 and 1 mM EDTA. Sample loads of 2 ml were washed using buffer B4 and CTFs were eluted using serial hyperbolic step gradients, 0 to 1.0 M NaCl in buffer B4 at a flow rate 2 ml/min. CTFs were identified as previously described and eluted at a conductance of 27.3 mS. Translocation in vitro competent fractions were pooled, dialyzed against 50 mM Tris HCl pH 7.4, 1% sucrose overnight at 4°C, and then concentrated using YM-10 Microcon Centrifugal Filters (Amicon) according to manufacturer's directions. Protein concentration was determined as previously described. Controls indicated that the purified CTF complex had no intrinsic ADP-ribosyltransferase activity.
Purification ofNLY22~ CTF Complex
Yeast crude cytosolic extract was isolated using the same procedure described above for HUT 102/6TG cells except NLY22" cells were lysed by vortexing cells with 212-300 micron glass beads (Sigma). Cell lysis was monitored by decrease in exclusion of Trypan Blue Dye (Gibco BRL). Controls indicated that the purified CTF complex had no intrinsic -ADP- ribosyltransferase activity In vitro Translocation Assay (TA) Translocation of the C domain was carried out using protocol modified by Lemichez et al, supra: 25 μl reaction mixtures containing 4 μl early endosomes in translocation buffer (TB; 50 mM Tris-HCl pH 7.4, 25 mM EDTA). For reducing conditions, TB contained 20 mM DTT. For non- reducing conditions, TB contained 10 μM NADPH (Alexis). ATP and cytosol were added to 2 mM and 5.0 to 0.09 μg/μl as indicated, respectively. Translocation mixtures were incubated at 37°C for 30 min., and the supernatant fluid and pellet were separated by ultra-centrifugation at 180,000 x g at 4°C for 20 min. The pellet fraction was resuspended in 25 μl TB containing 0.2% Triton x-100 (Sigma) and both the lysed pellet and supernatant fluid were boiled for 5 min. The inhibitors geldanamycin (Alomone Labs), radicicol (Sigma), cis- 13 -retinoic acid (Sigma), and trans- 13 -retinoic acid (Sigma) were added as indicated. hrHsp 90 (StressGen), brTrR-1 (American Diagnostica Inc.), and hrTrx (American Diagnostica Inc.) were added as indicated. The membrane integrity of purified early endosomes in the assay system was verified using horse radish peroxidase as previously described by Lemichez et al, supra.
In vitro ADP-Ribosylation Assay (RA) The in vitro NAD dependent ADP-ribosylation of EF-2 was performed according to a protocol by Chung et al, supra. Reaction mixtures contained 3 pM [32P]-NAD+ (800 μCi/mmol, Dupont-NEN), and when indicated 1 mM ATP and/or 0.5 mg/ml crude HUT102/6TG cytosol. Where indicated, autoradiographic signals on X-OMAT AR film (Kodak) were analyzed by ImageQuant Software (molecular Dynamics) and Kodak ID Software (Kodak) according to manufacturer's directions. Immunoprecipitation and Affinity Chromatography Immunoprecipitation of human Hsp 90 (both α and β), yeast Hsp 82 and human TrR-1 were performed according to standard protocols using rabbit IgG polyclonal anti-human Hsp 90 antibodies (Santa Cruz Biotechnology), rabbit polyclonal anti-Hsp82 antiserum (a gift from S. Lindquist), and rabbit polyclonal anti-human- TrR-1 antibodies (Upstate). Antibody was first crosslinked to Protein A-Agarose (Santa Cruz Biotechnology) prior to IP. In each instance, 2-4 μg rabbit polyclonal was incubated with 100 μl or 200 μl of resuspended volume of Protein A-Agarose in 50 mM Tris HCl, 1 mM EDTA containing 1% NP-40 and 100 mM NaCl on a rocker overnight at 4°C. Bound antibody was collected by centrifugation at 1,000 x g for 5 min at 4°C, and washed 2x with 10 c.v. with 0.2 M sodium borate (Sigma) pH 9.0 for 5 min at 25 C. Dimethyl Pimelimidate.2HCl (Sigma) were added to a final concentration of 20 mM and the reaction mixture incubated for 30 min. at 25 C. Crosslinked antibody was pelleted by centrifugation at 1,000 x g for 5 min at 4°C, and the pellet was washed 2x with 10 c.v. 0.2 M ethanolamine (Sigma) for 30 min. at 25 C, and 2x with PBS for 30 min. at 25 C. Immunoprecipitations using the cross-linked antibody agarose conjugates were performed according to standard protocols. Briefly, 200 μl of MONO-Q partially purified CTFs (approximately 0.1 μg/μl) in 50 mM Tris HCl, 1% sucrose, containing 1% NP-40 and 25 mM NaCl was incubated with 20 μl of antibody-agarose conjugate on a rocker overnight at 4°C. IPs were collected by centrifugation at 1,000 x g for 5 min at 4°C, and supernatant fluid was evaluated in the in vitro TA. Pellet was washed 3x with 100 μl cold 50 mM Tris HCl, 1 mM EDTA containing 1% NP-40 and 50 mM NaCl, and resuspended in 50 μl lx SDS-PAGE loading buffer and boiled for 5 min. Antibody-agarose beads were pelleted by centrifugation at 1,000 x g for 5 min at 25 C and the supernatant was analyzed by 10% SDS-PAGE, stained with colloidal Coomassie, and selected bands were evaluated by MS as described below. Yeast TrR-1 was affinity purified using 2 ',5' ADP-Sepharose agarose (Amersham Biosciences) using a protocol modified from Hunt et al, Eur. J. Biochem. 131:303-11, 1983. Briefly, 20 μg of 2',5' ADP-Sepharose agarose was washed 2x with 200 μl 50 mM Tris HCl, 1 mM EDTA for 20 min. MONO-Q partially purified CTFs (200 μl of approximately 0.1 μg/μl) in 50 mM Tris HCl, 1 mM EDTA, 1% Sucrose, 25 mM NaCl was incubated with 2',5' ADP-Sepharose on a rocker overnight at 4°C. Affinity purified TrR-1 was collected by centrifugation at 1,000 x g for 5 min at 4°C. The supernatant fluid was assayed for translocation activity in vitro. The pellet was washed 2x in 100 μl 50 mM Tris HCl pH 7.5, 1 mM EDTA, 1% sucrose, and then resuspended in 50 μl 50 mM Tris HCl pH 7.5, 1 mM EDTA, 1% sucrose, containing 20 μM NADPH and incubated for 2 hours at 25 C. The supernatant fluid was collected following centrifugation at 1,000 x g for 5 min at 4°C and the supernatant fluid was analyzed by 10% SDS-PAGE, stained with colloidal Coomassie, and selected bands were evaluated by MS as described below.
Western Blots
Confirmation of CTF identification by MS was performed by western blot analysis according to standard protocols. In addition to using antibodies described previously, horse polyclonal anti-DT antibody (Massachusetts Antitoxin and Vaccine Laboratories) was used. Briefly, samples were analyzed by 7-12% SDS-PAGE, transferred to Immobilon-P (Millipore), probed with the appropriate primary and secondary antibodies, and detected using either 3,3'- Diaminobenzidine (Sigma) or ECL (Amersham-Pharmacia) according to the manufacturer's directions. In gel' Reduction, Alkylation, and Digestion of Partially Purified CTFs
The preparation of partially purified CTFs for identification by MS was performed using a modified procedure from Shevchenko et al, Anal. Chem. 68:850-8, 1996. Briefly, partially purified CTFs were separated by 10% SDS- PAGE, stained with colloidal Coomassie, and selected bands were excised and chopped into small pieces. Gel pieces were washed 3x in 50 mM ammonium bicarbonate (Sigma) in 50%> acetonitrile (ACN; Acros) for 20 min at 25 C. Gel pieces were washed with 100% ACN for 10 min at 25 C. Supernatant was discarded, and the gel pieces were dried in a SpeedVac for 15 min. Gel pieces were reduced in 20 mM DTT, 50 mM ammonium bicarbonate, and 5%> ACN for 1 hr at 55°C. Supernatant was discarded and the pieces were washed with 100 μl 50 mM ammonium bicarbonate for 10 min at 25 C and subsequently with 100 μl 100% ACN for 10 min. at 25 C.
Gel pieces were alkylated in 100 μl 100 mM iodoacetamide (ICN Biomedicals Inc), 50 mM ammonium bicarbonate for 30 min. in the dark at 25 C. Supernatant fluid was discarded and the pelleted pieces were washed with 100 μl 50 mM ammonium bicarbonate for 10 min at 25 C and subsequently dried with 100 μl 100% ACN for 10 min. 25 C. The wash and drying steps were repeated before drying the pieces in a SpeedVac for 15 min. Gel pieces were rehydrated in digestion buffer [50 mM ammonium bicarbonate] and MS Sequencing Grade Trypsin (Roche Diagnostics Gmbh) at an estimated 1:100 enzyme to substrate ratio on ice for 45 min. Ammonium bicarbonate (50 mM) was added when necessary to keep the gel pieces wet. Digestions were incubated for 6-8 hours at 37°C. Peptides were extracted from the gel pieces using 100 μl 20 mM ammonium bicarbonate for 20 min, followed by 2x 200 μl 1% TFA in 50% ACN for 20 min, and finally lx 100 μl 100% ACN for 10 min. Supernatant fluids were pooled and dried in a SpeedVac. The pellets were resuspended in 0.1% TFA and desalted using ZipTipcι8 pipette tips (Millipore) according to manufacturer's directions. Capillary HPLC of tryptic peptides HPLC was performed using an LC Packings (Dionex Corp.) capillary LC system composed of a Famous autosampler, a Switchos microcolumn switching unit and an Ultimate pump. Sample loads of 5 μL were preconcentrated and desalted online with a "small molecule" C18 CapTrap (Michrome Bioresources) using a solution of 5% FA, 0.1% TFA at a flow rate of 50 μL per min. for 4 min. Capillary HPLC columns were prepared in house as follows: 300 μm ID x 15 cm fused silica capillaries were pressure bomb- packed (Mass Evolution, Inc) at 2000 PSI with Magic C18-3 μm-200 A pore reversed phase packing material (Michrome Bioresources) using 2-propanol as a carrier solvent. Columns were washed with 10% acetic acid, followed by methanol, then the HPLC mobile phase prior to use at a flow rate of 2 μL per min. Elution was by linear gradient; 95% A (5% ACN, 0.1% formic acid) to 55% B (85% ACN, 10% 2-propanol, 0.1% formic acid) over 50 min. followed by 60 min. of column regeneration.
MALDI and ESI Mass Spectrometry (MS), tandem MS, and LC-MS/MS MALDI MS were acquired in positive polarity on a Bruker Reflex IV mass spectrometer with delayed extraction in the reflectron mode using a UV nitrogen laser. A laser power of 28-45% was used and 50 to 100 laser shots were summed for each spectrum. The matrix used was 2,5-dihydroxybenzoic acid (Sigma). Data was analyzed using Bioanalyst™ (Applied Biosystems Inc.) reconstruction algorithms. For initial screening and searches, acquired mass values were compared to theoretical protein digests using the Mascot search engine (Matrix Science Ltd). Reported scores, based on a probability of match, were statistically significant for each protein identified in Table 1. ESI MS and MS/MS were performed using an Applied Biosystems
QSTARi Pulsar ESI quadrupole/orthogonal acceleration time-of-flight mass spectrometer (QoTOF MS). MS and MS/MS were acquired in the positive polarity mode over the range of m/z 320-1800 (MS) and m/z 100-1800 (MS/MS) with resolution > 1 :9,000 (fwhm) and better than 50 ppm mass accuracy (external calibration). For nanospray, a Protana source was employed using uncoated glass nanospray tips pulled in house to 1 μm ID using a capillary puller (Sutter Instrument Co.) ESI was initiated at ~ 1200 V via a Pt wire inserted into the glass tip. Tandem mass spectra were acquired using Ar as the collision gas and sufficient collision energy to obtain complete sequence information of the precursor. Pulsed ion enhancement of product ions was used for MS/MS of low S/N precursors. For LCMS, the LC was coupled to the mass spectrometer using 50 μm ID distal coated nanospray tips pulled to 15 μm ID, 75 μm OD at the tip (New Objectives Inc.). ESI was carried out at 4500 V. Information dependent acquisition (IDA) was used to obtain MS/MS spectra of peaks during elution from the LC system. MS peaks which exceeded a threshold of 10 counts/s were subjected to MS/MS using preset collision energies proportional to the m/z value of the precursor (ca. 18-60 V, lab frame). Pulsed ion enhancement was used for all LC MS/MS spectra.
Cytotoxicity Assays Cytotoxicity assays for the fusion protein toxins were performed essentially as described by vanderSpek et al, J. Biol. Chem. 269: 21455- 21459, 1994. Cytotoxicity assays to evaluate the affects of geldanamycin, radicicol, and retinoic acid upon DAB389IL-2 intoxication were modified as follows: Cells were seeded at 5 x 104 cells per well and pre-incubated with inhibitors geldanamycin, radicicol, cis- 13 -retinoic acid, for 30 min. at 37°C, 5% C02 and subsequently incubated with varying concentrations of DAB389IL- 2 and inhibitor for 15 min. at 37°C, 5% C02. Cells were pelleted and washed free of toxin with media containing inhibitor and incubated for eight to twelve hours at 37°C, 5% C02. Cells were then washed and pulsed with minimal media (leucine depleted; BioWhittaker) containing [14C]-leucine (New England Nuclear) for 2 hours at 37°C, 5% C02, and protein synthesis was analyzed according to vanderSpek et al, J. Biol. Chem. 269: 21455-21459, 1994. Media alone and media plus inhibitor alone served as controls. Assays were performed in quadruplicate.
Compound synthesis The compounds of formula I may be synthesized by either solid or liquid phase methods described and referenced in standard textbooks, or by a combination of both methods. These methods are well known to those skilled in the art, (see, for example, Bodanszky, In "The Principles of Peptide
Synthesis", Hafher, Rees, Trost, Lehn, Schleyer, Zahradnik, Eds., Springer- Verlag, Berlin, 1984; Stewart and Young, Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, 111., 1984 ; and U.S. Pat. Nos. 4,105,603; 3,972,859; 3,842,067; and 3,862,925). During the synthesis of the compounds of the present invention, the functional groups of the amino acid derivatives used in these methods are protected by blocking groups to prevent cross reaction during the amide bond- forming procedure. Examples of suitable blocking groups and their use are described in "The Peptides: Analysis, Synthesis, Biology," Academic Press, Gross & Meienhofer, Eds., Vol. 3 (1981) and Vol. 9 (1987). The reaction products are isolated and purified by conventional methods, typically by solvent extraction into a compatible solvent, or by using a washing protocol for resin bound intermediates. The products may be further purified by column chromatography or other appropriate methods, including medium pressure or high pressure liquid chromatography. In one example, compounds of the invention can be conveniently prepared using solid phase synthesis methodology (Merrifield, J. Am. Chem. Soc. 85:2149, 1964; Houghten, Proc. Natl. Acad. Sci. USA, 82:5132, 1985). Solid phase synthesis begins at the carboxy terminus of the compound by attaching a protected amino acid, or other carboxylic acid-containing compound, to an inert solid support. The inert solid support can be any macromolecule capable of serving as an anchor for the C-terminus of the initial amino acid. Typically, the macromolecular support is a cross-linked polymeric resin (e.g. a polyamide or polystyrene resin). In one embodiment, the C- terminal amino acid is coupled to a polystyrene resin to form a benzyl ester. Particularly useful benzyl-type resins, such as trityl resin, chlorotrityl resin, and Wang resin, are those in which the linkage of the carboxy group (or carboxamide) to the resing is acid-lable. A macromolecular support is selected such that the peptide anchor link is stable under the conditions used to deprotect the α-amino group of the blocked amino acids in peptide synthesis. If a base-labile α-protecting group is used, then it is desirable to use an acid- labile link between the peptide and the solid support. For example, an acid- labile ether resin is effective for base-labile Fmoc-amino acid peptide synthesis. Alternatively, a peptide anchor link and α-protecting group that are differentially labile to acidolysis can be used. For example, an aminomethyl resin such as the phenylacetamidomethyl (Pam) resin works well in conjunction with Boc-amino acid peptide synthesis.
After the initial amino acid is coupled to an inert solid support, the α- amino protecting group of the initial amino acid is removed with, for example, trifluoroacetic acid (TFA) in methylene chloride and neutralizing in, for example, triethylamine (TEA). Following deprotection of the initial amino acid's α-amino group, the next . α-amino and side chain protected amino acid in the synthesis is added. The remaining α-amino protected and, if necessary, side chain protected amino acids are then coupled sequentially in the desired order by condensation to obtain an intermediate compound connected to the solid support. Alternatively, some amino acids may be coupled to one another to form a fragment of the desired peptide followed by addition of the peptide fragment to the growing solid phase peptide chain. The condensation reaction between two amino acids, or an amino acid and a peptide, or a peptide and a peptide an be carried out according to the usual condensation methods such as the axide method, mixed acid anhydride method, DCC (N,N'-dicyclohexylcarbodiimide) or DIG (N,N'~ diisopropylcarbodiimide) methods, active ester method, p-nitrophenyl ester method, BOP (benzotriazole-l-yl-oxy-tris[dimethylamino] phosphonium hexafluorophosphate)method, N-hydroxysuccinicacid imido ester method, O- benzotriazolyl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) or 0-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and Woodward reagent K method.
Alternatively, other functional groups, can be introduced on the liberated N-terminus of the resin. For example, reaction with commercially available carboxylic acids or acid chlorides, sulfonyl chlorides, or isocyanates under standard conditions known in the art produce compounds of the invention containing an amide, sulfonamide, or urea bond, respectively. An alternative to using isocyanates in preparing urea- containing compounds is to activate the deprotected amine terminus with, for example, phosgene, triphosgene, carbonyl di-imidazole, or p-N02 phenylchloroformate followed by reaction with primary or secondary amines employed in excess. It is common in both solid-phase and solution-phase synthesis to protect any reactive side-chain groups of the amino acid with suitable protecting groups. Ultimately, these protecting groups are removed after the desired compounds have been sequentially assembled. Also common is the protection of the α-amino group on an amino acid or a fragment while that entity reacts at the carboxy group followed by the selective removal of the α-amino protecting group to allow subsequent reaction to take place at that location. Accordingly, it is common that an intermediate compound is produced which contains each of the amino acid residues located in the desired sequence in the peptide chain with various of these residues having side chain protecting groups attached. These protecting groups are then commonly removed at substantially the same time so as to produce the desired product following cleavage from the resin. Protecting groups and procedure for their use in peptide synthesis are reviewed in Protective Groups in Organic Synthesis, 3d ed., Greene, T. W. and Wuts, P. G. M., Wiley & Sons (New York: 1999). Suitable protecting groups for α-amino and side chain amino groups are exemplified by benzyloxycarbonyl (abbreviated Z), isonicotinyloxycarbonyl (iNOC), o-chlorobenzyloxycarbonyl [Z(2C1)], p-nitrobezyloxycarbonyl [Z(N02)], p-methoxybenzyloxycarbonyl [Z(OMe)], t-butoxycarbonyl (Boc), t- amyloxycarbonyl(Aoc), isobornyloxycarbonyl, adamantyloxycarbonyl, 2-(4- biphenyl)-2-propyloxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl(Fmoc), methylsulfonyethoxycarbonyl (Msc), trifluoroacetyl, phthalyl, formyl, 2- nitrophenylsulfenyl (NPS), diphenylphosphinothioyl (Ppt), and dimethylphosphinothioyl (Mpt) groups, and the like.
Protective groups for the carboxy functional group are exemplified by benzyl ester, (OBz), cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb), t-butyl ester (OtBu), 4-pyridylmethyl ester (OPic), and the like. It is often desirable that amino acids such as arginine, cysteine, and serine possessing a functional group other than amino and carboxy groups be protected by a suitable protecting group. For example, the guanidino group may be protected with nitro, p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl, p- methoxybenzenesulfonyl, 4-methoxy-2, 6-dimethylbenzenesulfonyl (Nds), 1,3,5-trimethylphenysulfonyl (Mts), and 2,3,6-trimethyl-4- methoxyphenylsulfonyl (Mtr), and the like. The thiol group can be protected with p-methoxybenzyl, trityl, and the like. In one embodiment, the compounds of the invention are synthesized with the help of blocking groups that protect the side chain amide bond- forming substituents of the N-terminal and C-terminal flanking residues. The protecting group or groups used for the side chain amide bond-forming substituents of the N-terminal and C-terminal flanking residues can be the same or different than the protecting group or groups used to block the side chain functional groups of other residues in the peptide. In a preferred embodiment, the protecting group or groups used to block the side chain amide bond- forming substituents is (are) differentially removable with respect to the protecting groups used for other side chain functional groups, i.e. the side chain amide bond-forming substituents can be deprotected without deprotecting the other side chain functional groups, in addition to being differentially removable with respect to the α-amino protecting group used in peptide synthesis. In another preferred embodiment, the side chain amide bond- forming substituents of the flanking residues are orthogonally protected with respect to each other such that the side chain amide bond- forming substituent of one flanking residue can be deprotected without deprotecting the side chain amide bond-forming substituent of the other flanking residue.
Suitable protecting groups for use in orthogonally protecting the side chain amide bond- forming substituents of the flanking residues with respect to other functional groups and/or with respect to each other include pairs of differentially removable carboxy protective groups, such as a reduction-labile carboxy protective group, e.g. allyl or benzyl esters, paired with a base-labile carboxy protective group, e.g. fluorenylmethylesters, methyl or other primary alkyl esters. Fluorenylmethyl, methyl or other primary alkyl groups or other base-labile carboxy protective groups can be removed from their corresponding esters to yield a free carboxy group (without deprotecting allyl or benzyl esters or other reduction-labile esters) by saponification of the esters with a suitable base such as piperidine and sodium hydroxide in a suitable solvent such as dimethylacetamide, or methanol and water, for a period of 10 to 120 minutes, and preferably 20 minutes, 0 °C to 50°C. The allyl or benzyl or other reduction-labile esters can be removed when desired by reduction in the presence of a suitable transition metal catalyst, such as Pd(PPh3)4, Pd(PPh3)2Cl2, Pd(OAc)2 or Pd on carbon with a source of hydrogen, e.g. H2 gas, in a suitable solvent such as dimethylacetamide, dimethylformamide, N- methylpyrrolidinoneor methanol for a period of 10 to 500 minutes, and preferably 100 minutes, at 0 °C to 50°C. For the sake of simplicity and convenience, all carboxy protective groups that are removable by Pd-catalyzed methods which result in the reduction of the protected carboxy substirutent are included n the term "reduction-labile protective groups" as used herein, even though such Pd-catalyzed deprotection methods may not result in the reduction of the protective group in question. In embodiments wherein Pd catalysis involves the formation of intermediates of Pd derivatized with reduction-labile protecting groups, e.g. Pd- allyl derivatives, the Pd catalyst can be restored by reaction with a suitable nucleophile, such as piperidine or tributyltin hydride. When such reduction- labile groups are used to provide orthogonal protection in combination with base-labile protecting groups, it is preferable to either (1) utilize a synthetic scheme that calls for the removal of the base-labile protecting groups before the removal of the reduction-labile protecting groups or (2) restore the Pd catalyst with a nucleophile that does not deprotect the base-labile protecting groups. Alternatively, the carboxy substituents of the flanking residues can be orthogonally protected with respect the other functional groups and/or with respect to each other by using an acid-labile protecting group, such as a tertiary alkyl ester, e.g. t-butyl ester, in combination with a reduction-labile protecting group, such as the allyl or benzyl esters described above. The tertiary alkyl or other acid-labile ester group can be removed by acidolysis, e.g. with trifluoroacetic acid in methylene chloride, and the allyl or benzyl or other reduction-labile esters can be removed by reduction in the presence of a transition metal catalyst as described above. In another embodiment, the carboxy substituents of the flanking residues can be orthogonally protected with respect to other functional groups and/or with respect to each other by using a fluoride ion-labile protecting group, such as 2-(trimethylsilyl)ethyl and silyl esters, in combination with a reduction- labile protecting group, such as the allyl or benzyl esters described above, or in combination with a base-labile protecting group, such as the fluorenylmethyl, methyl or other primary alkyl esters described above, without deprotecing the reduction-labile or base-labile esters. The 2-(trimethylsilyl)ethyl, silyl or other fluoride-labile ester group can be removed by reaction with a suitable fluoride ion source, such as tetrabutylammonium fluoride in the presence of a suitable solvent, such as dimethylacetamide(DMA), dimethylformamide (DMF), tetrahydrofuran (THF), or acetonitrile. Suitable protecting groups for use in orthogonally protecting the side chain amide bond- forming substituents of the flanking residues with respect to other functional groups and/or with respect to each other also include pairs of differentially removable amino protective groups, such as an allyloxycarbonyl or other reduction-labile amino protective group paired with a t-butoxycarbonyl (Boc) or other acid-labile amino protective group, and a reduction-labile amino protective group paired with a fluorenylmethoxycarbonyl (Fmoc) or other base- labile amino protective group. An allyloxycarbonyl (or other reduction-labile blocking group) protected amino group can be deprotected by reduction using a transition metal catalyst as in the procedure for removing reduction-labile carboxy protective groups described above, without deprotecting a Boc or Fmoc protected amino group. Likewise, an acid-labile amino protective group and a base-labile amino protective group can be removed by acidolysis and base saponifϊcation, respectively, without removing a reduction-labile amino protective group. For the sake of simplicity and convenience, all amino protective groups that are removable by Pd-catalyzed methods which result in the reduction of the protected amino substitutent are included in the term "reduction-labile protective groups" as used herein, even though such Pd- catalyzed deprotection methods may not result in the reduction of the protective group in question. In another embodiment, the amino substituents of the flanking residues can be orthogonally protected with respect to other functional groups and/or with respect to each other by using a fluoride-labile protecting group, such as 2-trimethylsilylethylcarbamate (Teoc), in combination with a reduction-labile protecting group, such as allyloxylcarbonyl, or in combination with a base- labile protecting group, such as fluorenylmethoxycarbonyl, as described above. The Teoc or other fluoride-labile group can be removed by reaction a with a suitable fluoride ion source, such as tetrabutylammonium fluoride, as in the procedures for removal of fluoride-labile carboxy protective groups described above, without deprotecting an allyloxycarbonyl or fluorenylmethoxycarbonyl protected amino group. Likewise, a reduction-labile amino protective group and a base-labile amino protective group can be removed by reduction and base saponification, respectively, without removing a fluoride-labile amino protective group. In embodiments that use a carboxy substituent as the side chain amide bond- forming substituent of one flanking residue and that use an amino substituent as the side chain amide bond- forming substituent of the other flanking residue, the carboxy substituent and the amino substituent can be orthogonally protected with respect to each other by using a reduction-labile protecting group to block one substituent, e.g. allyl ester or allyloxycarbonyl, and a fluoride-labile, acid-labile or base-labile protecting group to block other substituent, e.g. silyl ester, t-butyl ester, fluorenylmethyl ester, Teoc, Boc, or Fmoc. In a preferred embodiment, a reduction-labile protecting group is used to block the side chain amide bond-forming substituent of one flanking residue and the protecting group for the side chain amide bond- forming substituent of the other flanking residue is selected such that it provides orthogonal protection with respect to both the reduction-labile protecting group and the α-amino protecting group used in the synthesis. For example, in an embodiment using Fmoc chemistry for peptide synthesis, orthogonal protection of the side chain amide bond-forming substituents would be provided by a reduction-labile protecting group and an acid-labile protecting group. Likewise, in an embodiment using Boc chemistry for peptide synthesis, orthogonal protection of the side chain amide bond-forming substituents would be provided by a reduction-labile protecting group and a base-labile protecting group. In yet another preferred embodiment, the side chain amide bond- forming substituents of the flanking residues are orthogonally protected with respect to each other, with respect to α-amino protecting group used in the synthesis, and with respect to the protecting groups used to block other side chain functional groups in the peptide chain.
In still another preferred embodiment, the side chain amide bond- forming substituents of the flanking residues are orthogonally protected with respect to each other, and with respect to α-amino protecting group, but only one of the side chain amide bond- forming substituents is orthogonally protected with respect to the protecting groups used to block other side chain functional groups. In this embodiment, it is preferable to use the side chain amide bond-forming substituent with fully orthogonal protection as the target for initial attachment of the compound to the difunctional linker. Since the side chain amide bond-forming substituent with fully orthogonal protection can be deprotected without deprotecting other functional groups, the amide bond- forming reaction will be specific to the desired side chain amide bond- forming substituent, and will reduce the production of unwanted difunctional linker derivatives. Although cyclization will require the deprotection of the side chain amide bond- forming substituent of the other flanking residue, and may cause concomitant deprotection of other side chain functional groups, unwanted derivatives are less likely to form given that the peptide chains are anchored to a solid support and that the linker length will regioselectively favor a amide bond- forming reaction between the unbound functional group of the linker and the side chain amide bond-forming substituent of the other flanking residue. If further peptide chain synthesis is desired after cyclization, any side chain functional groups on other amino acid residues left unprotected by the cyclization reactions can be reprotected before chain synthesis is resumed.
Many of the blocked amino acids described above can be obtained from commercial sources such as Novabiochem (San Diego, Calif), Bachem Calif. (Torrence, Calif.) or Peninsula Labs (Belmont, Calif). Alternatively, functionalized or protected amino acids, including unnatural amino acids, can be prepared by methods known in the art.
In addition, the compounds of the invention can be prepared by, or in conjunction with, solution phase peptide synthesis, for example, the solution phase peptide synthesis methods described in Principles of Peptide Synthesis, 2d ed, M. Bodanszky, Springer- Verlag (1993) or in The Practice of Peptide Synthesis, 2d ed, M. Bodanszky and A. Bodanszky, Springer- Verlag (1994). It will be appreciated that solution phase peptide synthesis methods can be easily modified to incorporate the desired flanking residues, with or without orthogonally-protected side chain amide bond-forming substituents, into the compound of interest, using procedures similar to those used in the solid phase synthesis methods described herein. All publications and patents cited in this specification are hereby incorporated by reference herein as if each individual publication or patent specifically and individually indicated to be incorporated by reference. Although ihe foregoing invention has been described in some detail by way of illustration and exam le for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit r scope of the appended claims. What is claimed is:

Claims

1. A compound of formula I,
X- AA210- AA211 - AA212- AA 13 - AA214- AA215- AA216- AA217- AA218- AA219- AA220- AA221 - AA222-Y (I), wherein
X is H or a chain of amino acids of from 1 to 5 residues substituted at the N- terminus with a nitrogen protecting group, R1-C(0)-, or H; Y is OH, NH2, NHR2, NHR2R3, OR4, or a chain of amino acids of from 1 to 76 residues substituted at the C-terminus with OH, NH2, NHR2, NHR2R3, or OR4, wherein R1 is a d-6 alkyl, C6 or C10 aryl, d-9 heterocyclyl, Cι-6 alkoxy, C7-16 aralkyl, C2- 15 heterocyclylalkyl, C7-16 aralkoxy, C2-ι5 heterocyclyloxy, or a polyethylene 9 " glycol moiety; each of R and R is, independently, H, a Cι-6 alkyl, C6 or Cι0 aryl, C1.9 heterocyclyl, C7_ι6 aralkyl, C2-15 heterocyclylalkyl, or a polyethylene glycol moiety; R4 is H, d-6 alkyl, C6 or Cι0 aryl, C 1- heterocyclyl, d-6 alkoxy, C7-i6 aralkyl, C2-ι5 heterocyclylalkyl, a carboxyl protecting group, or a 10 91 1 919 polyethylene glycol moiety; AA is Arg or Lys; AA is Asp or Glu; AA is Lys or Arg; AA213 is Thr, Ser, Ala, Gly, Val, Asn, or Gin; AA214 is Lys or Arg; AA215 is Thr, Ser, Ala, Gly, Val, Asn, or Gin; AA216 is Lys or Arg; AA217 is lie, Leu, or Val; AA218 is Glu or Asp; AA219 is Ser, Ala, or Gly; AA220 is Leu, lie, or Val; AA221 is Lys or Arg; and AA222 is Glu or Asp.
2. The compound of claim 1 , wherein; Y is - AA223 - AA224 - AA225 - AA226 - AA227 - AA228 - AA229 -Ya, wherein Ya is OH, NH2, NHR2, NHR R3, or OR4; AA223 is His, Phe, or Tyr; AA224 is Gly, Ala, or Ser; AA225 is Pro; AA226 is lie, Leu, Val; AA227 is Lys or Arg; AA228 is Asn or Gin; and AA229 is Lys or Arg.
3. The compound of claim 2, wherein AA210 is Arg; AA211 is Asp; AA212 is Lys; AA213 is Thr; AA214 is Lys; AA215 is Thr; AA216 is Lys; AA217 is lie; AA218 is Glu; AA219 is Ser; AA220 is Leu; AA221 is Lys; AA222 is Glu; AA223 is His; AA224 is Gly; AA225 is Pro; AA226 is He; AA227 is Lys; AA228 is Asn; and AA229 is Lys.
4. The compound of claim 1 , wherein X is X - AA205 - AA206 - AA207 - AA208 - AA209 - and Y is - AA223 - AA224- Ya, wherein Xa is R!-C(0)- or H, Ya is OH, NH2, NHR2, NHR2R3, or OR4; AA205 is Asp or Glu; AA206 is Tip, Tyr, or Phe; AA207 is Asp or Glu; AA208 is Val, Leu, He, Thr, Ser, or Ala; AA209 is He, Leu, or Val; AA223 is His, Tyr, or Phe; and AA224 is Gly, Ala, or Ser.
5. The compound of claim 4, wherein AA205 is Asp; AA206 is Trp; AA207 is Asp; AA208 is Val; AA209 is He; AA210 is Arg; AA211 is Asp; AA212 is Lys; AA213 is Thr; AA214 is Lys; AA215 is Thr; AA216 is Lys; AA217 is He; AA218 is Glu; AA219 is Ser; AA220 is Leu; AA221 is Lys; AA222 is Glu; AA223 is His; and AA224is Gly.
6. The compound of any of the claims 1 to 5, wherein Y is H or a chain of amino acids of from 1 to 5 residues and Y is OH or a chain of amino acids of from 1 to 76 residues.
7. The compound of claim 6, wherein X is a chain of amino acids from 1 to 5 residues corresponding to the peptide sequence: Asp-Trp- Asp- Val-Ile-.
8. The compound of claim 6, wherein X is a chain of amino acids from 1 to 76 residues corresponding to the peptide sequence: -Arg-Asp-Lys-Thr-Lys- Thr-Lys-Ile-Glu-Ser-Leu-Lys-Glu-His-Glu-Pro-Ile-Lys-Asn-Lys-Met-Ser-Glu- Ser-Pro-Asn-Lys-Thr-Val-Ser-Glu-Glu-Lys-Ala-Lys-Gln-Tyr-Leu-Glu-Glu- Phe-His-Gln-Thr-Ala-Leu-Glu-His-Pro-Glu-Leu-Ser-Glu-Leu-Lys-Thr-Val- Thr-Gly-Thr-Asn-Pro-Val-Phe-Ala-Gly-Ala-Asn-Tyr-Ala-Ala-Trp-Ala-Val- Asn-Val-Ala-Gln- Val-Ile- Asp-Ser-Glu-Thr-Ala-Asp-Asn-Leu-Glu-Lys.
9. A compound of any of the claims 1-5, wherein R1, R2, or R4 is a polyethylene glycol moiety selected from the group consisting of: H3C(OCH2CH2)ccOCH2C(0)-, H(OCH2CH2)ccOCH2C(0)-, H3C(OCH2CH2)ccOC(0)-, H(OCH2CH2)ccOC(0)-, H3C(OCH2CH2)ccNHC(0)-, H(OCH2CH2)ccNHC(0)-, H3C(OCH2CH2)ccNHC(S)-, H(OCH2CH2)ccNHC(S)-, H3C(OCH2CH2)ccC(0)-, H(OCH2CH2)ccC(0)-, H3C(OCH2CH2)ccNHCH2C(0)-, H(OCH2CH2)ccNHCH2C(0)-, H3C(OCH2CH2)ccOC(0)C(CH3)2-, and H(OCH2CH2)ccOC(0)C(CH3)2-, wherein cc is a range of numbers that results in an average molecular weight of said polyethylene glycol moiety of between 1,000-40,000.
10. The compound of claim 9, wherein cc is a range of numbers that results in an average molecular weight of said polyethylene glycol moiety of 20,000.
11. The compound of claim 9, wherein cc is a range of numbers that results in an average molecular weight of said polyethylene glycol moiety of 40,000.
12. The compound of any of the claims 1-5, wherein R1, R2, or R4 is a polyethylene glycol moiety selected from the group consisting of: maleimide-(CH2)bbC(0)NHCH2CH2(OCH2CH2)aaOCH2C(0)-, maleimide- (CH2)bbC(0)NHCH2CH2(OCH2CH2)aaNHCH2C(0)-, maleimide- (CH2)bbC(0)NHCH2CH2(OCH2CH2)aaNHC(S)-, maleimide-(CH2)bbNHC(S), maleimide-(CH2)bbC(0)-, or maleimide-(CH2)bb-, wherein aa is 1-10 and bb is 1-4.
13. The compound of claim 12, wherein said compound is further reacted with a monoclonal antibody, or fragment thereof, to form a covalent bond between a sulfur atom of said antibody and said maleimide group of said compound.
14. Use of a compound of any of the claims 1 - 13 in the manufacture of a medicament for inhibiting cell death in a mammal.
15. The use of claim 14, wherein said compound inhibits the translocation of a viral or bacterial toxin from the lumen of an endosome to the cytosol of said cell.
16. The use of claim 15, wherein said toxin is an AB toxin.
17. The use of claim 15 , wherein said toxin is selected from the group consisting of: Diphtheria toxin, a Botulinum toxin, Anthrax toxin LF, and Anthrax toxin EF.
18. The use of claim 14, wherein said compound inhibits the translocation of a viral or retroviral transcription factor.
19. The use of claim 18, wherein said factor is human immunodeficiency virus reverse transcriptase.
20. The use of claim 18, wherein said factor is Tat.
21. A compound having a nucleic acid sequence encoding the peptide sequence of a compound of claim 6.
22. The compound of claim 21, wherein said nucleic acid sequence encodes a peptide sequence selected from the group consisting of: -Arg-Asp-Lys-Thr- Lys-Thr-Lys-Ile-Glu-Ser-Leu-Lys-Glu-His-Gly-Pro-He-Lys-Asn-Lys-; -Asp- Trp- Asp- Val-Ile- Arg- Asp-Lys-Thr-Lys-Thr-Lys-Ile-Glu-Ser-Leu-Lys-Glu-His- Gly-; and -Arg-Asp-Lys-Thr-Lys-Thr-Lys-Ile-Glu-Ser-Leu-Lys-Glu-His-Gly- Pro-Ile-Lys- Asn-Lys- .
23. The compound of claim 21, wherein said nucleic acid sequence encodes the peptide sequence: Arg-Asp-Lys-Thr-Lys-Thr-Lys-Ile-Glu-Ser-Leu-Lys- Glu-His-Glu-Pro-He-Lys-Asn-Lys-Met-Ser-Glu-Ser-Pro-Asn-Lys-Thr-Val-Ser- Glu-Glu-Lys-Ala-Lys-Gln-Tyr-Leu-Glu-Glu-Phe-His-Gln-Thr-Ala-Leu-Glu- His-Pro-Glu-Leu-Ser-Glu-Leu-Lys-Thr-Val-Thr-Gly-Thr-Asn-Pro-Val-Phe- Ala-Gly-Ala-Asn-Tyι--Ala-Ala-Trp-Ala-Val-Asn-Val-Ala-Gln- Val-Ile- Asp- Ser-Glu-Thr-Ala-Asp-Asn-Leu-Glu-Lys-.
24. The compound of any of the claims 21 to 23, wherein the nucleic acid sequence is operably linked to an inducible promoter.
25. The compound of claim 24, wherein expression of said peptide sequence is moderated by treating said cell with an agent selected from the group consisting of: doxycycline; retinal; cyclosporin or analogs thereof; FK506; FK520; and rapamycin or analogs thereof.
26. Use of a compound of any of the claims 21 to 23 in the manufacture of a medicament for inhibiting cell death in a mammal.
27. The use of claim 14 or 26, wherein said mammal is a human.
28. The use of claim 14 or 26, wherein said medicament further comprises a pharmaceutically acceptable vehicle.
29. A method of identifying a compound that inhibits cell death in a mammal comprising the following steps: a) isolating endosomes from said cell; b) placing said endosomes in a cytosolic buffer; c) contacting said endosomes with a fusion protein-toxin, wherein said protein comprises a binding moiety for a component of the cell membrane of said cell and said toxin comprises a fragment of Diphtheria toxin; d) contacting said endosomes with a cytosolic translocation factor complex; e) contacting said endosomes with said compound; and f) measuring translocation of said toxin, wherein a decreased level of said translocation relative to that observed in the absence of said compound indicates that said compound inhibits said cell death.
30. The method of claim 29, wherein said endosomes are early endosomes.
31. The method of claim 29, wherein said protein is IL-2.
32. The method of claim 29, wherein said fusion protein-toxin is DAB389IL-
2.
33. The method of claim 29, wherein said cytosolic translocation factor comprises Hsp 90 and thioredoxin reductase.
34. The method of claim 29, wherein measuring said translocation comprises measuring the ADP-ribosylation of elongation factor-2.
35. A method of identifying a compound that promotes cell death in a mammal comprising the following steps: a. isolating endosomes from said cell; b. placing said endosomes in a cytosolic buffer; c. contacting said endosomes with a fusion protein-toxin, wherein said protein comprises a binding moiety for a component of the cell membrane of said cell and said toxin comprises a fragment of Diphtheria toxin; d. contacting said endosomes with a cytosolic translocation factor complex; e. contacting said endosomes with said compound; and f measuring translocation of said toxin, wherein an increased level of said translocation relative to that observed in the absence of said compound indicates that said compound promotes said cell death.
36. The method of claim 35, wherein said endosomes are early endosomes.
37. The method of claim 35, wherein said protein is IL-2.
38. The method of claim 35, wherein said fusion protein-toxin is DAB389IL-
2.
39. The method of claim 35, wherein said cytosolic translocation factor comprises Hsp 90 and thioredoxin reductase.
40. The method of claim 35, wherein measuring said translocation comprises measuring the ADP-ribosylation of elongation factor-2.
PCT/US2004/009829 2003-03-31 2004-03-31 Novel compositions and methods for promoting, inhibiting, and detecting protein entry into cells WO2005014798A2 (en)

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