EP2111413A2 - Htra1-pdz- und htra3-pdz-modulatoren - Google Patents

Htra1-pdz- und htra3-pdz-modulatoren

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Publication number
EP2111413A2
EP2111413A2 EP08729968A EP08729968A EP2111413A2 EP 2111413 A2 EP2111413 A2 EP 2111413A2 EP 08729968 A EP08729968 A EP 08729968A EP 08729968 A EP08729968 A EP 08729968A EP 2111413 A2 EP2111413 A2 EP 2111413A2
Authority
EP
European Patent Office
Prior art keywords
pdz domain
htral
htra3
polypeptide
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP08729968A
Other languages
English (en)
French (fr)
Inventor
Brent A. Appleton
Steven T. Runyon
Sachdev S. Sidhu
Nicholas J. Skelton
Christian Wiesmann
Yingnan Zhang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genentech Inc
Genetech Inc
Original Assignee
Genentech Inc
Genetech Inc
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Filing date
Publication date
Application filed by Genentech Inc, Genetech Inc filed Critical Genentech Inc
Publication of EP2111413A2 publication Critical patent/EP2111413A2/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)

Definitions

  • PDZ domains are common modular protein domains that mediate a wide range of specific protein-protein interactions by binding in a sequence-specific manner to the C- termini of their biological partner or in some instances to internal hairpin motifs (Sheng, M., and SaIa, C. (2001) Annual Review ofNeuroscience 24(1), 1-29). Specificity of PDZ domains for their ligands was originally classified in two groups based on the presence of a Ser/Thr residue (type I) or a hydrophobic residue (type II) at the position -2 (using the standardized PDZ ligand nomenclature in which the C terminus is designated residue 0 and the remaining residues are numbered with negative integers whose absolute value increases towards the N terminus).
  • Erbin-PDZ domain selectivity are more complex, with binding determinants potentially constituted by 3 to 6 C-terminal ligand residues.
  • the binding profile of Erbin-PDZ is extremely specific ([D/E][T/S]WVCOOH) and that of ZOl-PDZl is similar ([R/K/S/T][T/S][W/Y][V/I/L]COOH), but exhibits increased promiscuity for three of the last four ligand residues.
  • Both Erbin-PDZ and ZOl-PDZl also employ auxiliary ligand interactions upstream of position -3 that modulate binding affinity (Appleton, B. A., Zhang, Y., Wu, P., Yin, J. P., Hunziker, W.,
  • HtrA The HtrA family of serine proteases has four known members in humans with extensive homology to bacterial high-temperature requirement A protease (HtrA). This bacterial protease acts as a chaperone under normal temperature conditions, and is essential for survival at high temperatures where the proteolytic function mediates the degradation of denatured proteins. Although all human HtrA proteins share homologous trypsin-like serine protease (SP) domains and C-terminal PDZ domains, HtrAl and HtrA3 also contain a signal sequence for secretion as well as an insulin-like growth factor binding-protein domain and a Kazal-type SP inhibitor domain in the N-terminal region.
  • SP homologous trypsin-like serine protease
  • HtrAl and HtrA3 also contain a signal sequence for secretion as well as an insulin-like growth factor binding-protein domain and a Kazal-type SP inhibitor domain in the N-terminal region.
  • HtrAl was originally identified as a gene that is down-regulated in a human fibroblast cell line after transfection with the oncogenic virus SV40 (Zumbrunn, J., and Trueb, B. (1996) FEBS Lett 398(2-3), 187-192). HtrAl has since been implicated in a number of malignancies. HtrAl is down-regulated in cancerous as compared to normal tissue and over-expression results in the inhibition of tumor growth and proliferation (Baldi, A., De Luca, A., Battista, T., Felsani, A., Baldi, F., Catricala, C, Amantea, A., Noonan, D.
  • HtrAl is up-regulated in cartilage of osteoarthritic joints and may contribute to the development of this and other arthritic diseases (Hu, S.-L, Carozza, M., Klein, M., Nantermet, P., Luk, D., and Crowl, R. M. (1998) J. Biol Chem. 273(51), 34406-34412). HtrAl has also been implicated in amyloid precursor protein processing (Grau, S., Baldi, A., Bussani, R., Tian, X., Stefanescu, R., Przybylski, M.,
  • HtrA3 plays a role in placental development (Nie et al, Biol Reprod. 2006 Feb;74(2):366-74) and has also been implicated, alongside HtrAl, in endometrial cancer (Bowden et al., Gynecol Oncol. 2006 Oct;103(l):253-60).
  • HtrAl has been shown to bind to TGF- ⁇ family proteins in mouse, and is proposed to mediate suppression of TGF- ⁇ signaling (Oka, C, Tsujimoto, R., Kajikawa, M., Koshiba- Takeuchi, K., Ina, J., Yano, M., Tsuchiya, A., Ueta, Y., Soma, A., Kanda, H., Matsumoto, M., and Kawaichi, M. (2004) Development 131(5), 1041-1053). While the TGF- ⁇ suppression is dependent on the protease activity of HtrAl, the PDZ domain seems to play a direct role in regulating the protease activity.
  • Peptides derived from the C-terminus of TGF- ⁇ family members such as CoBaI bind to the PDZ domain of HtrAl and stimulate protease activity (Murwantoko, Yano, M., Ueta, Y., Murasaki, A., Kanda, H., Oka, C, and Kawaichi, M. (2004) Biochem J381(Pt 3), 895-904).
  • HtrA3 also shows comparable protease and TGF- ⁇ signal inhibitory activities (Tocharus, J., Tsuchiya, A., Kajikawa, M., Ueta, Y., Oka, C, and Kawaichi, M. (2004) Development, Growth and Differentiation 46(3), 257-274).
  • HtrAl and HtrA3 The important molecular functions ascribed to HtrAl and HtrA3 above, in particular those mediated through the protein-protein interaction between HtrAl PDZ domain or HtrA3 PDZ and ligand, suggest that the HtrAl and HtrA3 PDZ domains represent significant therapeutic targets. It would therefore be beneficial to elucidate the mechanistic aspects of the interaction between ligand and the HtrAl or HtrA3 PDZ domains and provide compositions and methods targeted at modulating its associated functional activities. The present invention provides this and other benefits.
  • compositions, and methods of using these compositions for modulating activity of the PDZ domains of each of the HtrAl and HtrA3 proteins. Because of the important functions associated with HtrAl and HtrA3, compositions and methods of the invention present significant clinical and therapeutic utilities.
  • the invention is based in part on analysis and characterization of binding partners (ligands) of HtrAl PDZ domain and HtrA3 PDZ domain, said analysis resulting in novel and unexpected findings as described herein.
  • HtrAl PDZ domain and HtrA3 PDZ domain Two groups of peptide ligands to each of HtrAl PDZ domain and HtrA3 PDZ domain were separately generated from phage-displayed libraries, with peptides fused either to the C- terminus or N-terminus of Ml 3 p8 protein representing peptide binders that require a free carboxyl group and those that do not.
  • Peptide ligands of HtrAl PDZ domain or HtrA3 PDZ domain that comprise a free carboxyl terminus or those that do not comprise a free carboxy terminus are herein described.
  • HtrAl PDZ domain ligands and HtrA3 PDZ domain ligands are capable of binding to HtrAl PDZ domain or HtrA3 PDZ domain, respectively without a free carboxyl terminus.
  • Ligands without a free carboxyl terminus represent N-terminus and/or internal HtrAl PDZ domain ligand or HtrA3 PDZ domain ligand sequences that are N terminal or internal sequences of polypeptides.
  • binding specificities of a series of peptide ligands were assessed by measuring their relative affinities.
  • Alanine scanning analysis was performed on the individual residues of an exemplary peptide ligand to elucidate the energetic contribution of different residues at each ligand position.
  • Molecular modeling was also performed to dock a specific exemplary ligand to each of HtrAl PDZ domain and HtrA3 PDZ domain to further assess the binding specificities on a structural basis.
  • a phage-based combinatorial scanning approach was also used to identify the residues in HtrAl PDZ domain that contribute energetically to ligand PDZ interaction, providing further insight regarding structure and energetic components of HtrAl PDZ domain interaction with its ligands.
  • the invention provides an isolated polypeptide that binds specifically to an HtrAl PDZ domain, wherein the polypeptide comprises a sequence having a tryptophan at position -2 relative to the C-terminus.
  • the polypeptide further comprises a small amino acid at position 0.
  • the amino acid at position 0 is selected from leucine and valine.
  • the polypeptide further comprises an amino acid comprising a bulky side chain at position -1.
  • the amino acid at position -1 is selected from tryptophan, isoleucine, and phenylalanine.
  • the amino acid at position -1 is tryptophan.
  • the polypeptide further comprises a threonine or isoleucine at position -3.
  • the amino acid at position -4 is charged.
  • the amino acid at position -4 is selected from glutamic acid, lysine, arginine, and aspartic acid.
  • the amino acid at position -4 is selected from lysine and arginine.
  • the invention provides an isolated polypeptide that binds specifically to an HtrAl PDZ domain, wherein the amino acid at position 0 relative to the C- terminus is selected from leucine and valine; wherein the amino acid at position -1 is selected from tryptophan, isoleucine, and phenylalanine; wherein the amino acid at position -2 is tryptophan; wherein the amino acid at position -3 is selected from threonine and isoleucine; and wherein the amino acid at position-4 is selected from glutamic acid, lysine, arginine, and aspartic acid.
  • the amino acid at position 0 is leucine; the amino acid at position -1 is tryptophan; the amino acid at position -3 is selected from threonine and isoleucine; and the amino acid at position-4 is selected from lysine and arginine.
  • the invention provides an isolated polypeptide that binds specifically to an HtrAl PDZ domain, wherein the polypeptide comprises a sequence selected from the sequences of HtrAl PDZ domain-binding peptides set forth in Table 1.
  • the polypeptide comprises the sequence DIETWLL (SEQ ID NO: 3), GWKTWIL (SEQ ID NO: 4), DSRIWWV (SEQ ID NO: 5), or WDKIWHV (SEQ ID NO: 6).
  • the polypeptide comprises the sequence DSRIWWV (SEQ ID NO: 5).
  • the invention provides an isolated polypeptide that binds specifically to an HtrAl PDZ domain, wherein the polypeptide directly interacts with at least one specific HtrAl -PDZ domain residue.
  • the C-terminal carboxylate group of the isolated polypeptide is coordinated by at least one HtrAl PDZ domain residue selected from Ile383, Ile385, and Gly384.
  • the amino acid at position -1 of the isolated polypeptide dynamically interacts with at least one HtrAl PDZ domain residue selected from Tyr382, Arg386, and Ile418.
  • the tryptophan at position -2 of the isolated polypeptide interacts with at least one HtrAl PDZ domain residue selected from Ala445, Met387, and Gln446.
  • the amino acid at position -3 of the isolated polypeptide interacts with at least one HtrAl PDZ domain residue selected from Ile415 and Arg386.
  • the isolated polypeptide interacts with and/or is coordinated by at least one HtrAl PDZ domain residue selected from Tyr382, Ile383, Gly384, Met387, and S389.
  • the invention provides an isolated polypeptide that binds specifically to an HtrAl PDZ domain, wherein the polypeptide comprises a sequence having a tryptophan at position -2 relative to an acidic amino acid.
  • the polypeptide comprises a sequence according to the formula X1-X2-W-X3-X4, wherein Xl is selected from valine and leucine; wherein X2 is selected from serine, threonine, arginine, alanine, and valine; wherein X3 is selected from glycine, serine, phenylalanine, and leucine; and wherein X4 is an acidic amino acid.
  • X3 is glycine and X4 is selected from glutamic acid and aspartic acid.
  • the invention provides an isolated polypeptide that binds specifically to HtrAl PDZ domain and comprises a C-terminal, N-terminal, or internal amino acid sequence comprising the amino acid sequence of a peptide selected from the amino acid sequences set forth in Tables 1, 2, and 3.
  • the C terminal amino acid sequence is selected from DIETWLL (SEQ ID NO: 3), GWKTWIL (SEQ ID NO: 4), DSRIWWV(SEQ ID NO: 5), and WDKIWHV(SEQ ID NO: 6).
  • the invention provides an isolated polypeptide comprising an amino acid sequence that competes with any of the above-described polypeptides for binding to HtrAl PDZ domain. In another embodiment, the invention provides an isolated polypeptide that binds to the same epitope on HtrAl PDZ domain as any of the above- described polypeptides. In another embodiment, the invention provides a variant HtrAl PDZ domain.
  • an isolated polypeptide comprising an HtrAl PDZ variant sequence wherein at least one HtrAl PDZ domain residue selected from Ile383, Ile385, Gly384, Tyr382, Arg386, Ile418, Ala445, Met387, GIn 446, Ile415, Arg386, Ser389, Lys380, Lys381, G1411, Tyr413, Ile414, Val417, Thr421, and Pro422 is substituted with another amino acid is provided.
  • Ile418 is substituted with another amino acid.
  • the other amino acid is alanine.
  • the invention provides an isolated polypeptide comprising an amino acid sequence that competes with any of the above-described HtrAl PDZ domain variants for binding to a ligand of HtrAl PDZ domain.
  • the invention provides an isolated polypeptide that binds to the same epitope on a ligand of HtrAl PDZ domain as any of the above-described HtrAl PDZ domain variants.
  • the invention provides methods of using the above-described HtrAl PDZ domain variant polypeptides and HtrAl PDZ domain-binding polypeptides.
  • the invention provides methods of identifying a compound capable of modulating an HtrAl PDZ domain-ligand interaction, comprising contacting a sample comprising HtrAl PDZ domain, a fragment of HtrAl PDZ domain and/or a functional equivalent thereof and at least one of the HtrAl PDZ domain-binding polypeptides described above with a candidate compound, and determining the amount of HtrAl PDZ domain-ligand interaction in the presence of the candidate compound as compared to the absence of the candidate compound; whereby a change in the amount of HtrAl PDZ domain-ligand interaction in the presence of the candidate compound as compared to the amount in the absence of the candidate compound indicates that the candidate compound is a compound capable of modulating HtrAl PDZ domain-ligand interaction
  • the invention provides methods of rationally designing a modulator of HtrAl PDZ domain-ligand interaction comprising designing the modulator to comprise or mimic the function of a tryptophan located at position -2 relative to the C-terminus or relative to an acidic residue in a peptide, wherein the modulator is capable of specifically binding to HtrAl PDZ domain.
  • the peptide is at the carboxy terminus.
  • the amino acid at position 0 is selected from leucine and valine
  • the amino acid position -1 is selected from tryptophan, isoleucine, and phenylalanine
  • the amino acid at position -3 is selected from threonine and isoleucine
  • the amino acid at position -4 is selected from glutamic acid, aspartic acid, lysine, and arginine.
  • the amino acid at position 0 is selected from leucine and valine
  • the amino acid at position -1 is tryptophan
  • the amino acid at position -3 is selected from threonine and isoleucine
  • the amino acid at position -4 is selected from glutamic acid and aspartic acid.
  • the invention provides methods of treating a pathological condition associated with dysregulation of HtrAl protein activity comprising administering to a subject in need thereof an effective amount of an HtrAl PDZ domain ligand described above.
  • the invention provides methods of treating a pathological condition associated with dysregulation of HtrAl protein activity comprising administering to a subject in need thereof an effective amount of an HtrAl PDZ domain-ligand modulator, wherein the modulator is capable of modulating an interaction between HtrAl PDZ domain and any of the above-described HtrAl PDZ domain-binding polypeptides.
  • the invention provides methods of treating a pathological condition associated with dysregulation of HtrAl protein activity comprising administering to a subject in need thereof an effective amount of a variant HtrAl PDZ domain polypeptide described above, wherein the variant HtrAl PDZ domain polypeptide is capable of modulating an interaction between HtrAl PDZ domain and an HtrAl PDZ domain ligand.
  • the modulating is inhibiting interaction between HtrAl PDZ domain and a ligand.
  • the modulating is enhancing interaction between HtrAl PDZ domain and a ligand.
  • the pathological condition is selected from malignant and benign tumors or cancers, non- leukemias and lymphoid malignancies; neurodegenerative disorders; inflammatory and immunologic disorders; and intraocular neovascular diseases.
  • the pathological condition is selected from cancer (including, but not limited to, ovarian and endometrial cancer), Alzheimer's disease, rheumatoid arthritis, and age-related (wet) macular degeneration.
  • the invention provides methods of treating a pathological condition associated with dysregulation of HtrAl protein activity comprising administering to a subject in need thereof an effective amount of an agonist of the interaction of any of the HtrAl PDZ domain-binding proteins described above and HtrAl PDZ domain.
  • the invention provides methods of treating a pathological condition associated with dysregulation of HtrAl protein activity comprising administering to a subject in need thereof an effective amount of an antagonist of the interaction of any of the HtrAl PDZ domain-binding proteins described above and HtrAl PDZ domain.
  • the pathological condition is selected from malignant and benign tumors or cancers, non-leukemias and lymphoid malignancies; neurodegenerative disorders; inflammatory and immunologic disorders; and intraocular neovascular diseases.
  • the pathological condition is selected from cancer (including, but not limited to, ovarian and endometrial cancer), Alzheimer's disease, rheumatoid arthritis, and age-related (wet) macular degeneration.
  • the invention provides methods of detecting the presence, amount, or function of HtrAl or HtrAl PDZ domain in a sample using one or more of the polypeptides, polynucleotides, and/or antibodies described above.
  • the invention provides an isolated polypeptide that binds specifically to an HtrA3 PDZ domain, wherein the polypeptide comprises a sequence having a tryptophan at position -1 relative to the C-terminus.
  • the polypeptide has overall hydrophobic character.
  • the polypeptide further comprises an amino acid at position 0 selected from valine, isoleucine, and alanine.
  • the polypeptide further comprises an amino acid at position -3 selected from glycine and serine.
  • the amino acid at position 0 is selected from valine, isoleucine, and alanine; and wherein the amino acid at position -3 is selected from glycine and serine.
  • the amino acid at position 0 is valine and the amino acid at position -3 is selected from glycine and serine.
  • the invention provides an isolated polypeptide that specifically binds to HtrA3 PDZ domain, wherein the polypeptide comprises a sequence selected from the sequences of HtrA3 PDZ domain-binding peptides set forth in Table 1 and Table 3.
  • the polypeptide comprises the sequence PGRWV (SEQ ID NO: 7), SGKGIWV (SEQ ID NO: 8), GFWV (SEQ ID NO: 9), IFDGRWI (SEQ ID NO: 10), FGRWV (SEQ ID NO: 11), RSWWV (SEQ ID NO: 12), FGRWI (SEQ ID NO: 13), FGRWL (SEQ ID NO: 14), GRWV (SEQ ID NO: 15), WV, FLRWV (SEQ ID NO: 16), FERWV (SEQ ID NO: 17), FYRWV (SEQ ID NO: 18), FGAWV (SEQ ID NO: 19), and
  • the polypeptide comprises the sequence FGRWV (SEQ ID NO: 11).
  • the invention provides an isolated polypeptide that specifically binds to HtrA3 PDZ domain, wherein the polypeptide directly interacts with at least one specific HtrA3-PDZ domain residue.
  • the C-terminal carboxylate group of the HtrA3 PDZ domain-binding polypeptide is coordinated by at least one HtrA3 PDZ domain residue selected from Phe356, Ile357, and Gly358.
  • the tryptophan at position -1 of the HtrA3 PDZ domain-binding polypeptide interacts with at least one HtrA3 PDZ domain residue selected from Glu390 and Ala392.
  • the amino acid at position -2 of the HtrA3 PDZ domain-binding polypeptide interacts with the HtrA3 PDZ domain residue Gln423.
  • the amino acid at position -3 of the HtrA3 PDZ domain-binding polypeptide interacts with the HtrA3 PDZ domain residue Arg360.
  • the HtrA3 PDZ domain-binding polypeptide interacts with and/or is coordinated by at least one HtrA3 PDZ domain residue selected from Phe356, Ile357, Gly358, Glu390, Ala392, Gln423, and Arg360.
  • the invention provides an isolated polypeptide that binds specifically to an HtrA3 PDZ domain, wherein the polypeptide comprises a sequence having a conserved acidic residue preceded by one or more hydrophobic residues.
  • the polypeptide comprises the sequence WVL.
  • the polypeptide comprises the sequence GVVVDEWMLSLL (SEQ ID NO: 21), GVVVDEWVLSLL (SEQ ID NO: 22), ELLVDGYVLELL (SEQ ID NO: 23), or GVVVNEWVLSLL (SEQ ID NO: 24).
  • the invention provides an isolated polypeptide that binds specifically to an HtrA3 PDZ domain, wherein the polypeptide comprises a sequence selected from the HtrA3 PDZ domain-binding sequences set forth in Table 2.
  • the invention provides an isolated polypeptide that binds specifically to HtrA3 PDZ domain and comprises a C-terminal, N-terminal, or internal amino acid sequence comprising the amino acid sequence of a peptide selected from the amino acid sequences set forth in Tables 1, 2, and 3.
  • the C-terminal amino acid sequence is selected from PGRWV (SEQ ID NO: 7), SGKGIWV (SEQ ID NO: 8), GFWV (SEQ ID NO: 9), IFDGRWI (SEQ ID NO: 10), FGRWV (SEQ ID NO: 11), RSWWV (SEQ ID NO: 12), FGRWI (SEQ ID NO: 13), FGRWL (SEQ ID NO: 14), GRWV (SEQ ID NO: 15), WV, FLRWV (SEQ ID NO: 16), FERWV (SEQ ID NO: 17), .FYRWV (SEQ ID NO: 18), FGAWV (SEQ ID NO: 19), and FARWV (SEQ ID NO: 20).
  • the invention provides an isolated polypeptide comprising an amino acid sequence that competes with any of the above-described HtrA3 PDZ domain- binding polypeptides for binding to HtrA3 PDZ domain. In another embodiment, the invention provides an isolated polypeptide comprising an amino acid sequence that binds to the same epitope on HtrA3 PDZ domain as any of the above-described HtrA3 PDZ domain binding polypeptides.
  • the invention provides an isolated variant HtrA3 PDZ domain polypeptide.
  • the isolated polypeptide comprises an HtrA3 PDZ variant sequence wherein at least one HtrA3 PDZ domain residue selected from Phe356, Ile357, Gly358, Glu390, Ala392, Gln423, and Arg360 is substituted with another amino acid.
  • Glu390 and/or Ala392 of the isolated variant polypeptide is substituted with another amino acid.
  • the other amino acid is alanine.
  • the invention provides an isolated polypeptide comprising an amino acid sequence that competes with any of the above-described variant HtrA3 PDZ domain polypeptides for binding to a ligand of HtrA3 PDZ domain.
  • the invention provides an isolated polypeptide that binds to the same epitope on a ligand of HtrA3 PDZ domain as any of the variant HtrA3 PDZ domain polypeptides described above.
  • the invention provides methods of using the above-described
  • the invention provides methods of identifying a compound capable of modulating an HtrA3 PDZ domain-ligand interaction, comprising contacting a sample comprising HtrA3 PDZ domain, a fragment of HtrA3 PDZ domain and/or a functional equivalent thereof and at least one of the HtrA3 PDZ domain-binding polypeptides described above with a candidate compound, and determining the amount of HtrA3 PDZ domain-ligand interaction in the presence of the candidate compound as compared to the absence of the candidate compound; whereby a change in the amount of HtrA3 PDZ domain-ligand interaction in the presence of the candidate compound as compared to the amount in the absence of the candidate compound indicates that the candidate compound is a compound capable of modulating HtrA3 PDZ domain-ligand interaction.
  • the invention provides methods of rationally designing a modulator of HtrA3 PDZ domain-ligand interaction comprising designing the modulator to comprise or mimic the function of a tryptophan located at position -1 relative to the C-terminus or at position-2 relative to leucine in a peptide, wherein the modulator is capable of specifically binding to HtrA3 PDZ domain.
  • the amino acid at position 0 is selected from valine, isoleucine, and alanine
  • the amino acid at position -1 is tryptophan
  • the amino acid at position -3 is selected from glycine and serine.
  • the amino acid at position 0 is valine
  • the amino acid at position -1 is tryptophan
  • the amino acid at position -3 is selected from glycine and serine.
  • the modulator comprises the amino acid sequence WVL.
  • the invention provides methods of treating a pathological condition associated with dysregulation of HtrA3 protein activity comprising administering to a subject in need thereof an effective amount of an HtrA3 PDZ domain ligand described above.
  • the invention provides methods of treating a pathological condition associated with dysregulation of HtrA3 protein activity comprising administering to a subject in need thereof an effective amount of an HtrA3 PDZ domain-ligand modulator, wherein the modulator is capable of modulating an interaction between HtrA3 PDZ domain and any of the above-described HtrA3 PDZ domain-binding polypeptides.
  • the invention provides methods of treating a pathological condition associated with dysregulation of HtrA3 protein activity comprising administering to a subject in need thereof an effective amount of a variant HtrA3 PDZ domain polypeptide described above, wherein the variant HtrA3 PDZ domain polypeptide is capable of modulating an interaction between HtrA3 PDZ domain and an HtrA3 PDZ domain ligand.
  • the modulating is inhibiting interaction between HtrA3 PDZ domain and a ligand.
  • the modulating is enhancing interaction between HtrA3 PDZ domain and a ligand.
  • the pathological condition is selected from malignant and benign tumors or cancers, non- leukemias and lymphoid malignancies and placental dysfunction.
  • the pathological condition is cancer (including, but not limited to, endometrial cancer).
  • the invention provides methods of treating a pathological condition associated with dysregulation of HtrA3 protein activity comprising administering to a subject in need thereof an effective amount of an agonist of the interaction of any of the HtrA3 PDZ domain-binding proteins described above and HtrA3 PDZ domain.
  • the invention provides methods of treating a pathological condition associated with dysregulation of HtrA3 protein activity comprising administering to a subject in need thereof an effective amount of an antagonist of the interaction of any of the HtrA3 PDZ domain-binding proteins described above and HtrA3 PDZ domain.
  • the pathological condition is selected from malignant and benign tumors or cancers, non-leukemias and lymphoid malignancies and placental dysfunction.
  • the pathological condition is cancer (including, but not limited to, endometrial cancer).
  • the invention provides methods of detecting the presence, amount, or function of HtrA3 or HtrA3 PDZ domain in a sample using one or more of the polypeptides, polynucleotides, and/or antibodies described above.
  • the invention provides an isolated polynucleotide encoding any of the above-described HtrAl PDZ domain-binding polypeptides, HtrA3 PDZ domain- binding polypeptides, variant HtrAl PDZ domains, or variant HtrA3 PDZ domains, or a complement thereof.
  • the invention provides a vector comprising one or more such polynucleotides.
  • the invention provides a host cell comprising one or more such vectors.
  • the invention provides a method of producing a polypeptide comprising culturing such host cells under conditions in which the polynucleotide is expressed and optionally recovering and/or purifying the polypeptide.
  • the invention provides a transgenic nonhuman mammal expressing one or more such polynucleotides.
  • the transgenic nonhuman mammal further has at least one inactivated HtrAl or HtrA3 gene.
  • the invention provides antibodies that specifically bind to one or more of the above-described HtrAl PDZ domain-binding polypeptides, HtrA3 PDZ domain-binding polypeptides, variant HtrAl PDZ domains, or variant HtrA3 PDZ domains.
  • the invention provides kits including one or more of the molecules of the invention. In one aspect, such a kit comprises at least one of the above-described HtrAl PDZ domain-binding polypeptides, HtrA3 PDZ domain-binding polypeptides, variant HtrAl PDZ domains, or variant HtrA3 PDZ domains.
  • such a kit comprises at least one isolated polynucleotide encoding one of the above-described HtrAl PDZ domain-binding polypeptides, HtrA3 PDZ domain-binding polypeptides, variant HtrAl PDZ domains, or variant HtrA3 PDZ domains.
  • such a kit comprises at least one antibody that specifically binds to at least one of the above-described HtrAl PDZ domain-binding polypeptides, HtrA3 PDZ domain-binding polypeptides, variant HtrAl PDZ domains, or variant HtrA3 PDZ domains.
  • Figure 1 depicts a sequence alignment of human HtrA family proteins, with the sequence numbering to the left of each sequence. Elements of secondary structure are indicated above the sequences. Residues that are identical in all four members are highlighted with broken-line boxes, and residues that are similar in all four members are boxed with solid lines. Residues that are directly involved in ligand recognition and important for ligand specificity are indicated below the sequences as filled stars (for site 0), filled triangles (site -1 and site -3) or filled circles (site -2).
  • FIG. 1 The ligand-binding specificity profile for HtrAl, HtrA2, and HtrA3 as determined by phage display and synthetic peptide affinity assays is summarized at the bottom right of the figure, where X indicates no preference and ⁇ indicates a preference for hydrophobic amino acids.
  • Figure 2 depicts experimental calorimetric data of the binding of peptide Hl_c3 to HtrAl-PDZ.
  • the top panel shows the raw heat data obtained during a single titration experiment in which 10 ⁇ L injections of peptide (284 ⁇ M) are titrated into HtrAl-PDZ domain (5.5 ⁇ M) in PBS buffer with 1 mM sodium azide.
  • the integrated heat signals of the data from the top panel give rise to the binding curve shown in the bottom panel.
  • the solid line represents a non-linear least squares fit of the data, after having subtracted the heats of dilution, based on a single binding site model.
  • Figure 3 depicts the structure of HtrAl-PDZ bound to phage-derived peptide ligands, as described in Example 3 (a).
  • Figure 3 A shows an ensemble of structures for HtrAl-PDZ in complex with peptide H 1 3. Only the backbone N, Ca, and C atoms are shown as lines. The HtrAl-PDZ structure is shown in light grey, while the peptide H 1 3 is shown in dark grey. Selected peptide side chain heavy atoms are included. Root mean square deviation to the mean structure was 0.52 ⁇ 0.1 A for the backbone heavy atoms of residues 378-389, 411-463, and 468-475.
  • Figure 3B shows a ribbon view of HtrAl-PDZ bound to the H1 3 peptide (the peptide is shown in stick representation). Elements of regular secondary structure and peptide residues are labeled.
  • Figure 4 A depicts a dimer of the HtrA3-PDZext structure, as described in Example 3(b).
  • the PDZ domains are shaded dark grey and the pentapeptides are shown in light grey.
  • the peptide is linked to the C terminus of the PDZ domain via three glycine residues.
  • the HtrA3-PDZext structure contains two molecules per asymmetric unit, and both copies are well-defined in the electron density with the exception of a disordered region between strands ⁇ l and ⁇ 2.
  • Figure 4B shows a ribbon view of HtrA3-PDZext, with the peptide shown in stick representation. Elements of regular secondary structure and peptide residues are labeled.
  • Figure 5 depicts the interactions of certain PDZ domains with particular peptide ligands, as described in Example 3(c).
  • Figure 5A shows HtrAl-PDZ bound to peptide Hl_c3.
  • Figure 5B shows HtrA2-PDZ bound to peptide H2_cl (WTMFWVCOO H ) (SEQ ID NO: 170).
  • Figure 5 C sho ws HtrA3 -PDZ bound to peptide H3_cl.
  • Figures 5D-5F show unliganded DegP-PDZl (Figure 5D, pdb entry 1KY9), unliganded DegS-PDZ ( Figure 5E, pdb entry ISOT), and DegS-PDZ bound to OmpC peptide ( Figure 5F, pdb entry ISOZ).
  • the structures are shown in the same relative orientation.
  • the peptides shown in panels A, B, C, and F appear at the center of each figure.
  • Side chain nitrogen atoms are shown in dark grey.
  • Peptide ligand side chains are labeled in grey with three letter amino acid code and ligand position numbers in superscript, while side chains for the PDZ domains are labeled in black with single letter amino acid code and residue number.
  • Figure 6 shows heteronuclear NOE measurements for the HtrAl-PDZ complex, as described in Example 3(c).
  • Figure 7 depicts the mapping of shotgun alanine scanning for peptide Hl_c3 ( Figure
  • the invention provides molecules and methods for identifying and using molecules capable of modulating binding interactions between the PDZ domain of the HtrAl or HtrA3 protein and its cellular binding partner(s).
  • these molecules are generated by a combinatorial approach that results in the identification of peptide binders capable of binding to HtrAl PDZ or HtrA3 PDZ domains at various affinities.
  • Oligonucleotides, polynucleotides, peptides, polypeptides and small molecules employed or described in the present invention can be generated using standard techniques known in the art.
  • Isolated when referred to a molecule, refers to a molecule that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that interfere with diagnostic or therapeutic use.
  • Control sequences are DNA sequences that enable the expression of an operably-linked coding sequence in a particular host organism.
  • Prokaryotic control sequences include promoters, operator sequences, and ribosome binding sites.
  • Eukaryotic control sequences include promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is "operably-linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably-linked to a coding sequence if it affects the transcription of the sequence, or a ribosome-binding site is operably-linked to a coding sequence if positioned to facilitate translation.
  • a promoter or enhancer is operably-linked to a coding sequence if it affects the transcription of the sequence
  • a ribosome-binding site is operably-linked to a coding sequence if positioned to facilitate translation.
  • operably-linked means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous.
  • an "active" polypeptide, or fragments thereof, retains a biological activity of native or naturally-occurring counterpart of the active polypeptide.
  • Biological activity refers to a function mediated by the native or naturally-occurring counterpart of the active polypeptide.
  • binding or protein-protein interaction constitutes a biological activity.
  • antibody and “immunoglobulin” are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies ⁇ e.g., bispecific antibodies so long as they exhibit the desired biological activity) and may also include certain antibody fragments (as described in greater detail herein).
  • the light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
  • antibodies can be assigned to different classes.
  • immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-I, IgG-2, IgA-I, IgA-2, and etc.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.
  • Antibody fragments comprise only a portion of an intact antibody, wherein the portion preferably retains at least one, preferably most or all, of the functions normally associated with that portion when present in an intact antibody.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non- human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • a "human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Methods of producing a human antibody are well known in the art and include as a nonlimiting example xenomouse technology (e.g., as described in WO96/33735).
  • affinity matured antibody is one with one or more alterations in one or more CDRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s).
  • Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen.
  • Affinity matured antibodies are produced by procedures known in the art. Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described by: Barbas et al Proc Nat. Acad.
  • an “epitope tagged” polypeptide refers to a chimeric polypeptide fused to a "tag polypeptide". Such tags provide epitopes against which Abs can be made or are available, but do not substantially interfere with polypeptide activity. To reduce anti-tag antibody reactivity with endogenous epitopes, the tag polypeptide is usually unique. Suitable tag polypeptides generally have at least six amino acid residues, usually between about 8 and 50 amino acid residues, preferably between 8 and 20 amino acid residues. Examples of epitope tag sequences include HA from Influenza A virus, GD, and c-myc, poly-His and FLAG.
  • Polynucleotide or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include, but are not limited to, DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non- nucleotide components.
  • a polynucleotide may be further modified after synthesis, such as by conjugation with a label.
  • Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals,
  • any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports.
  • the 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping groups moieties of from 1 to 20 carbon atoms.
  • Other hydroxyls may also be derivatized to standard protecting groups.
  • Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-O-methyl-, 2'-O- allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, . alpha. -anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.
  • One or more phosphodiester linkages may be replaced by alternative linking groups.
  • linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S("thioate"), P(S)S ("dithioate"), "(O)NR.sub.2 ("amidate"), P(O)R, P(O)OR', CO or CH. sub.2 ("formacetal"), in which each R or R is independently H or substituted or unsubstituted alkyl (1-20 C.) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
  • Oligonucleotide generally refers to short, generally single stranded, generally synthetic polynucleotides that are generally, but not necessarily, less than about 200 nucleotides in length.
  • oligonucleotide and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.
  • peptide generally refers to a contiguous and relatively short sequence of amino acids linked by peptidyl bonds. Typically, but not necessarily, a peptide has a length of about 2 to 50 amino acids, 4-40 amino acids or 10-30 amino acids. Although the term “polypeptide” generally refers to longer forms of a peptide, the two terms can be and are used interchangeably in some contexts herein.
  • a “region,” of a polypeptide is a contiguous sequence of 2 or more amino acids. In other embodiments, a region is at least about any of 3, 5, or 10 contiguous amino acids.
  • the "C-terminal region”, or variants thereof refers to a region of a polypeptide that includes the 1-5 residues located closest to the C terminus of the polypeptide.
  • the "N-terminal region”, or variants thereof refers to a region of a polypeptide that includes the 1-5 residues located closest to the N terminus of the polypeptide.
  • An “internal” region of a polypeptide refers to a region of a polypeptide that is located neither at the N-terminus of the polypeptide nor at the C -terminus of the polypeptide.
  • PDZ domains generally bind to short carboxyl-terminal peptide sequences located on the carboxyl-terminal end of interacting proteins. Usually, PDZ domains comprise two ⁇ helixes and six ⁇ sheets.
  • “HtrAl PDZ domain”, “HtrAl PDZ”, and variations thereof, refer to part or all of the sequence of SEQ ID NO: 1
  • HtrA3 PDZ domain “HtrA3 PDZ” and variations thereof, refer to part or all of the sequence of SEQ ID NO: 2
  • a "ligand” refers to a naturally-occurring or synthetic molecule or moiety that is capable of a binding interaction with a specific site on a protein or other molecule.
  • An HtrAl PDZ domain ligand is a molecule or moiety that specifically interacts with HtrAl PDZ domain.
  • An HtrA3 PDZ domain ligand is a molecule or moiety that specifically interacts with HtrA3 PDZ domain. Examples of ligands include proteins, peptides, and small organic and inorganic molecules.
  • a “fusion protein” refers to a polypeptide having two portions covalently linked together, where each of the portions is derived from different proteins.
  • the two portions may be linked directly by a single peptide bond or through a peptide linker containing one or more amino acid residues.
  • the two portions and the linker will be in reading frame with each other and are produced using recombinant techniques.
  • a “disorder” or “pathological condition” is any condition that would benefit from treatment with a substance/molecule or method of the invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.
  • HtrAl -related disorders to be treated herein include malignant and benign tumors or cancers, non-leukemias and lymphoid malignancies; neurodegenerative disorders; inflammatory and immunologic disorders; and intraocular neovascular diseases.
  • Non-limiting examples of HtrA3 -related disorders include malignant and benign tumors or cancers, non-leukemias and lymphoid malignancies and placental dysfunction.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation.
  • examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, melanoma, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
  • a "neurodegenerative disorder” includes, but is not limited to a disease or disorder of the central and/or peripheral nervous system in mammals that is typically characterized by deterioration of nervous tissue or deterioration of communication between cells in nervous tissue.
  • Examples of neurodegenerative disorders include, but are not limited to, neurodegenerative diseases (including, but not limited to, Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, tauopathies (including, but not limited to, Alzheimer disease and supranuclear palsy), prion diseases
  • spongiform encephalopathy including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt- Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia
  • bulbar palsy including, but not limited to, Canavan disease, Huntington's disease, neuronal ceroid-lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome
  • dementia including, but not limited to, Pick's disease, and spinocerebellar ataxia.
  • an "intraocular neovascular disease” includes, but is not limited to, proliferative retinopathies, e.g., diabetic retinopathy, macular degeneration (for example, age- related macular degeneration (AMD) or "dry” macular degeneration), neovascular glaucoma, and immune rejection of transplanted corneal tissue and other tissues.
  • AMD age-related macular degeneration
  • AMD age-related macular degeneration
  • drusen a progressive neovascular lesion characterized by a broad spectrum of clinical and pathologic findings, such as pale yellow spots known as drusen, disruption of the retinal pigment epithelium (RPE), choroidal neovascularization (CNV), and disciform macular degeneration.
  • RPE retinal pigment epithelium
  • CNV choroidal neovascularization
  • disciform macular degeneration The manifestations of the disease are classified into two forms: non exudative (dry) and exudative (wet or neovascular).
  • Drusen are the characteristic lesions of the dry form, and neovascularization characterizes the wet form.
  • Disciform AMD is the fibrotic stage of the neovascular lesion.
  • Dry AMD is characterized by changes in the RPE and Bruch's membrane. It is thought that the RPE, compromised by age and other risk factors, deposits lipofuscin and cellular debris on Bruch's membrane. These changes may be seen ophthalmoscopically as drusen, which are scattered throughout the macula and posterior retinal pole. There are also variable degrees of atrophy and pigmentation of the RPE. Dry AMD may be asymptomatic or accompanied by variable and usually minimal visual loss and is considered to be a prelude to development of wet AMD. Wet AMD is characterized by CNV of the macular region. The choroidal capillaries proliferate and penetrate Bruch's membrane to reach the RPE and may extend into the subretinal space.
  • the increased permeability of the newly formed capillaries leads to accumulation of serous fluid or blood under the RPE and/or the neurosensory retina or within the neurosensory retina.
  • the fovea becomes swollen or detached, decreases in vision result. Fibrous metaplasia and organization may ensue, resulting in an elevated subretinal mass called a disciform scar that constitutes end-stage AMD and is associated with permanent vision loss (D'Amico DJ. N Engl J Med ' 331:95-106 (1994)).
  • Older therapeutic physical treatment for wet AMD included photodynamic therapy (PDT, e.g. with VISUDYNE).
  • an "inflammatory and immunologic disorder” includes, but is not limited to disorders caused by aberrant immunologic mechanisms and/or aberrant cytokine signaling.
  • inflammatory and immunologic disorders include, but are not limited to, autoimmune diseases, immunologic deficiency syndromes, and hypersensitivity.
  • autoimmune disease herein is a non-malignant disease or disorder arising from and directed against an individual's own tissues.
  • the autoimmune diseases herein specifically exclude malignant or cancerous diseases or conditions, especially excluding B cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hairy cell leukemia and chronic myeloblasts leukemia.
  • Examples of autoimmune diseases or disorders include, but are not limited to, inflammatory responses such as inflammatory skin diseases including psoriasis and dermatitis (e.g.
  • atopic dermatitis atopic dermatitis
  • systemic scleroderma and sclerosis responses associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions such as eczema and asthma and other conditions involving infiltration of T cells and chronic inflammatory responses; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; systemic lupus erythematosus (SLE) (including but not limited to lupus nephritis, cutaneous lupus); diabetes mellitus (e.g.
  • Type I diabetes mellitus or insulin dependent diabetes mellitus multiple sclerosis; Reynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenile onset diabetes; and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes typically found in tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis; pernicious anemia (Addison's disease); diseases involving leukocyte diapedesis; central nervous system (CNS) inflammatory disorder; multiple organ injury syndrome; hemolytic anemia (including, but not limited to cryoglobinemia or Coombs positive anemia) ; myasthenia gravis; antigen-antibody complex mediated diseases; anti-glomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; pemphigoid bull
  • the HtrAl PDZ domain and HtrA3 PDZ domain modulatory compounds of the invention may be used in combination with one or more additional agents to treat or prevent one or more HtrAl -related disorder or HtrA3-related disorder.
  • the polypeptides of the invention may be used in combination with one or more cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, anti-inflammatory agents, anti-angiogenic agents, and physical treatments to treat or prevent one or more HtrAl -related disorder or HtrA3 -related disorder, as will be understood by one of ordinary skill in the art.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g., At 211 , 1 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu), chemotherapeutic agents e.g.
  • methotrexate adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below.
  • a tumoricidal agent causes destruction of tumor cells.
  • chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
  • examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topote
  • calicheamicin especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAM YCIN ® doxorubicin
  • morpholino-doxorubicin including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin
  • epirubicin including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin
  • epirubicin including esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin
  • anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin,
  • chemotherapeutic agents include the cytotoxic agents useful as antibody drug conjugates, such as maytansinoids (DMl, for example) and the auristatins MMAE and MMAF, for example. Also included in this definition are anti-hormonal agents that act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer, and are often in the form of systemic, or whole-body treatment. They may be hormones themselves.
  • anti-estrogens and selective estrogen receptor modulators include, for example, tamoxifen (including NOLVADEX® tamoxifen), EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LYl 17018, onapristone, and FARESTON® toremifene; anti-progesterones; estrogen receptor down-regulators (ERDs); agents that function to suppress or shut down the ovaries, for example, leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRON® and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetate and tripterelin; other anti-androgens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the
  • SERMs selective
  • chemotherapeutic agents includes bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid/zoledronate, FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, or ACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTEC
  • a “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell either in vitro or in vivo.
  • the compound or composition inhibits growth of a tumor cell.
  • the growth inhibitory agent may be one which significantly reduces the percentage of cells (for example, tumor cells) in S phase.
  • growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce Gl arrest and M-phase arrest.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
  • Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells. "Doxorubicin” is an anthracycline antibiotic.
  • doxorubicin The full chemical name of doxorubicin is (8S-cis)-10-[(3-amino-2,3,6-trideoxy- ⁇ -L-lyxo-hexapyranosyl)oxy]-7, 8,9,10- tetrahydro-6,8,l l-trihydroxy-8-(hydroxyacetyl)-l-methoxy-5,12-naphthacenedione.
  • An "anti-angiogenic agent” as used herein is an agent that prevents or inhibits angiogenesis.
  • anti-angiogeneic agents include those known in the art, e.g., antibodies to VEGF (for example an anti-VEGF neutralizing antibody or fragment (e.g., humanized A4.6.1, AVASTIN ® (Genentech, South San Francisco, CA), Y0317, M4, G6, B20, 2C3, etc. and Lucentis®). See, e.g., U.S.
  • small molecules that block VEGF receptor signaling e.g., PTK787/ZK2284, SU6668, SUTENT®/SU11248 (sunitinib malate), AMG706
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • modulatory compounds of the invention are used to delay development of a disease or disorder.
  • an “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • a “therapeutically effective amount” of a substance/molecule of the invention, agonist or antagonist may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.
  • the prophylactically effective amount will be less than the therapeutically effective amount.
  • Modulators of HtrAl PDZ domain-ligand or HtrA3 PDZ domain-ligand Interaction The invention provides modulators, and methods for identifying modulators of HtrAl
  • HtrAl PDZ domain-ligand interaction or HtrA3 PDZ domain-ligand interaction in vivo.
  • One way to modulate the interaction between HtrAl PDZ domain or HtrA3 PDZ domain and its ligand is to inhibit the interaction.
  • Any molecule that disrupts HtrAl PDZ domain-ligand interaction or HtrA3 PDZ domain-ligand interaction can be a candidate inhibitor. Screening techniques well known to those skilled in the art can identify these molecules.
  • inhibitors include: (1) small organic and inorganic compounds, (2) small peptides, (3) antibodies and derivatives, (4) peptides closely related to HtrAl PDZ domain ligand or HtrA3 PDZ-domain ligand (5) nucleic acid aptamers.
  • HtrAl PDZ-domain-ligand interaction inhibitor includes any molecule that partially or fully blocks, inhibits, or neutralizes the interaction between HtrAl PDZ domain and its ligand.
  • Molecules that may act as such inhibitors include peptides that bind HtrAl PDZ domain, such as the peptide binders listed in Tables 1, 2, and 3 (for example and in particular peptides DIETWLL (SEQ ID NO: 3), GWKTWIL (SEQ ID NO: 4), DSRIWWV (SEQ ID NO: 5), or WDKIWHV (SEQ ID NO: 6)), antibodies (Ab's) or antibody fragments, fragments or variants of endogenous HtrAl PDZ domain ligands, cognate HtrAl PDZ- containing polypeptides; variants of HtrAl PDZ-containing polypeptides (e.g., wherein the HtrAl PDZ domain sequence comprises one or
  • HtrA3 PDZ-domain-ligand interaction inhibitor includes any molecule that partially or fully blocks, inhibits, or neutralizes the interaction between HtrA3 PDZ domain and its ligand.
  • Molecules that may act as such inhibitors include peptides that bind HtrA3 PDZ domain, such as the peptide binders listed in Tables 1, 2, and 3 (for example and in particular peptides PGRWV (SEQ ID NO: 7), SGKGIWV (SEQ ID NO: 8), GFWV (SEQ ID NO: 9), IFDGRWI (SEQ ID NO: 10), FGRWV (SEQ ID NO: 11), RSWWV (SEQ ID NO: 12), FGRWI (SEQ ID NO: 13), FGRWL (SEQ ID NO: 14), GRWV (SEQ ID NO: 15), WV, FLRWV (SEQ ID NO: 16), FERWV (SEQ ID NO: 17), FYRWV (SEQ ID NO: 18
  • Small molecules can be useful modulators of HtrAl PDZ domain-ligand interaction and/or HtrA3 PDZ domain-ligand interaction. Small molecules that inhibit either interaction are potentially useful inhibitors.
  • small molecule modulators include small peptides, pep tide-like molecules, preferably soluble, and synthetic, non-pep tidyl organic or inorganic compounds.
  • a "small molecule” refers to a composition that has a molecular weight of preferably less than about 5 kD, preferably less than about 4 kD, and preferably less than 0.6 kD.
  • Small molecules can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules.
  • a cell-free assay comprises contacting HtrAl PDZ domain or HtrA3 PDZ domain with a known binder compound (such as one or more of the binder peptides described herein) to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with HtrAl PDZ domain or HtrA3 PDZ domain or the binder compound, where determining the ability of the test compound to interact with HtrAl PDZ domain or HtrA3 PDZ domain or the binder compound comprises determining whether a detectable characteristic of HtrAl PDZ domain/binder complex or HtrA3 PDZ domain/binder complex is modulated.
  • a known binder compound such as one or more of the binder peptides described herein
  • the binding interaction of HtrAl PDZ domain and the binder compound can be indicative of whether the test compound is able to modulate the interaction between HtrAl PDZ domain and the binder compound.
  • the binding interaction of HtrA3 PDZ domain and the binder compound as determined by the amount of complex that is formed, can be indicative of whether the test compound is able to modulate the interaction between HtrA3 PDZ domain and the binder compound.
  • the amount of complex can be assessed by methods known in the art, some of which are described herein, for example ELISA (including competitive binding ELISA), yeast two-hybrid, Biacore® assays, and proximity (e.g., fluorescent resonance energy transfer, enzyme-substrate) assays.
  • ELISA including competitive binding ELISA
  • yeast two-hybrid yeast two-hybrid
  • Biacore® assays yeast two-hybrid
  • proximity e.g., fluorescent resonance energy transfer, enzyme-substrate
  • One aspect of the invention pertains to isolated peptide/polypeptide modulators of the interaction between HtrAl PDZ domain or HtrA3 PDZ domain and their cellular and/or physiological binding partner(s).
  • the binder peptides described herein, and peptide modulators obtained by methods described herein are also suitable for use as immunogens to raise antibody modulators of this interaction.
  • modulators (such as peptides and antibodies) can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • the modulators are produced by recombinant DNA techniques.
  • modulators can be synthesized chemically using standard peptide synthesis techniques.
  • HtrAl PDZ domain binder peptides and HtrA3 PDZ domain binder peptides of the invention include those described in Tables 1, 2, and 3.
  • the invention also provides a mutant or variant protein any of which residues may be changed from the corresponding residues of these peptides, while still encoding a peptide that maintains modulatory activity.
  • a variant of a binder peptide/polypeptide/ligand has at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% amino acid sequence identity with the sequence of a reference binder peptide/polypeptide/ligand.
  • variants of the invention exhibits substantially the same or greater binding affinity than the reference binder peptide/polypeptide/ligand, e.g., at least 0.75X, 0.8X, 0.9X, 1.0X, 1.25X or 1.5X the binding affinity of the reference binder peptide/polypeptide/ligand, based on an art-accepted binding assay quantitation unit/metric.
  • variants of the invention include variants in which residues at a particular position in the sequence have been substituted by other amino acids, and further includes the possibility of inserting an additional residue or residues between two residues of the parent protein/peptide as well as the possibility of deleting one or more residues from the parent sequence or adding one or more residues to the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In certain circumstances, the substitution is a conservative substitution as described herein.
  • Percent (%) amino acid sequence identity is defined as the percentage of amino acid residues that are identical with amino acid residues in a reference (parent) polypeptide sequence when the two sequences are aligned. To determine % amino acid identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum % sequence identity; conservative substitutions are not considered as part of the sequence identity. Amino acid sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align peptide sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B can be calculated as:
  • % amino acid sequence identity X/Y ' 100 where X is the number of amino acid residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and
  • Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
  • an "isolated” or “purified” peptide, polypeptide, protein or biologically active fragment is separated and/or recovered from a component of its natural environment.
  • Contaminant components include materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous materials.
  • Preparations having preferably less than 30% by dry weight of non-desired contaminating material (contaminants), preferably less than 20%, 10%, and preferably less than 5% contaminants are considered to be substantially isolated.
  • An isolated, recombinantly-produced peptide/polypeptide or biologically active portion thereof is preferably substantially free of culture medium, i.e., culture medium represents preferably less than 20%, preferably less than about 10%, and preferably less than about 5% of the volume of a peptide/polypeptide preparation.
  • culture medium represents preferably less than 20%, preferably less than about 10%, and preferably less than about 5% of the volume of a peptide/polypeptide preparation.
  • contaminants include cell debris, culture media, and substances used and produced during in vitro synthesis of the peptide/polypeptide.
  • Substantial modifications in the biological properties of the peptide/polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side-chain properties:
  • hydrophobic norleucine, met, ala, val, leu, ile
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • Variants of antibody modulators of HtrAl PDZ domain-ligand interaction or HtrA3 PDZ domain-ligand interaction can also be made based on information known in the art, without substantially affecting the activity of antibody.
  • antibody variants can have at least one amino acid residue in the antibody molecule replaced by a different residue.
  • the sites of greatest interest for substitutional mutagenesis generally include the hypervariable regions, but framework region (FR) alterations are also contemplated.
  • one type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody).
  • a parent antibody e.g. a humanized or human antibody.
  • the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated.
  • a convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g. 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibodies thus generated are displayed from filamentous phage particles as fusions to the gene III product of M 13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. binding affinity) as herein disclosed.
  • alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen.
  • Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.
  • Nucleic acid molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the antibody. It may be desirable to introduce one or more amino acid modifications in an Fc region of the immunoglobulin polypeptides of the invention, thereby generating a Fc region variant.
  • the Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions including that of a hinge cysteine.
  • the Fc region variant may display altered neonatal Fc receptor
  • Such variant Fc regions may comprise an amino acid modification at any one or more of amino acid positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439 or 447 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat.
  • Fc region variants with reduced binding to an FcRn may comprise an amino acid modification at any one or more of amino acid positions 252, 253, 254, 255, 288, 309, 386, 388, 400, 415, 433, 435, 436, 439 or 447 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat.
  • the above-mentioned Fc region variants may, alternatively, display increased binding to FcRn and comprise an amino acid modification at any one or more of amino acid positions 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat.
  • the Fc region variant with reduced binding to an Fc ⁇ R may comprise an amino acid modification at any one or more of amino acid positions 238, 239, 248, 249, 252, 254, 265,
  • the Fc region variant may display reduced binding to an Fc ⁇ RI and comprise an amino acid modification at any one or more of amino acid positions 238, 265,
  • the Fc region variant may display reduced binding to an Fc ⁇ RII and comprise an amino acid modification at any one or more of amino acid positions 238, 265, 269, 270, 292, 294, 295, 298, 303, 324, 327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat.
  • the Fc region variant of interest may display reduced binding to an Fc ⁇ RIII and comprise an amino acid modification at one or more of amino acid positions 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 293, 294, 295, 296, 301, 303, 322, 327, 329, 338, 340, 373, 376, 382, 388, 389, 416, 434, 435 or 437 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat.
  • Fc region variants with altered (i.e. improved or diminished) CIq binding and/or Complement Dependent Cytotoxicity (CDC) are described in WO99/51642.
  • Such variants may comprise an amino acid substitution at one or more of amino acid positions 270, 322, 326, 327, 329, 331, 333 or 334 of the Fc region.
  • CDC Complement Dependent Cytotoxicity
  • Desired polynucleotide sequences may be isolated and sequenced from appropriate source cells.
  • Source cells for antibodies would include antibody producing cells such as hybridoma cells.
  • polynucleotides can be synthesized using a nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the peptide or polypeptide are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a host cell.
  • Many vectors that are available and known in the art can be used for the purpose of the present invention. Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components, depending on its function
  • the vector components generally include, but are not limited to: an origin of replication (in particular when the vector is inserted into a prokaryotic cell), a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert and a transcription termination sequence.
  • plasmid vectors containing replicon and control sequences which are derived from a species compatible with the host cell are used in connection with these hosts.
  • the vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells.
  • E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species.
  • pBR322 contains genes encoding ampicillin (Amp) and tetracycline (T et) resistance and thus provides easy means for identifying transformed cells.
  • pBR322 its derivatives, or other microbial plasmids or bacteriophage may also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of endogenous proteins.
  • phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts.
  • bacteriophage such as ⁇ GEM.TM.-l 1 may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.
  • Either constitutive or inducible promoters can be used in the present invention, in accordance with the needs of a particular situation, which can be ascertained by one skilled in the art.
  • a large number of promoters recognized by a variety of potential host cells are well known.
  • the selected promoter can be operably linked to cistron DNA encoding a polypeptide described herein by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of choice.
  • Both the native promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of the target genes. However, heterologous promoters are preferred, as they generally permit greater transcription and higher yields of expressed target gene as compared to the native target polypeptide promoter.
  • Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the ⁇ - galactamase and lactose promoter systems, a tryptophan (trp) promoter system and hybrid promoters such as the tac or the trc promoter.
  • trp tryptophan
  • other promoters that are functional in bacteria such as other known bacterial or phage promoters
  • Their nucleotide sequences have been published, thereby enabling a skilled worker operably to ligate them to cistrons encoding the target light and heavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers or adaptors to supply any required restriction sites.
  • each cistron within a recombinant vector comprises a secretion signal sequence component that directs translocation of the expressed polypeptides across a membrane.
  • the signal sequence may be a component of the vector, or it may be a part of the target polypeptide DNA that is inserted into the vector.
  • the signal sequence selected for the purpose of this invention should be one that is recognized and processed (i.e. cleaved by a signal peptidase) by the host cell.
  • prokaryotic host cells that do not recognize and process the signal sequences native to the heterologous polypeptides, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group consisting of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PeIB, OmpA and MBP.
  • Prokaryotic host cells suitable for expressing polypeptides include, but are not limited to, Archaebacteria and Eubacteria, such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B.
  • subtilis subtilis
  • Enterobacteria Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus.
  • gram-negative cells are used.
  • the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may desirably be incorporated in the cell culture.
  • Host cells are transformed or transfected with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • Transfection refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, CaPO 4 precipitation and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell.
  • Transformation means introducing DNA into the prokaryotic host so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant.
  • transformation is done using standard techniques appropriate to such cells.
  • the calcium treatment employing calcium chloride is generally used for bacterial cells that contain substantial cell-wall barriers.
  • Another method for transformation employs polyethylene glycol/DMSO.
  • Yet another technique used is electroporation. Further techniques are known in the art.
  • Prokaryotic cells used to produce the polypeptides of the invention are grown in media known in the art and suitable for culture of the selected host cells.
  • suitable media include Luria broth (LB) plus necessary nutrient supplements.
  • the media also contains a selection agent, chosen based on the construction of the expression vector, to selectively permit growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to media for growth of cells expressing ampicillin resistant gene.
  • any necessary supplements besides carbon, nitrogen, and inorganic phosphate sources may also be included at appropriate concentrations introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source.
  • the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, dithioerythritol and dithiothreitol.
  • the prokaryotic host cells are cultured at suitable temperatures.
  • the preferred temperature ranges from about 20°C to about 39°C, more preferably from about 25°C to about 37°C, even more preferably at about 30°C.
  • the pH of the medium may be any pH ranging from about 5 to about 9, depending mainly on the host organism.
  • the pH is preferably from about 6.8 to about 7.4, and more preferably about 7.0.
  • an inducible promoter is used in the expression vector, protein expression is induced under conditions suitable for the activation of the promoter.
  • a PhoA promoter is used for controlling transcription
  • the transformed host cells may be cultured in a phosphate-limiting medium for induction.
  • inducers may be used, according to the vector construct employed, as is known in the art.
  • Polypeptides described herein expressed in a microorganism may be secreted into and recovered from the periplasm of the host cells. Protein recovery typically involves disrupting the microorganism, generally by such means as osmotic shock, sonication or lysis. Once cells are disrupted, cell debris or whole cells may be removed by centrifugation or filtration. The proteins may be further purified, for example, by affinity resin chromatography. Alternatively, proteins can be transported into the culture media and isolated therefrom. Cells may be removed from the culture and the culture supernatant being filtered and concentrated for further purification of the proteins produced.
  • the expressed polypeptides can be further isolated and identified using commonly known methods such as fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G- 75; hydrophobic affinity resins, ligand affinity using a suitable antigen immobilized on a matrix and Western blot assay.
  • eukaryotic host cell systems are also well established in the art. Suitable hosts include, but are not limited to, mammalian cell lines such as CHO, and insect cells such as those described below.
  • Polypeptide/peptide Purification Polypeptides/peptides that are produced may be purified to obtain preparations that are substantially homogeneous for further assays and uses. Standard protein purification methods known in the art can be employed.
  • HtrAl PDZ domain and HtrA3 PDZ domain modulators can be identified by any number of methods known in the art.
  • the modulatory characteristics of modulators can be assessed by determining the ability of the modulators to modulate the interaction between HtrAl PDZ domain and its cellular binding partners or
  • HtrA3 PDZ domain and its cellular binding partners One of the important characteristics is binding affinity.
  • candidate modulators e.g. peptides
  • binding characteristics of candidate modulators (e.g. peptides) of interest can be assessed in any of a number of ways known in the art.
  • An initial step in the process can include generating one or more candidate peptides comprising sequences of interest, which are then displayed under conditions suitable to determine their HtrAl PDZ domain or HtrA3 PDZ domain binding characteristics.
  • candidate peptides can be displayed as carboxyl-terminal (C-terminal) display libraries of peptides on the surface of a phage or phagemid, for example a filamentous phage(mid) using protein fusions with a coat protein such as p3 or p8.
  • C-terminal display is known in the art. See, e.g., Jespers et al, Biotechnology (N Y). 13:378-82 and WO 00/06717.
  • N-terminal phage(mid) display libraries of peptides on the surface of a phage or phagemid.
  • Methods of N-terminal phage(mid) display include those described herein, and those that are well known in the art, e.g., as described in US Pat. No. 5,750,373 (and references cited therein).
  • a phage display library with the displayed candidate HtrAl PDZ domain binding peptides or HtrA3 PDZ domain binding peptides is contacted with HtrAl PDZ domain proteins or fusion proteins or HtrA3 PDZ domain proteins or fusion proteins in vitro to determine those members of the library that bind to an HtrAl PDZ domain or HtrA3 PDZ domain target.
  • Any method, known to the skilled artisan, may be used to assay for in vitro protein binding. For example, 1, 2, 3 or 4 rounds or more of binding selection may be performed, after which individual phage are isolated and, optionally, analyzed in a phage ELISA.
  • Binding affinities of pep tide-displaying phage particles to immobilized PDZ target proteins may be determined using a phage ELISA (Barrett et al., Anal Biochem. 204:357-64 (1992)).
  • the appropriate binding competition conditions are provided.
  • screening/selection/biopanning can be performed in the presence of one or more concentrations of the known HtrAl PDZ domain binder or HtrA3 PDZ domain binder.
  • candidate binders isolated from the library can be subsequently assessed in a competitive ELISA assay in the presence of the known HtrAl PDZ domain binder or HtrA3 PDZ domain binder.
  • HtrAl PDZ domains or HtrA3 PDZ domains may be produced conveniently as protein fragments containing the domain or as fusion polypeptides using conventional synthetic or recombinant techniques. Fusion polypeptides are useful in phage(mid) display wherein HtrAl PDZ domain or HtrA3 PDZ domain is the target antigen, in expression studies, cell-localization, bioassays, ELISAs (including binding competition assays), etc.
  • An HtrAl PDZ domain or HtrA3 PDZ domain "chimeric protein" or "fusion protein” comprises HtrAl PDZ domain or HtrA3 PDZ domain fused to a non-PDZ domain polypeptide.
  • a non- PDZ domain polypeptide is not substantially homologous to the PDZ domain.
  • An HtrAl PDZ domain fusion protein or an HtrA3 PDZ domain fusion protein may include any portion to the entire PDZ domain, including any number of the biologically active portions.
  • the fusion protein can then be purified according to known methods using affinity chromatography and a capture reagent that binds to the non-PDZ domain polypeptide.
  • HtrAl PDZ domain or HtrA3 PDZ domain may be fused to an affinity sequence, e.g. the C-terminus of the GST (glutathione S-transferase) sequences.
  • Such fusion proteins facilitate the purification of the recombinant HtrAl PDZ domain or HtrA3 PDZ domain using, e.g., glutathione bound to a solid support and/or attachment to solid support (e.g., a matrix for peptide screening/selection/biopanning). Additional exemplary fusions are presented in Table B, including some common uses for such fusions.
  • Fusion proteins can be easily created using recombinant methods.
  • a nucleic acid encoding HtrAl PDZ domain (or portion thereof) or HtrA3 PDZ domain (or portion thereof) can be fused in- frame with a non-PDZ domain encoding nucleic acid, at the PDZ domain N - terminus, C-terminus or internally.
  • Fusion genes may also be synthesized by conventional techniques, including automated DNA synthesizers. PCR amplification using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (Ausubel et al., Current protocols in molecular biology. John Wiley & Sons, New York 1987) is also useful.
  • Many vectors are commercially available that facilitate sub-cloning the HtrAl PDZ domain or HtrA3 PDZ domain in-frame to a fusion protein.
  • GST-HtrAl PDZ fusion may be prepared from a gene of interest in the following manner. With the full- length gene of interest as the template, the PCR is used to amplify DNA fragments encoding the PDZ domain using primers that introduce convenient restriction endonuclease sites to facilitate sub-cloning.
  • Each amplified fragment is digested with the appropriate restriction enzymes and cloned into a similarly digested plasmid, such as pGEX6P-3 or pGEX-4T-3, that contains GST and is designed such that the sub-cloned fragments will be in-frame with the GST and operably linked to a promoter, resulting in plasmids encoding GST-HtrAl PDZ fusion proteins or GST-HtrA3 PDZ fusion proteins.
  • a similarly digested plasmid such as pGEX6P-3 or pGEX-4T-3
  • the bacteria are pelleted by centrifugation, resuspended in PBS and lysed by sonication.
  • the suspension is centrifuged, and GST-HtrAl PDZ domain fusion proteins or GST-HtrA3 PDZ domain fusion proteins are purified from the supernatant by affinity chromatography on 0.5 ml of glutathione-Sepharose.
  • HtrAl PDZ domain protein or HtrA3 PDZ domain protein may be used in this invention.
  • fusions of the HtrAl PDZ domain or the HtrA3 PDZ domain and an epitope tag may be constructed as described above and the tags used to affinity purify the
  • HtrAl PDZ domain or HtrA3 PDZ domain proteins/pep tides may also be prepared without any fusions; in addition, instead of using the microbial vectors to produce the protein, in vitro chemical synthesis may instead be used.
  • Other cells may be used to produce HtrAl PDZ domain or HtrA3 PDZ domain proteins/peptides, such as other bacteria, mammalian cells (such as COS), or baculoviral systems.
  • a wide variety of polynucleotide vectors to produce a variety of fusions are also available.
  • HtrAl PDZ domain or HtrA3 PDZ domain fusion protein will generally depend on the fusion partner; for example, a poly-histidine tag fusion can be purified on nickel columns.
  • Phage(mid) that bind to HtrAl PDZ domain or HtrA3 PDZ domain with the desired characteristics (and optionally, does not bind to unrelated sequences), can be subjected to sequence analysis.
  • the phage(mid) particles displaying the candidate binding peptides are amplified in host cells, the DNA isolated, and the appropriate portion of the genome (encoding the candidate peptide) sequenced using any appropriate known sequencing technique.
  • HtrA3 PDZ domain-ligand binding is to incorporate rational drug design; that is, to understand and exploit the biology of the PDZ interaction.
  • the critical residues in a PDZ ligand are determined, as is, optionally, the optimal peptide length.
  • small molecules are designed with this information in hand; for example, if a tryptophan is found to be a critical residue for binding to a PDZ domain, then small molecules that contain a tryptophan residue will be prepared and tested as inhibitors. Generally 2,3, 4 or 5 amino acid residues will be determined to be critical for binding and candidate small molecule inhibitors will be prepared containing these residues or the residue sidechains.
  • the test compounds are then screened for their ability to inhibit HtrAl PDZ domain-ligand or HtrA3 PDZ domain-ligand interactions using protocols well-known in the art, for example, a competitive inhibition assay.
  • HtrAl PDZ domain-ligand binding interactions or HtrA3 PDZ domain-ligand binding interactions are useful to treat diseases and conditions that are associated with dysregulation of binding interactions of HtrAl PDZ domains or HtrA3 PDZ domains.
  • Diseases and conditions that are associated with regulation of HtrAl PDZ domain interactions include malignant and benign tumors or cancers, non-leukemias and lymphoid malignancies; neurodegenerative disorders; inflammatory and immunologic disorders; and intraocular neovascular diseases.
  • HtrA3 PDZ domain interactions include malignant and benign tumors or cancers, non- leukemias and lymphoid malignancies and placental dysfunction. 1. Determining critical residues in an HtrAl PDZ domain binding polypeptide or an HtrA3 PDZ domain binding polypeptide
  • Alanine scanning an HtrAl PDZ domain binding peptide sequence or an HtrA3 PDZ domain binding peptide sequence can be used to determine the relative contribution of each residue in the ligand to PDZ binding.
  • residues are substituted with a single amino acid, typically an alanine residue, and the effect on PDZ domain binding is assessed. See US 5,580,723; US 5,834,250; and the Examples.
  • Truncations (deletion series) Truncation of an HtrAl PDZ domain binding peptide or an HtrA3 PDZ domain binding peptide can elucidate not only binding critical residues, but also determine the minimal length of peptide to achieve binding. In some cases, truncation will reveal a ligand that binds more tightly than the native ligand; such a peptide is useful to modulate HtrAl PDZ domain:PDZ ligand interactions or HtrA3 PDZ domain:PDZ ligand interactions.
  • HtrAl PDZ-domain binding peptide truncations or HtrA3 PDZ domain binding peptide truncations are prepared.
  • One series will truncate the amino terminal amino acids sequentially; in another series, the truncations will begin at the carboxy terminus.
  • the peptides may be synthesized in vitro or prepared by recombinant methods.
  • a modulator peptide can be designed to include 2 appropriate-spaced hydrophobic moieties, (d) Binding assays
  • HtrAl PDZ domain binding peptide and HtrAl PDZ domain or a complex of an HtrA3 PDZ domain binding peptide and HtrA3 PDZ domain facilitates separation of the complexed from the uncomplexed forms thereof and from impurities.
  • HtrAl PDZ domain:binding ligand complexes or HtrA3 PDZ domain:binding ligand complexes can be formed in solution or where one of the binding partners is bound to an insoluble support.
  • the complex can be separated from a solution, for example using column chromatography, and can be separated while bound to a solid support by filtration, centrifugation, etc. using well-known techniques. Binding the PDZ domain containing polypeptide or the ligand therefor to a solid support facilitates high throughput assays.
  • Test compounds can be screened for the ability to modulate (e.g., inhibit) the interaction of a binder polypeptide with HtrAl PDZ domain or HtrA3 PDZ domain in the presence and absence of a candidate binding compound, and screening can be accomplished in any suitable vessel, such as microtiter plates, test tubes, and microcentrifuge tubes.
  • Fusion proteins can also be prepared to facilitate testing or separation, where the fusion protein contains an additional domain that allows one or both of the proteins to be bound to a matrix.
  • GST-PDZ-binding peptide fusion proteins or GST-PDZ domain fusion proteins can be adsorbed onto glutathione sepharose beads (SIGMA Chemical, St.
  • the test compound or the test compound and either the nonadsorbed HtrAl PDZ domain protein or HtrA3 PDZ domain protein or PDZ-binding peptide, and the mixture is incubated under conditions allowing complex formation (e.g., at physiological conditions of salt and pH).
  • the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, and the complex determined either directly or indirectly.
  • the complexes can be dissociated from the matrix, and the level of binding or activity determined using standard techniques.
  • HtrAl PDZ binding peptide or HtrAl PDZ domain or an HtrA3 PDZ binding peptide or HtrA3 PDZ domain can be immobilized using biotin- avidin or biotin-streptavidin systems. Biotinylation can be accomplished using many reagents, such as biotin-N-hydroxy-succinimide (NHS; PIERCE Chemicals, Rockford, IL), and immobilized in wells of streptavidin coated 96 well plates (PIERCE Chemical).
  • antibodies reactive with HtrAl PDZ domain binding peptides or HtrAl PDZ domain or with HtrA3 PDZ domain binding peptides or with HtrA3 PDZ domain but which do not interfere with binding of a binding peptide to its target molecule can be derivatized to the wells of the plate, and unbound HtrAl PDZ domain or binder peptide or unbound HtrA3 PDZ domain or binder peptide trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the binder peptides or HtrAl PDZ domain or HtrA3 PDZ domain.
  • Assay for binding Competition ELISA
  • competition binding assays may be used, where the ability of the ligand to bind HtrAl PDZ domain or HtrA3 PDZ domain (and the binding affinity, if desired) is assessed and compared to that of a compound known to bind the PDZ domain, for example, a high-affinity binder peptide determined by phage display as described herein.
  • binding affinities can be determined as IC50 values using competition ELISAs.
  • the IC50 value is defined as the concentration of binder which blocks 50% of HtrAl PDZ domain binding or HtrA3 PDZ domain binding to a ligand.
  • assay plates may be prepared by coating microwell plates (preferably treated to efficiently adsorb protein) with neutravidin, avidin or streptavidin.
  • Non-specific binding sites are then blocked through addition of a solution of bovine serum albumin (BSA) or other proteins (for example, nonfat milk) and then washed, preferably with a buffer containing a detergent, such as Tween-20.
  • BSA bovine serum albumin
  • a biotinylated known HtrAl PDZ binder or HtrA3 PDZ binder (for example, the phage peptides as fusions with GST or other such molecule to facilitate purification and detection) is prepared and bound to the plate.
  • Serial dilutions of the molecule to be tested with HtrAl PDZ domain or HtrA3 PDZ domain are prepared and contacted with the bound binder.
  • the plate coated with the immobilized binder is washed before adding each binding reaction to the wells and briefly incubated. After further washing, the binding reactions are detected, often with an antibody recognizing the non-PDZ fusion partner and a labeled (such as horseradish peroxidase (HRP), alkaline phosphatase (AP), or a fluorescent tag such as fluorescein) secondary antibody recognizing the primary antibody.
  • a labeled such as horseradish peroxidase (HRP), alkaline phosphatase (AP), or a fluorescent tag such as fluorescein
  • the plates are then developed with the appropriate substrate (depending on the label) and the signal quantified, such as using a spectrophotometric plate reader.
  • the absorption signal may be fit to a binding curve using a least squares fit.
  • PDZ domain binders may be chemically-linked to a substrate, or simply adsorbed. 2. PDZ domain peptide ligands found during phage display
  • PDZ domain peptide ligands are potential useful inhibitors of the HtrAl PDZdomain- ligand interaction or the HtrA3 PDZ domain-ligand interaction, including those described in the Examples (and Tables 1, 2, and 3).
  • the competitive binding ELISA is a useful means to determine the efficacy of each phage-displayed PDZ-domain binding peptide.
  • Aptamers are short oligonucleotide sequences that can be used to recognize and specifically bind almost any molecule.
  • the systematic evolution of ligands by exponential enrichment (SELEX) process (Ausubel et al., Current protocols in molecular biology. John Wiley & Sons, New York (1987); Ellington and Szostak, Nature. 346:818-22 (1990); Tuerk and Gold, Science. 249:505-10 (1990)) can be used to find such aptamers.
  • Aptamers have many diagnostic and clinical uses; for almost any use in which an antibody has been used clinically or diagnostically, aptamers too may be used.
  • aptamers are less expensive to manufacture once they have been identified and can be easily applied in a variety of formats, including administration in pharmaceutical compositions, bioassays and diagnostic tests (Jayasena, Clin Chem. 45:1628-50 (1999)).
  • the screen for candidate aptamers includes incorporating the aptamers into the assay and determining their ability to modulate HtrAl PDZ domain:ligand binding or HtrA3 PDZ domain:ligand binding.
  • Any antibody that modulates (e.g., inhibits) ligand:HtrAl PDZ domain binding or ligand:HtrA3 PDZ domain binding can be a modulator (e.g., inhibitor) of HtrAl PDZ domain-ligand interaction or HtrA3 PDZ domain-ligand interaction, respectively.
  • suitable antibodies include polyclonal, monoclonal, single-chain, anti-idiotypic, chimeric Abs, or humanized versions of such antibodies or fragments thereof.
  • Antibodies may be from any suitable source, including of synthetic origin and any species in which an immune response can be raised. Screening methods
  • This invention encompasses methods of screening compounds to identify those that modulate HtrAl PDZ domain-ligand interaction or HtrA3 PDZ domain-ligand interaction. Screening assays are designed to identify compounds that bind or complex with HtrAl PDZ domain and/or ligand or HtrA3 PDZ domain and/or ligand, or otherwise interfere with the interaction of HtrAl PDZ domain or HtrA3 PDZ domain and cellular factors.
  • One approach to determining the ability of a candidate compound to be a modulator is to assess the activity of the candidate compound in a competitive inhibition assay in the presence of a known HtrAl PDZ domain binder or a known HtrA3 PDZ domain binder, such as any of the binder peptides (e.g., the high affinity binders described in the Examples) disclosed herein.
  • Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates.
  • the assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art.
  • All assays for modulators are common in that they call for contacting the drug candidate with HtrAl PDZ domain (or equivalent thereof) and/or binding ligand that is involved in the binding interaction of HtrAl PDZ domain and the binding ligand, or HtrA3 PDZ domain (or equivalent thereof) and/or binding ligand that is involved in the binding interactions of HtrA3 PDZ domain and the binding ligand, under conditions and for a time sufficient to allow these two components to interact.
  • a candidate substance or molecule is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non- covalent attachments.
  • Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the substance/molecule and drying.
  • an immobilized affinity molecule such as an antibody, e.g., a monoclonal antibody, specific for the substance/molecule to be immobilized can be used to anchor it to a solid surface.
  • the assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component.
  • the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected.
  • the detection of label immobilized on the surface indicates that complexing occurred.
  • complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.
  • the candidate compound interacts with but does not bind to HtrAl PDZ domain or HtrA3 PDZ domain or the binding partner of either HtrAl PDZ domain or HtrA3 PDZ domain
  • its interaction with the polypeptide can be assayed by methods well known for detecting protein-protein interactions.
  • assays include traditional approaches, such as, e.g., cross-linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns.
  • protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers (Fields and Song, Nature (London). 340:245-246 (1989); Chien et al, Proc. Natl.
  • yeast GAL4 Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, the other one functioning as the transcription-activation domain.
  • yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain.
  • GALl-lacZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for ⁇ -galactosidase.
  • a complete kit (MATCHMAKERTM) for identifying protein- protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.
  • Candidate compounds can be generated by combinatorial libraries and/or mutations of known binders based on information described herein, in particular information relating to contributions and importance to HtrAl PDZ domain-ligand binding interactions of individual residues and moieties within a ligand or HtrA3 PDZ domain-ligand binding interactions of individual residues and moieties within a ligand or HtrAl PDZ domain or HtrA3 PDZ domain sequence itself.
  • HtrAl PDZ domain and binding ligand or HtrA3 PDZ domain and binding ligand can be tested as follows: usually a reaction mixture is prepared containing HtrAl PDZ domain or HtrA3 PDZ domain and a ligand under conditions and for a time allowing for the interaction and binding of the PDZ domain and the ligand. To test the ability of a candidate compound to inhibit the binding interaction, the reaction is run in the absence and in the presence of the test compound. In addition, a placebo may be added to a third reaction mixture, to serve as positive control.
  • the binding (complex formation) between the test compound and HtrAl PDZ domain and/or binding ligand present in the mixture or HtrA3 PDZ domain and/or binding ligand present in the mixture is monitored as described hereinabove.
  • the formation of a complex in the control reaction(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of HtrAl PDZ domain and binding ligand or the interaction of HtrA3 PDZ domain and binding ligand.
  • a substance/molecule of the invention can be a peptide.
  • Methods of obtaining such peptides are well known in the art, and include screening peptide libraries for binders to a target antigen.
  • suitable target antigens would comprise HtrAl PDZ domain or HtrA3 PDZ domain (or portion thereof that comprises binding site for an HtrAl PDZ domain ligand or an HtrA3 PDZ domain ligand), which is described in detail herein.
  • Libraries of peptides are well known in the art, and can also be prepared according to art-known methods. See, e.g., Clark et al., U.S. Pat. No. 6,121,416.
  • a peptide having ability to block HtrAl PDZ domain protein-protein interaction or HtrA3 PDZ domain protein- protein interaction comprises the amino acid sequence of any of the binder peptides disclosed herein.
  • a peptide having ability to block HtrAl PDZ domain protein- protein interaction or HtrA3 PDZ domain protein-protein interaction comprises the amino acid sequence of a binder peptide obtained from a modulator screening assay as described above.
  • the peptide has the ability to compete with one or more of the binder peptides disclosed herein (see Examples) for binding to HtrAl PDZ domain or to HtrA3 PDZ domain. In one embodiment, the peptide binds to the same epitope on HtrAl PDZ domain or to the same epitope on HtrA3 PDZ domain to which one or more of the binder peptides disclosed herein (see Examples) bind.
  • Variants of a first peptide binder can be generated by screening mutants of the peptide to obtain the characteristics of interest (e.g., enhancing target binding affinity, enhanced pharmacokinetics, reduced toxicity, improved therapeutic index, etc.). Mutagenesis techniques are well known in the art. Furthermore, scanning mutagenesis techniques (such as those based on alanine scanning) can be especially helpful to assess structural and/or functional importance of individual amino acid residues within a peptide.
  • Determination of the ability of a candidate substance/molecule of the invention, such as a peptide comprising the amino acid sequence of a binder peptide disclosed herein, to modulate HtrAl PDZ domain activity or HtrA3 PDZ domain activity can be performed by testing the modulatory capability of the substance/molecule in in vitro or in vivo assays, which are well established in the art, e.g., as described in Martins et al. (J Biol. Chem.
  • HtrAl PDZ domain binders and modulators of HtrAl PDZ domain- ligand interaction or HtrA3 PDZ domain binders and modulators of HtrA3 PDZ domain- ligand interaction
  • HtrA3 PDZ domain peptide binders as described herein provide valuable insights into the cellular functions of the HtrAl protein or the HtrA3 protein, respectively, and provides compositions and methods for modulating the in vivo interactions between these important cellular proteins and their binding partner(s).
  • these peptides and their homologs can be utilized to interfere with the in vivo binding interactions involving HtrAl PDZ domain or HtrA3 PDZ domain.
  • Homologs can be generated conveniently based on their binding and/or functional characteristics relative to the well-characterized peptides provided herein.
  • binding partners of HtrAl PDZ domain or HtrA3 PDZ domain can be located both in the conventional C-terminal region and also the heretofore unknown N-terminal and/or internal regions of a polypeptide.
  • HtrAl PDZ domain or HtrA3 PDZ domain can be further used to elucidate important structural characteristics of HtrAl PDZ domain or HtrA3 PDZ domain itself.
  • Other variants can be similarly identified.
  • HtrAl PDZ domain-binding partner modulators or HtrA3 PDZ domain-binding partner modulators developed based on the ligand peptides described herein can be used to achieve the modulatory effect of interest.
  • manipulation may include inhibition of the association between HtrAl PDZ domain and its cognate binding protein or inhibition of the association between HtrA3 PDZ domain and its cognate binding protein.
  • such manipulation may include agonistic effects through, for example, induction of cellular functions as a result of binding of the modulators to HtrAl PDZ domain or HtrA3 PDZ domain or through enhancement of association between HtrAl PDZ domain and its cognate binding protein by the modulators or between HtrA3 PDZ domain and its cognate binding protein by the modulators.
  • modulators of HtrAl PDZ domain or HtrA3 PDZ domain include diagnostic assays for diseases related to HtrAl or HtrA3 and its associating partners, the use of the HtrAl PDZ domain or HtrA3 PDZ domain and ligands of HtrAl PDZ domain or
  • HtrA3 PDZ domain respectively, in fusion proteins as purification handles and anchors to substrates.
  • HtrAl protein is implicated in important biological processes, including for example processing of amyloid precursor protein, and downregulation of HtrAl has been implicated in cancer development (i.e., endometrial cancer, ovarian cancer, and melanoma, among others) while upregulation of HtrAl has been implicated in arthritis and age-related (wet) macular degeneration.
  • HtrA3 protein is implicated in important biological processes, including placental development, and dysregulation of HtrA3 has been implicated in cancer development (i.e., endometrial cancer, among others).
  • the HtrAl and HtrA3 proteins each contain a PDZ domain, which is a domain reported to be essential in protein-protein binding interactions.
  • Modulatory compounds e.g., inhibitory or agonistic
  • Modulatory compounds such as peptides/ligands, may be delivered into live cells or animal models which are models for a disease (i.e. mimic certain properties of a disease) to determine if disruption of HtrAl PDZ domain-ligand interaction or HtrA3 PDZ domain- ligand interaction by the modulatory compound of interest provides an outcome consistent with expectations for therapeutic benefit.
  • these methods can be used to assess the ability of a molecule, such as a synthetic peptide, to modulate the binding interaction of HtrAl PDZ domain protein and its cognate ligand or HtrA3 PDZ domain protein and its cognate ligand in vivo.
  • Therapeutic/prophylactic applications Compounds that have the property of increasing or decreasing HtrAl PDZ domain or
  • HtrA3 PDZ domain protein activity are useful. This increase in activity may come about in a variety of ways, for example by administering to a subject in need thereof an effective amount of one or more of the modulators described herein.
  • Antagonists or negative modulators include any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of HtrAl PDZ domain and/or its endogenous ligand(s) or HtrA3 PDZ domain and/or its endogenous ligand(s).
  • agonists or positive modulators include any molecule that mimics or enhances a biological activity of HtrAl PDZ domain and/or its endogenous ligand(s) or HtrA3 PDZ domain and/or its endogenous ligand(s).
  • Molecules that can act as agonists or antagonists include the modulators of HtrAl PDZ domain-binder/ligand interaction or HtrA3 PDZ domain-binder/ligand interaction described herein, including but not limited to Abs or antibody fragments, fragments or variants of HtrAl PDZ domain/ligands/binders, peptides, small organic molecules, etc or HtrA3 PDZ domain/ligands/binders, peptides, small organic molecules, etc.
  • the invention provides various methods based on the discovery of various binding molecules capable of interacting specifically with HtrAl PDZ domain or HtrA3 PDZ domain, and the identification of unique characteristics of the binding interactions between HtrAl PDZ domain and ligand binding peptides or between HtrA3 PDZ domain and ligand binding peptides.
  • compositions including peptides, etc.
  • therapeutic agents may be employed as therapeutic agents. These substances or molecules can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the product hereof is combined in admixture with a pharmaceutically acceptable carrier vehicle.
  • Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as
  • EDTA EDTA
  • sugar alcohols such as mannitol or sorbitol
  • salt-forming counterions such as sodium
  • nonionic surfactants such as TWEENTM, PLURONICSTM or PEG.
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution.
  • Therapeutic compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the route of administration is in accord with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, subcutaneous, intraarterial or intralesional routes, topical administration, or by sustained release systems.
  • Dosages and desired drug concentrations of pharmaceutical compositions of the present invention may vary depending on the particular use envisioned.
  • the determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician. Animal experiments provide reliable guidance for the determination of effective doses for human therapy.
  • Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. "The use of interspecies scaling in toxicokinetics” In Toxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Press, New York 1989, pp. 42-96.
  • normal dosage amounts may vary from about 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day, preferably about 1 ⁇ g/kg/day to 10 mg/kg/day, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. It is anticipated that different formulations will be effective for different treatment compounds and different disorders, that administration targeting one organ or tissue, for example, may necessitate delivery in a manner different from that to another organ or tissue.
  • microencapsulation of the substance or molecule is contemplated.
  • Microencapsulation of recombinant proteins for sustained release has been successfully performed with human growth hormone (rhGH), interferon- (rhIFN- ), interleukin-2, and MN rgpl20. Johnson et al., Nat. Med., 2:795-799 (1996); Yasuda, Biomed.
  • the sustained-release formulations of these proteins were developed using poly- lactic-coglycolic acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties.
  • PLGA poly- lactic-coglycolic acid
  • the degradation products of PLGA, lactic and glycolic acids, can be cleared quickly within the human body.
  • the degradability of this polymer can be adjusted from months to years depending on its molecular weight and composition.
  • Lewis “Controlled release of bioactive agents from lactide/glycolide polymer," in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41.
  • a modulator molecule/substance of the invention can be incorporated into compositions, which in some embodiments are suitable for pharmaceutical use.
  • Such compositions typically comprise the nucleic acid molecule, peptide/protein, small molecule and/or antibody, and an acceptable carrier, for example one that is pharmaceutically acceptable.
  • an acceptable carrier for example one that is pharmaceutically acceptable.
  • a “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration (Gennaro, Remington: The science and practice of pharmacy. Lippincott, Williams & Wilkins, Philadelphia, PA (2000)).
  • Such carriers or diluents include, but are not limited to, water, saline, Finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. Except when a conventional media or agent is incompatible with an active compound, use of these compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. 1. General considerations
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration, including intravenous, intradermal, subcutaneous, oral ⁇ e.g., inhalation), transdermal ⁇ i.e., topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic. 2. Injectable formulations
  • compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL TM (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid so as to be administered using a syringe.
  • Such compositions should be stable during manufacture and storage and must be preserved against contamination from microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures.
  • Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants.
  • Various antibacterial and antifungal agents for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal, can contain microorganism contamination.
  • Isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride can be included in the composition.
  • Compositions that can delay absorption include agents such as aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound ⁇ e.g., any modulator substance/molecule of the invention) in the required amount in an appropriate solvent with one or a combination of ingredients as required, followed by sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium, and the other required ingredients.
  • Sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying that yield a powder containing the active ingredient and any desired ingredient from a sterile solutions.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included.
  • Tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch; a lubricant such as magnesium stearate or STEROTES; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch
  • a lubricant such as magnesium stearate or STEROTES
  • a glidant such as colloidal silicon dioxide
  • the compounds can be delivered as an aerosol spray from a nebulizer or a pressurized container that contains a suitable propellant, e.g. , a gas such as carbon dioxide. 5.
  • a suitable propellant e.g. , a gas such as carbon dioxide. 5.
  • Systemic administration can also be transmucosal or transdermal.
  • penetrants that can permeate the target barrier(s) are selected.
  • Transmucosal penetrants include, detergents, bile salts, and fusidic acid derivatives.
  • Nasal sprays or suppositories can be used for transmucosal administration.
  • the active compounds are formulated into ointments, salves, gels, or creams.
  • the compounds can also be prepared in the form of suppositories (e.g., with bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable or biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such materials can be obtained commercially from ALZA Corporation (Mountain View, CA) and NOVA Pharmaceuticals, Inc. (Lake Elsinore, CA), or prepared by one of skill in the art.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as in (Eppstein et al, US Patent No. 4,522,811, 1985).
  • Unit dosage form refers to physically discrete units suited as single dosages for the subject to be treated, containing a therapeutically effective quantity of active compound in association with the required pharmaceutical carrier.
  • the specification for the unit dosage forms are dictated by, and directly dependent on, the unique characteristics of the active compound and the particular desired therapeutic effect, and the inherent limitations of compounding the active compound.
  • the nucleic acid molecules can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (Nabel and Nabel, US Patent No. 5,328,470, 1994), or by stereotactic injection (Chen et al, Proc Natl Acad Sd USA. 91:3054-7 (1994)).
  • the pharmaceutical preparation of a gene therapy vector can include an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
  • the pharmaceutical composition and method may further comprise other therapeutically active compounds that are usually applied in the treatment of HtrAl protein- related or HtrA3 protein-related (specifically HtrAl PDZ domain-related or HtrA3 PDZ domain-related) conditions.
  • HtrAl protein- related or HtrA3 protein-related specifically HtrAl PDZ domain-related or HtrA3 PDZ domain-related
  • an appropriate dosage level will generally be about 0.01 to 500 mg per kg patient body weight per day which can be administered in single or multiple doses.
  • the dosage level will be about 0.1 to about 250 mg/kg per day; more preferably about 0.5 to about 100 mg/kg per day.
  • a suitable dosage level may be about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day. Within this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg/kg per day.
  • compositions are preferably provided in the form of tablets containing 1.0 to 1000 milligrams of the active ingredient, particularly 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • the compounds may be administered on a regimen of 1 to 4 times per day, preferably once or twice per day.
  • the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.
  • compositions e.g., pharmaceutical compositions
  • the different components of the composition may be packaged in separate containers and admixed immediately before use. Such packaging of the components separately may permit long-term storage without losing the active components' functions.
  • Kits may also include reagents in separate containers that facilitate the execution of a specific test, such as diagnostic tests or tissue typing.
  • kits can be supplied in containers of any sort such that the life of the different components are preserved and are not adsorbed or altered by the materials of the container.
  • sealed glass ampules may contain lyophilized modulator substance/molecule and/or buffer that have been packaged under a neutral, non-reacting gas, such as nitrogen.
  • Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc., ceramic, metal or any other material typically employed to hold reagents.
  • suitable containers include simple bottles that may be fabricated from similar substances as ampules, and envelopes, that may consist of foil-lined interiors, such as aluminum or an alloy.
  • Containers include test tubes, vials, flasks, bottles, syringes, or the like.
  • Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle.
  • Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix.
  • Removable membranes may be glass, plastic, rubber, etc.
  • Kits may also be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zip disc, videotape, laserdisc, audio tape, etc. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit, or supplied as electronic mail.
  • the following examples are included to demonstrate preferred embodiments of the present invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
  • FIG. 1 A comparison of the HtrAl, HtrA2 and HtrA3 amino acid sequences ( Figure 1) shows that these HtrA proteins have a high degree of sequence homology, with greater than 30% identity within the PDZ domain. While this level of sequence conservation is expected to translate into structural conservation, key sequence difference in proximity to the ligand binding site might be expected to impart differences in ligand specificity. Thus, the binding specificity profiles of the PDZ domains of human HtrAl and HtrA3 were assessed using phage-displayed peptide libraries.
  • GST-HtrAl-PDZ, GST-HtrA 1 -(1415 Q/I418A)-PDZ or GST-HtrA3-PDZ fusion proteins were separately used to screen a library of random decapeptides displayed in a high valency format by fusion to the C-terminus of the M 13 gene-8 major coat protein (libC) (Held, H. A., and Sidhu, S. S. (2004) Journal of Molecular Biology 340(3), and (b) 587-597).
  • the HtrAl-PDZ and HtrA3-PDZ GST fusion proteins were also used to screen a 12-mer peptide library fused to the N-terminus of the M13 major coat protein (libN).
  • the libraries were constructed with a degenerate codon encoding for all 20 natural amino acids (ATCGACAGCGCCCCCGGTGGCGGANNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNKNKNKN NKTGATAAACCGATACA (SEQ ID NO: 25)). Oligonucleotides were designed as described previously using equimolar DNA degeneracies (Sidhu, S. S., Lowman, H. B., Cunningham, B. C, and Wells, J. A. (2000) Methods Enzymol 328, 333-363).
  • the libraries further included a stop codon that permitted the display of truncated peptides.
  • individual clones were grown in a 96-well format in 500 ⁇ L of 2 YT broth supplemented with carbenicillin and M13-KO7, and the culture supernatants were used directly in phage ELISAs to detect peptides that bound specifically to HtrAl or HtrA3 PDZ. Specific binding clones were obtained for each PDZ domain. Culture supernatants containing phage particles were used as templates for PCRs that amplified DNA fragments containing the peptides. The DNA fragments were sequenced as described previously (Vajdos, F. F., Adams, C.
  • HtrAl-PDZ and HtrA3-PDZ prefer ligands with overall hydrophobic character.
  • HtrA2 PDZ domain had previously been shown to also prefer ligands with hydrophobic character (Zhang et al., (2007), in preparation)).
  • the specificity profile for HtrAl-PDZ was defined by the four residues at the C-terminus of the domain. The C-terminal residue (position 0) of this domain displayed a slight preference for leucine, with valine being the next most prevalent residue.
  • HtrA3-PDZ specificity profile as determined by the C-terminal library selectants, was defined by two residues at the C-terminus of the domain and also position -3.
  • the C- terminal residue (position 0) of this domain slightly preferred valine with isoleucine being the next most prevalent residue selected.
  • Position -1 was exclusively tryptophan.
  • HtrA3- PDZ showed no preference at position -2 and frequently selected non-hydrophobic amino acids at that position.
  • a preference for glycine or serine was observed at position -3. No preferences were observed at position -4 or any other position in the obtained HtrA3-PDZ- binding peptides from the LibC selection.
  • Binding selection with the N-terminal library was also successful for both HtrAl-PDZ and HtrA3-PDZ, and sequencing of clones revealed 16 and 8 unique sequences, respectively (Table 2). Alignment of the sequences was more difficult for the LibN selectants than for the LibC selectants, because the latter was facilitated by the C-terminal residue functioning as an anchor position.
  • HtrAl-PDZ a subset of the LibN selected sequences contained a conserved V-X-W-G-[D/E] sequence (SEQ ID NO: 141).
  • Example 2 To further refine the binding specificities of the HtrAl-PDZ domain and the HtrA3 PDZ domain, a panel of synthetic peptides was generated based on the information obtained in Example 1. Peptides were synthesized using standard 9-fluorenylmethoxycarbonyl (Fmoc) protocols, cleaved off the resin with 2.5% triisopropylsilane and 2.5% H 2 O in trifluoroacetic acid (TFA), and purified by reversed-phase high performance liquid chromatography. The purity and mass of each peptide were verified by liquid chromatography/mass spectrometry (LC/MS). The binding affinities of peptides for HtrAl-PDZ or HtrA3-PDZ were determined as
  • IC50 values using a solution-phase competition ELISA were prepared by immobilizing an N-terminally biotinylated peptide (biotin-GWKTWIL for HtrAl-PDZ, and biotin-RSWWV for HtrA3-PDZ) on MaxisorpTM plates (Nalge NUNC International (Naperville, IL) coated with NeutrAvidinTM (Pierce) (Rockford, IL) and blocked with BSA (Sigma).
  • HtrAl-PDZ and HtrA3-PDZ constructs with glutathione S-transferase (GST) fused to the N-terminus were prepared as described (Laura, R. P., Witt, A.
  • IC 50 value was defined as the concentration of peptide that blocked 50% of PDZ domain binding to immobilized peptide.
  • the obtained IC50 values for certain synthetic peptides are shown in Table 3.
  • HtrAl-PDZ the peptide Hl_c3 (DSRIWWV) (SEQ ID NO: 5) competed with biotin-GWKTWIL (SEQ ID NO: 4) for binding to GST-HtrAl-PDZ with an IC 50 of 0.9 ⁇ 0.1 ⁇ M, the lowest IC50 obtained amongst the synthetic peptides tested for binding to HtrAl- PDZ.
  • the dissociation constant for the H I_c3 -HtrAl-PDZ interaction was also measured using isothermal titration calorimetry. Briefly, ITC measurements were made at 28°C using a VP-ITC titration calorimeter (MicroCal).
  • Blocking the C-terminus by amidation abrogated the ability of the peptide to compete with biotin-GWKTWIL (SEQ ID NO: 4) binding to HtrAl-PDZ at concentrations up to 500 ⁇ M, indicating that the terminal carboxylate group was required for binding.
  • a comparison of peptides Hl cl to Hl_c5 confirmed the modest importance of a tryptophan at position -1 and suggested that basic residues (e.g., lysine and arginine) may provide an energetic advantage over acidic (e.g., glutamic acid or aspartic acid) residues at position -4.
  • basic residues e.g., lysine and arginine
  • acidic residues e.g., glutamic acid or aspartic acid
  • HtrA3-PDZ the peptide H3_cl competed with biotin-RSWWV (SEQ ID NO: 12) for binding to GST-HtrA3-PDZ with an IC 50 of 0.6 ⁇ /- 0.1 ⁇ M.
  • Peptides H3_c6-9 further confirmed the lack of importance of amino acid identity at the -3 and -4 positions.
  • Peptides corresponding to the C-termini of CoBaI and GM130 were synthesized.
  • Binding of the C-termini of CoBaI and GM130 to HtrAl-PDZ was compared to biotin-GWKTWIL (SEQ ID NO: 4) in a competition binding assay, as described above.
  • the CoBaI and GM130 peptides competed for binding to GST-HtrAl-PDZ with biotin-GWKTWIL (SEQ ID NO: 4) with IC 50 values of 15.7 ⁇ 3.3 ⁇ M and 24.1 ⁇ 8.4 ⁇ M, respectively (Table 3).
  • HtrA3-PDZ binding was detectable for both peptides tested (H3_nl and H3_n2), but both peptides bound with markedly reduced affinity compared to the peptides having a free C-terminus (Table 3).
  • peptide H3_nl was derived from the sequence having the highest frequency of selection in the N-terminal peptide phage selection, the affinity for HtrA3-PDZ is several hundred fold weaker than an optimized C-terminal peptide (Table 3).
  • Peptide H3_n2 bound with an affinity comparable to that of the non-optimized C-terminal dipeptides (compare with peptides H3_cle and H3_clf) (Table 3).
  • Example 3 Structure of the PDZ Domains of HtrAl and HtrA3 a. NMR Analysis To better understand the binding interaction between the HtrAl-PDZ domain and a peptide ligand, an NMR analysis was performed on a complex between HtrAl-PDZ and the peptide Hl_c3 (DSRIWWV) (SEQ ID NO: 5).
  • BLR(DE3)pLysS cells harboring the expression plasmid were grown in M9 minimal media supplemented with 15 N-ammonium chloride (>99%, Spectra Stable Isotopes) and 12 C- and/or 13 C c6 -D-glucose (>99%, Spectra Stable Isotopes).
  • QIAGEN Nickel-NTA Superflow column
  • the column was washed with 10 mM imidazole in 50 mM Tris-HCl pH 8.0, 500 mM NaCl (buffer A), and eluted with 500 mM imidazole in buffer A. Fractions were pooled, thrombin was added (1 unit/mg protein), and the sample was dialyzed overnight against 50 mM Tris HCl pH 7.5, 100 mM NaCl (buffer B) with 2 mM CaCl 2 at 4 °C. The protein sample was concentrated and purified over a Superdex-75 column (Pharmacia) in 25 mM Tris HCl pH 7.5, 300 mM NaCl.
  • the sample was further purified over a MonoQ (Pharmacia) anion exchange column in Tris pH 7.5 with a 0.1-1.0 M NaCl gradient. Protein samples were concentrated to approximately 2 mM in 25 mM sodium phosphate, pH 6.0 containing 10% deuterium oxide (D 2 O) and 1 mM sodium azide. "100%" D 2 O samples were prepared by lyophilizing 10% D 2 O samples and dissolving them in 99.996% D 2 O (Cambridge Isotope Labs, Inc.).
  • NMR spectra were acquired at 25 °C on either a Bruker DRX600 MHz or DRX800 MHz spectrometer equipped with triple resonance, triple axis actively shielded gradient probe. All NMR data was processed using NMRPipe (Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) JBiomolNMR 6(3), 277-293) and analyzed using the program Sparky (version 3.11, Goddard & Kneller, UCSF).
  • HtrA3-PDZ domain (residues 354-453) was crystallized in complex with a high- affinity penta-peptide sequence (FGRWVCOOH) (SEQ ID NO: 11) identified by phage display.
  • FGRWVCOOH high- affinity penta-peptide sequence
  • a previously described strategy was used to crystallize the PDZ-ligand complex (designated herein as HtrA3-PDZext), involving fusing the 5 -residue peptide sequence to the C-terminus of the PDZ domain via a tri-glycine linker (Appleton, B. A., Zhang, Y., Wu, P., Yin, J. P., Hunziker, W., Skelton, N.
  • HtrA3-PDZ HtrA3-PDZ-ext (extension, GGGFGRWV) (SEQ ID NO: 161).
  • the bacteria were pelleted by centrifugation at 4,000 x g for 15 minutes, washed twice with 20 mM Tris-HCl (pH 8.0), and frozen at -80 °C for 8 hours. The pellet was resuspended in 100 mL of buffer A (50 mM Tris-HCl pH 8.0 and 500 mM NaCl), and the bacteria were lysed by passage through the Micro fluidizer® Processing Equipment (model HOY, Micro fluidics Corp., Newton, MA, USA). The cell lysate was loaded onto a nickel-nitrilotriacetic acid-agarose column (Qiagen).
  • the column was washed with buffer A plus 20 mM imidazole, and the protein was eluted with 250 mM imidazole in buffer A. Fractions containing the protein of interest were pooled, thrombin was added (1 unit/mg of protein), and the sample was dialyzed overnight against PBS at 4 °C. The protein sample was concentrated and further purified over a Superdex-75 column in 50 mM Tris-HCl pH 8.0, 300 mM NaCl, and 5 mM ⁇ -mercaptoethanol.
  • Crystals were obtained by vapor diffusion in sitting drops at 19 °C by mixing equal volumes of protein (10 mg/mL) with 0.1 M Bis-Tris (pH 6.5), 0.2 M MgCl 2 , and 25% PEG
  • HtrA3-PDZext crystallized in space group P4i2i2 with two molecules per asymmetric unit.
  • Each PDZ domain in the asymmetric unit formed a crystallographic dimer with the ligand from an apposing molecule that is related by crystallographic symmetry.
  • the PDZ fold ( Figures 3B and 4B) consisted of a five-stranded ⁇ - sandwich ( ⁇ l- ⁇ 5) capped by two ⁇ -helices ( ⁇ l, ⁇ 3), as has been seen in other PDZ domain structures (Sheng, M., and SaIa, C. (2001) Annual Review ofNeuroscience 24(1), 1-29). Additionally, short ⁇ -strands were observed at the N- and C-termini ( ⁇ N and ⁇ C). Like the PDZ domain of the HtrA2 protein (Zhang et al. 2007, in preparation), HtrAl-PDZ and HtrA3-PDZ have a cyclically permuted fold as compared to the canonical PDZ fold
  • the peptide binds in an extended conformation in the cleft between strand ⁇ l and helix ⁇ 3, extending by one strand the antiparallel ⁇ -sheet formed by strands ⁇ l and ⁇ 2 ( Figures 3, 5B, and 5C).
  • the C-terminal carboxylate group is coordinated by the three main chain amides immediately preceding strand ⁇ l.
  • these amides are protected from solvent exchange in the complex, as determined by the hydrogen/deuterium exchange NMR experiments.
  • the amide hydrogen of ⁇ N: ⁇ l-2 (GIy 384) does not point directly towards the carboxylate group, and may be involved in a water-mediated hydrogen bond. While the hydrogen/deuterium exchange experiments and down-field chemical shifts observed for the three amide hydrogens suggested that hydrogen bonds are formed, the inherent lack of NOE restraints prevented precise definition of the ligand carboxylate and carboxylate binding loop or the use of specific hydrogen bond restraints in the structure calculation.
  • Trp "1 adopts a conformation nearly identical to that seen with other PDZ domains that prefer tryptophan at position -1 (e.g., HtrA2 (Zhang et al. 2007, in preparation) ) and Erbin (Skelton, N. J., Koehler, M. F., Zobel, K., Wong, W. L., Yeh, S., Pisabarro, M. T., Yin, J. P., Lasky, L. A., and Sidhu, S. S.
  • Trp "1 also extends across the backbone of strand ⁇ l, however, the orientation of the indole ring is significantly different from that of HtrA3-PDZ ( Figure 7A). Trp "1 is positioned between the side chains of ⁇ N-3 (Tyr 382) and ⁇ l-2 (Arg 386) and abuts the isoleucine side chain at position ⁇ 2: ⁇ 2-l (He 418).
  • HtrAl-PDZ does show a strong preference for tryptophan at position -2.
  • Trp " is particularly well-defined in the ensemble by 20 unique intermolecular NOEs assigned to its aromatic protons.
  • the indole ring packs against helix ⁇ 3, making favorable hydrophobic interactions with ⁇ 3-l (Ala 445), ⁇ l-3 (Met 387), and the methylene of ⁇ 3-2 (GIn 446) ( Figure 5A).
  • the indole ring is positioned directly above the alpha proton of GIn 446, inducing a large (-1 ppm) chemical shift perturbation due to the ring current effect.
  • the ligand-binding profile and synthetic peptide binding assays for HtrAl-PDZ suggest that it prefers an isoleucine residue at position -3.
  • the NMR structure shows that He- 3 packs into a hydrophobic patch created by ⁇ 2-3 (He 415) and ⁇ l-2 (Arg 386) ( Figure 5A).
  • the packing of He "3 of the peptide and ⁇ 2-3 of the protein against the side chain methylenes of ⁇ l-2 (Arg 386) are likely responsible for the protrusion of the Arg 386-Glu 416 salt bridge that affects the accessibility of position -1.
  • GIy "3 of peptide H3_cl does not contact HtrA3-PDZ directly, but adopts a positive Phi angle positioning the carbonyl oxygen of Phe-4 to make a hydrogen bond with the side chain at position ⁇ 2-2 (Arg 360) and also allowing the side chain of Phe-4 to interact via ⁇ - ⁇ stacking with the Arg 360 side chain ( Figure 5C).
  • HtrA3-PDZ there is a type I reverse turn stabilized by a hydrogen bond between the carbonyl oxygen of GIy "6 and the amide of GIy "3 , however the synthetic peptide binding assays do not suggest that this conformation is important for ligand binding.
  • a genome-wide search of potential extracellular ligands with C-termini matching the specificity profile for HtrAl-PDZ identified leucyl/cystinyl aminopeptidase isoform 1 (GenBank No. NP 005566) having the C-terminal peptide KNLKSLTWWL (SEQ ID NO: 162) and leucyl/cystinyl aminopeptidase isoform 2 (GenBank No.
  • NP_787116 having the C- terminal peptide KNLKSLTWWL (SEQ ID NO: 163).
  • HtrAl specifically interacts with TGF- ⁇ family members (Murwantoko et al., (2004) Biochem J.
  • HtrAl-PDZ does bind C-termini such as that of CoBaI and may therefore play a role in the metabolism of fibrillar collagen C-propeptides
  • the analysis also shows that a variety of other C-terminal sequences are able to bind with comparable or even higher affinity.
  • HtrAl-PDZ appears to recognize a range of targets with exposed hydrophobic C-termini rather than just a select few targets.
  • HtrA3-PDZ the striking preference for ligands containing tryptophan at position -1 suggests that despite the limited determinants for specificity, HtrA3 may preferentially bind to ligands containing tryptophan at this position.
  • a number of mammalian proteins localized to the extracellular matrix have a conserved tryptophan at the penultimate position, including human matrix metalloproteinase 15 (GenBank No. NP 002419) having the C-terminal sequence YCKRSMQEWV (SEQ ID NO: 164); matrix metalloproteinase 16 (GenBank No.
  • NP 005932 having the C-terminal sequence YCKRSMQEWV (SEQ ID NO: 165); and matrix metalloproteinase 24 (GenBank No. NP 006681) having the C-terminal sequence YYKRPVQEWV (SEQ ID NO: 166).
  • Melanoma-associated chondroitin sulfate (GenBank No. NP 001888) having the C-terminal sequence PALKNGQ YWV (SEQ ID NO: 167), and keratinocyte-associated protein 3 (GenBank No.
  • NP_776252 having the C- terminal sequence EIRASQRSWV (SEQ ID NO: 168) also have conserved tryptophans at the penultimate position and thus may be ligands for the HtrA3-PDZ domain.
  • Example 4 Shotgun Scanning of HtrAl-PDZ
  • HtrAl-PDZ domain The contribution of individual residues of the HtrAl-PDZ domain to ligand binding was assessed by combinatorial alanine scanning (see, for example, WO2001/044463).
  • Three libraries were constructed in which 64 positions in and around the peptide binding site were represented by trinucleotides that encoded either the wild-type amino acid or alanine (or, in the case when the wild-type amino acid was alanine, a non-alanine mutant).
  • the libraries were constructed as follows. HtrAlPDZ was displayed on the surface of M13 bacteriophage by modifying a previously described phagemid (pS2202b) ( Skelton, N. J., Koehler, M. F., Zobel, K., Wong, W.
  • the resulting phagemid contained an open reading frame that encoded the maltose binding protein secretion signal, followed by an epitope tag (amino acid sequence: SMADPNRFRGKDLGS (SEQ ID NO: 169)), followed by HtrAlPDZ and ending with the C-terminal domain of the M13 gene-8 minor coat protein.
  • E. coli harboring p ⁇ HtrAl were co-infected with M13-KO7 helper phage and grown at 37°C without IPTG induction, resulting in the production of phage particles that encapsulated p ⁇ HtrAl DNA and displayed HtrAlPDZ in a monovalent format.
  • library 1 positions 380-400; library 2, positions 401-422; library 3, positions 440-460.
  • Libraries 1, 2 and 3 contained 3.0 x 10 10 , 2.5 x 10 10 and 2.3 x 10 10 unique members, respectively.
  • Phage from the libraries described above were propagated in E.coli XLl -blue with the addition of M13-KO7 helper phage. After overnight growth at 37 °C, phage were concentrated by precipitation with PEG/NaCl and resuspended in PBS, 0.5% BSA, 0.1% Tween 20, as described previously (Sidhu, S. S., Lowman, H. B., Cunningham, B. C, and
  • Phage solutions (10 12 phage/mL) were added to 96-well MaxisorpTM immunoplates that had been coated with capture target and blocked with BSA.
  • Two different targets were used: for the display selection the target was an immobilized antibody that recognized the epitope tag fused to the N terminus of HtrAl PDZ, while for the functional selection a biotinylated peptide that bound to HtrAl -PDZ with high affinity (biotin-GWKTWIL (SEQ ID NO: 26) or biotin-DSRIWWV (SEQ ID NO: 5)) (Laura, R. P., Witt, A. S., Held, H.
  • SGCOUNT aligned each DNA sequence against the wild-type DNA sequence by using a Needleman-Wunch pairwise alignment algorithm, translated each aligned sequence of acceptable quality, and tabulated the occurrence of each natural amino acid at each position.
  • the number of analyzed clones are indicated in parentheses following the name of each library: for the functional selection against biotin-GWKTWIL (SEQ ID NO: 4): Ll (80), L2 (91), L3 (89); for the functional selection against biotin-DSRIWWV (SEQ ID NO: 5): Ll (82), L2(89), L3(93); and for the display selection, the following number of clones were analyzed: Ll (69), L2(78), L3(91).
  • HtrAl-PDZ The effects of alanine substitutions in HtrAl-PDZ upon binding to peptides Hl_c3 and Hl_c2 are mapped onto the structure of HtrAl-PDZ in Figures 7A and 7B, respectively.
  • some of the substitutions having a significant effect on binding reside at positions that are in direct contact with the peptide ligand (see, e.g., Y382, 1383, G384, M387, and S389), while others reside in positions that are greater than 4.5 A from the peptide ligand (see, e.g., K380, K381, G411, Y413, 1414, V417, T421, and P422).
  • the wt/mutant ratios were determined from the sequences of binding clones isolated after selection for binding to either a high affinity peptide ligand (functional selection) or an anti- gD-tag antibody (display selection).
  • a normalized frequency of occurrence (F) was derived by dividing the functional selection wr/mutant ratio by the display selection wt/mutant ratio. In the cases where a particular mutation was not observed amongst the functional selection sequences, only a lower limit could be defined for the wt/mutant ratio and the F value (indicated by a greater than sign).
  • the F values were determined for alanine substitutions and also for two additional substitutions (m2 and m3) in cases where the alanine-scan required a tetranomial codon.
  • the identities of non-alanine substitutions are shown in parentheses to the right of each F value.
  • Bold numbers indicate mutations having more than a 4-fold effect.
  • the wt/mutant ratios were determined from the sequences of binding clones isolated after selection for binding to either a high affinity peptide ligand (functional selection) or an anti- gD-tag antibody (display selection).
  • a normalized frequency of occurrence (F) was derived by dividing the functional selection wr/mutant ratio by the display selection wt/mutant ratio. In the cases where a particular mutation was not observed amongst the functional selection sequences, only a lower limit could be defined for the wt/mutant ratio and the F value (indicated by a greater than sign).
  • the F values were determined for alanine substitutions and also for two additional substitutions (m2 and m3) in cases where the alanine-scan required a tetranomial codon.
  • the identities of non-alanine substitutions are shown in parentheses to the right of each F value.
  • Bold numbers indicate mutations having more than a 4-fold effect.
  • HtrAl- and HtrA3-PDZ are a consequence of a limited number of sequence differences proximal to the peptide binding site, primarily at positions ⁇ 2-3, ⁇ 2: ⁇ 2- 1 and ⁇ 3-5.
  • the effect on ligand specificity of mutations in the context of HtrAl-PDZ was assessed.
  • Phage-displayed peptide libraries were used to define the binding specificity profiles of point mutations (I415Q, I418A, and S449Q) and a double mutant (I415Q/I418A) of HtrAl-PDZ, in which residues of HtrAl-PDZ were substituted with the corresponding residues of HtrA3-PDZ.
  • the mutants were sorted against a library of random heptapeptides displayed at the C-terminus of M 13 p8 coat protein. Specific binding clones were obtained only for the double mutant, HtrA 1(1415 Q/I418A)-PDZ.
  • HtrA3-PDZ This mutant was designed specifically to mimic the binding behavior of HtrA3-PDZ (see Table 1), and its specificity profile was essentially identical to that of HtrA3-PDZ.
  • HtrA 1(1415 Q/I418A)-PDZ showed a preference for valine and leucine residues, although other hydrophobic amino acids were also selected.
  • position 0 preference of HtrA 1(1415 Q/I418 A)-PDZ was subtly different from that of HtrA3-PDZ, preferences at the remaining ligand positions were indistinguishable from those observed for HtrA3-PDZ binding. Tryptophan was selected for exclusively at position -1, there was no preference at position -2, position -3 showed a preference for glycine and serine residues, and none of the other ligand positions showed any preference.

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EP08729968A 2007-02-16 2008-02-15 Htra1-pdz- und htra3-pdz-modulatoren Withdrawn EP2111413A2 (de)

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PCT/US2008/054081 WO2008101160A2 (en) 2007-02-16 2008-02-15 Htra1-pdz and htra3-pdz modulators

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DK1994155T4 (da) 2006-02-13 2022-07-25 Daiichi Sankyo Co Ltd Polynukleotid- og polypeptidsekvenser involveret i fremgangsmåden med knogleremodellering
US8168181B2 (en) 2006-02-13 2012-05-01 Alethia Biotherapeutics, Inc. Methods of impairing osteoclast differentiation using antibodies that bind siglec-15
TW201321414A (zh) 2011-10-14 2013-06-01 Genentech Inc 抗－ＨｔｒＡ１抗體及其使用方法
ES2723885T3 (es) 2012-07-19 2019-09-03 Daiichi Sankyo Co Ltd Anticuerpos anti-Siglec-15
CA3001362C (en) 2015-10-30 2020-10-13 Genentech, Inc. Anti-htra1 antibodies and methods of use thereof
JP2019507176A (ja) 2016-03-04 2019-03-14 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ Ｈｔｒａ１阻害剤としての新規ジフルオロケタミド誘導体
CN108699106B (zh) 2016-03-04 2021-11-30 豪夫迈·罗氏有限公司 作为htra1抑制剂的新型三氟甲基丙酰胺衍生物
JP2019522673A (ja) 2016-07-18 2019-08-15 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ Ｈｔｒａ１阻害剤としての新規なジフルオロケトアミド誘導体
EP3504197B1 (de) 2016-08-23 2021-11-17 F. Hoffmann-La Roche AG Neue trifluormethylpropanamidderivate als htra1-inhibitoren
JP2019526563A (ja) 2016-08-23 2019-09-19 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ Ｈｔｒａ１阻害剤としての新規ジフルオロケタミド誘導体
US11149067B2 (en) 2017-05-12 2021-10-19 Universität Duisburg-Essen Tailored cyclodepsipeptides as potent non-covalent serine protease inhibitors
EP3870596A1 (de) * 2018-10-22 2021-09-01 University of Copenhagen Viral exprimierte inhibitoren von pdz-domänen wie etwa pick1 und deren verwendungen
BR112022017673A2 (pt) * 2020-03-06 2022-11-08 Univ Copenhagen Inibidor de interação de proteína com c cinase ? 1, micela, e, composição farmacêutica

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US20090087878A9 (en) * 1999-05-06 2009-04-02 La Rosa Thomas J Nucleic acid molecules associated with plants

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AR065370A1 (es) 2009-06-03
WO2008101160A3 (en) 2009-02-19
CL2008000493A1 (es) 2008-06-27
TW200846358A (en) 2008-12-01
WO2008101160A2 (en) 2008-08-21

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