TW200846358A - HtrA1-PDZ and HtrA3-PDZ modulators - Google Patents

Abstract

The invention provides optimized HtrA1 PDZ domain and HtrA3 PDZ domain ligands. The invention further provides modulators of HtrA1 PDZ domain-ligand interaction and HtrA3 PDZ domain-ligand interaction, and methods of identifying and using these modulators.

Description

200846358 IX. INSTRUCTIONS: [Prior Art] The PDZ domain is a common modularized protein domain that binds to the C-terminus of its bioligand in a sequence-specific manner or, in some cases, to an internal hairpin-shaped motif. To mediate a variety of specific protein-protein interactions (Sheng, Μ· and Sala, C. (2001) Annual Review of 24(1), 1-29). The specificity of the PDZ domain for its ligand is initially based on position-2 (using the normalized PDZ ligand nomenclature, where the C-terminal φ is designated as a residue 〇 and the negative number is increased to the N-terminus by a negative integer to number the remaining residues The presence of the Ser/Thr residue (type I) or the hydrophobic residue (Π type) is divided into two categories. More recent studies have suggested that the determinants of PDZ domain selectivity are more complex, where the binding determinant may consist of 3 to 6 C-terminal ligand residues. For example, the binding profile of 'Erbin-PDZ is very specific ([D/E][T7S]WVC00H) and the binding profile of ZO-PDZ1 is similar ([R/K/S/T][T/S][W /Y][V/I/L]C00H), but exhibits increased chaos for three of the last four ligand residues. Erbin-PDZ and ZOl-PDZl φ also use an auxiliary ligand interaction that regulates the position of the binding affinity-3 upstream (Appleton, Β·A·, Zhang, Y·, Wu, P·, Yin, J. P ·, Hunziker, W., Skelton, N. J., Sidhu, S. S. & Wiesmann, C. (2006) J. 5沁/· Ckm. 281(31), 22312-22320). In addition, specific protein-protein interactions are important for the biological function of PDZ-containing proteins (Zhang, Y,, Yeh, S., Appleton, Β·A·, Held, H. A., Kausalya, P J., Phua, DC Y·, Wong, W. L., Lasky, L. A., Wiesmann, C., Hunziker, W. and Sidhu, S. S. (2006) J. 128788.doc 200846358 5ζ ·Μ·CTzem·, 281(31): 22299-311).

In humans, the Htr A family of serine proteases has four known members with broad homology to the high-temperature requirement A protease (Htr A). This bacterial protease acts as a concomitant protein under normal temperature conditions and is critical for survival at high temperatures where proteolytic function mediates denaturing protein degradation. Although all human HtrA proteins share a homologous trypsin-like serine protease (SP) domain and a C-terminal PDZ domain, HtrAl and HtrA3 also contain a secretory signal sequence and insulin-like growth factor binding in the N-terminal region. Protein domain and Kazal type SP inhibitor domain. HtrAl was originally identified as a gene that was down-regulated in human fibroblast cell lines after transfection with the oncogenic virus SV40 (Zumbrunn, J. ATrueb, B. (1996) FEES Lett 398 (2-3), 187-192). Since then, HtrAl has been implicated in a variety of malignancies. Compared with normal tissues, HtrAl is down-regulated in cancer, and excessive expression inhibits tumor growth and proliferation (Baldi, A·, De Luca, A·, Battista, T·, Felsani, A,, Baldi, F· , Catricala, C., Amantea, A., Noonan, D. M., Albini, A., Natali, P. G., Lombardi, D. and Paggi, M. G. (2002) 21(43), 6684 -6688). In contrast, HtrAl is up-regulated in the cartilage of osteoarthritic joints and can cause the development of such and other arthritic diseases (Hu, S.-I., Carozza, M., Klein, M., Nantermet, P., Luk) , D· and Crowl, R. (1998) «/. Call / 〇 / · CAem. 273 (51), 34406-34412). HtrAl is also involved in the processing of amyloid precursor proteins (Grau, S., Baldi, A., Bussani, R., Tian, Χ·5 Stefanescu, R., Przybylski, M., Richards, P., Jones, S. A·, Shridhar, V·, 128788.doc 200846358

Clausen, Τ· and Ehrmann, Μ (2005) PAMS 102(17), 6021-6026) and thus can play a role in Alzheimer's disease. Recently, single nucleotide polymorphisms in the promoter region of HtrAl have been identified in patients with wet age-related macular degeneration leading to increased HtrAl performance, confirming the critical role of HtrAl in the pathogenesis of this disease. (DeWan, A·, Liu, M·, Hartman, S·, Zhang, SS·-

M·,Liu,DT L·,Zhao,C·,Tam,Ρ·0·S·,Chan,W.M·, Lam, DS C·,Snyder,M,,Barnstable,C·,Pang,C. P· and Hoh, J. (2006) Science 314(5801), 989-992; Yang, Z., Camp, N. J., Sun, H., Tong, Z., Gibbs, D., Cameron, D J·, Chen, H,, Zhao, Y·, Pearson, E·, Li, X·, Chien, J., DeWan, A·, Harmon, J., Bernstein, P. S., Shridhar, V· , Zabriskie, NA, Hoh, J., Howes, K. and Zhang, K. (2006) iSc/ence 314 (5801), 992-993). HtrA3 plays a role in placental development (Nie et al., Biol Reprod. February 2006; 74(2): 366-74) and is also involved in endometrial cancer with HtrAl (Bowden et al., Gynecol Oncol. October 2006; 103(1): 253-60). It has been shown that HtrAl binds to TGF-β family proteins in mice and suggests that it mediates inhibition of TGF-β 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) Deve/opmeni 131(5), 1041-1053). Although inhibition of TGF-β is dependent on the protease activity of HtrAl, the PDZ domain appears to play a direct role in regulating protease activity. A peptide derived from the C-terminus of TGF-β family member 128788.doc 200846358 (such as Col3al) binds to the PDZ domain of HtrAl and stimulates protease activity (Murwantoko, Yano, M., Ueta, Y., Murasaki, A., Kanda) , H., Oka, C. and Kawaichi, Μ. (2004) Price oc/zem / 381 (Part 3), 895-904). Hti*A3 also displays comparable proteases and TGF-β signaling inhibitory activity (Tocharus, J., Tsuchiya, A., Kajikawa, M., Ueta, Y., Oka, C. and Kawaichi, M_ (2004) Growth and Differentiation 25Ί -2Ί. The above-mentioned important molecular functions attributed to HtrAl and HtrA3, especially their function mediated by the protein-protein interaction between HtrAl PDZ domain or HtrA3 PDZ and ligands suggest that HtTAl PDZ and HtrA3 PDZ domain represent Important therapeutic targets. Thus, it would be advantageous to clarify the mechanism of interaction between the ligand and the HtrAl or HtrA3 PDZ domain and to provide compositions and methods for targeted modulation of its related functional activities. The present invention provides this benefit and Other Advantages The present invention provides compositions for modulating the activity of the PDZ domain of each of the HtrAl and HtrA3 proteins and methods of using the same. Due to the important functions associated with HtrA1 and HtrA3, The compositions and methods of the invention have significant clinical and therapeutic utility. The binding partners (ligands) of the partial base & system, HtrAl PDZ domain and HtrA3 PDZ domain of the present invention> Analysis and characterization, the analysis yielded novel and unexpected results as described herein. Two groups of HtrAl PDZ domains and HtrA3 PDZ domains were independently generated by phage display libraries, with M13 stomach C- The terminal or Ν-terminally fused peptide represents the form of a free carboxyl group. # ^ + 128788.doc 200846358 and a peptide conjugate that does not require a free retinoyl group. The HtrA1 PDZ domain comprising a free or a carboxy terminal or a free carboxy terminus is described herein or Peptide ligands of the HtrA3 PDZ domain. These results demonstrate that unlike a ligand that requires a free carboxyl terminus to bind to most other PDZ domains of PDZ, a set of HtrAl PDZ domain ligands and HtrA3 PDZ domains The ligand can bind to the HtrAl PDZ domain or the HtrA3 PDZ domain, respectively, without a free carboxy terminus. The ligand without a free carboxy terminus represents the N-terminus and/or the internal HtrAl PDZ domain of the N-terminal or internal sequence of the polypeptide. Ligand or HtrA3 PDZ domain ligand sequence. The binding specificity of a series of peptide ligands is assessed by measuring the relative affinities of a series of peptide ligands as described below. Individual residues of an exemplary peptide ligand Alanine scanning analysis was performed to elucidate the energy contribution of different residues at each ligand position. Molecular modeling was also performed to interface specific excipient ligands with each of the HtrAl PDZ domain and the HtrA3 PDZ domain to structure based The binding specificity was further evaluated. Phage-based combinatorial scanning is also used to identify residues in the HtrAl PDZ domain that contribute energetically to the ligand PDZ interaction, thereby providing further structure and energy components for the interaction of the HtrAl PDZ domain with its ligands. Insight. In one embodiment, the invention provides an isolated polypeptide that specifically binds to a HtrAl PDZ domain, wherein the polypeptide comprises a sequence having tryptophan at position -2 relative to the C-terminus. In one aspect, the polypeptide further comprises a small amino acid at the position of the oxime. In another aspect, the amino acid at position 0 is selected from the group consisting of leucine and valine. In another aspect, the polypeptide further comprises an amino acid comprising a bulky side chain at position -1. In another aspect, position -18 128788.doc -10- 200846358 Amino acid is selected from the group consisting of tryptophan, isoleucine, and phenylalanine. In another aspect, the amino acid at position -1 is tryptophan. In another aspect, the polypeptide further comprises threonine or isoleucine at position -3. In another aspect, the amino acid at position -4 is charged. In another sad case, the amino acid at position -4 is selected from the group consisting of glutamic acid, lysine, arginine, and aspartic acid. In another aspect, the amino acid at position -4 is selected from the group consisting of lysine and arginine. In another embodiment, the invention provides an isolated polypeptide that specifically binds to a HtrAl PDZ domain, wherein the amino acid at position 0 relative to the C-terminus is selected from the group consisting of leucine and valine; wherein position -1 The amino acid is selected from the group consisting of tryptophan, isoleucine and phenylalanine; wherein the amino acid at position -2 is tryptophan; wherein the amino acid at position -3 is selected from the group consisting of sulphate and Leucine; and wherein the amino acid at position -4 is selected from the group consisting of glutamic acid, lysine, arginine, and aspartic acid. In one aspect, 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 the group consisting of threonine and isoleucine And the amino acid at position -4 is selected from the group consisting of lysine and arginine. In another embodiment, the invention provides an isolated polypeptide that specifically binds to the HtrAl PDZ domain, wherein the polypeptide comprises a sequence selected from the sequence of the HtrAl PDZ domain binding peptide set forth in Table 1. In one aspect, 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). In one aspect, the polypeptide comprises the sequence DSRIWWV (SEQ ID NO: 5). In another embodiment, the invention provides an isolated polypeptide that specifically binds to the HtrAl PDZ domain, wherein the polypeptide directly interacts with at least one specific HtrAl-128788.doc 11 200846358 PDZ domain residue. In one aspect, the C-terminal carboxylate group of the isolated polypeptide is coordinated to at least one HtrAl PDZ domain residue selected from the group consisting of Ile383, Ile385 and Gly384. In another aspect, the amino acid at position -1 of the isolated polypeptide interacts dynamically with at least one HtrAl PDZ domain residue selected from the group consisting of Tyr382, Arg386 and Ile418. In another aspect, the tryptophan at position-2 of the isolated polypeptide interacts with at least one HtrAl PDZ domain residue selected from the group consisting of Ala445, Met387 and Gln446. In another aspect, the amino acid at position -3 of the isolated polypeptide interacts with at least one HtrAl PDZ domain residue selected from the group consisting of Ile415 and Arg386. In another aspect, the isolated polypeptide is ligated and/or coordinated to at least one HtrAl PDZ domain residue selected from the group consisting of Tyr382, Ile383, Gly384, Met387, and S389. In another embodiment, the invention provides an isolated polypeptide that specifically binds to the HtrAl PDZ domain, wherein the polypeptide comprises a sequence having tryptophan at position -2 relative to the acidic amino acid. In one aspect, the polypeptide comprises a sequence of the formula XI-X2-W-X3-X4, wherein the XI is selected from the group consisting of lysine and leucine; wherein the X2 is selected from the group consisting of serine, threonine, and arginine And alanine and valine; wherein X3 is selected from the group consisting of glycine, serine, phenylalanine and leucine; and wherein X4 is an acidic amino acid. In another aspect, X3 is glycine and X4 is selected from the group consisting of glutamic acid and aspartic acid. In another embodiment, the invention provides an isolated polypeptide that specifically binds to a HtrAl PDZ domain, and which comprises an amino acid sequence comprising a peptide selected from the amino acid sequences set forth in Tables 1, 2 and 3 C-terminal, N-terminal or internal amino acid sequence. In one aspect, the C-terminal amino acid sequence is selected from the group consisting of 128788.doc -12. 200846358 DIETWLL (SEQ ID NO: 3), GWKTWIL (SEQ ID NO: 4), DSRIWWV (SEQ ID NO: 5), and WDKIWHV (SEQ ID NO: 6) In another embodiment, the invention provides an isolated polypeptide comprising an amino acid sequence that competes with any of the above polypeptides for binding to the HtrAl PDZ domain. In another embodiment, the invention provides an isolated polypeptide that binds to the same epitope on the HtrAl PDZ domain as any of the above polypeptides. In another embodiment, the invention provides a variant HtrAl PDZ domain. In one aspect, an isolated polypeptide comprising a HtrAl PDZ variant sequence is provided, wherein: at least one HtrAl PDZ domain residue selected from the following residues is substituted with another amino acid: Ile383, Ile385, Gly384, Tyr382, Arg386 Ile418, Ala445, Met387, Gln446, Ile415, Arg386, Ser389, Lys380, Lys381, G1411, Tyr413, Ile414, Val417, Thr421 and Pi*o422. In another aspect, Ile418 is substituted with another amino acid. In another aspect, the additional amino acid is alanine. In another embodiment, the invention provides an isolated polypeptide comprising an amino acid sequence-column that competes with any of the above Htr A1 PDZ domain variants for binding to a ligand of the HtrAl PDZ domain. In another embodiment, the invention provides an isolated polypeptide that binds to the same antigenic determinant on a ligand of the HtrAl PDZ domain with any of the above HtrAl PDZ domain variants. In another embodiment, the invention provides methods of using the HtrAl PDZ domain variant polypeptides described above and the HtrAl PDZ domain binding polypeptide. In one aspect, the invention provides a method of identifying a compound capable of modulating HtrAl PDZ domain-ligand interaction, comprising comprising a fragment comprising a HtrAl PDZ domain, a HtrAl PDZ domain, and/or a functional equivalent thereof, and at least one The above HtrAl PDZ domain 128788.doc •13- 200846358 The sample of the binding polypeptide is contacted with the candidate compound, and (4) the case of the presence of the candidate compound in the absence of the candidate compound. TM The amount of PDZ domain-ligand interaction ; Yu Tian and in the absence of candidate compounds

In the case of the amount of HtrAl PDZ domain-ligand interaction in the presence of candidate servants, kansas & compounds, the κ κ ming candidate compound is capable of modulating the HtrAl PDZ domain-coordination Compounds that interact with each other.

In another aspect, the invention provides a method of rationally designing a modulator of domain-ligand interaction comprising designing a modulator to comprise or mimic acidic residues located relative to or relative to the C-terminus The function of tryptophan at position ^, wherein the modulator is capable of specifically binding to the HtrAi domain. In one aspect, the peptide is located at the carboxy terminus. In another aspect, the amino acid at position 0 is selected from the group consisting of leucine and valine, and the amino acid at the position is selected from the group consisting of tryptophan, isoleucine, and phenylalanine, at position _3. The amino acid is selected from the group consisting of threonine and isoleucine, and the amino acid at position _4 is selected from the group consisting of glutamic acid, aspartic acid, lysine, and arginine. In another aspect, the amino acid at position 0 is selected from the group consisting of leucine and valine, the amino acid at position 丨 is tryptophan, and the amino acid at position _3 is selected from sulphate. The acid and isotonic acid 'and the amino acid at position _4 are selected from the group consisting of glutamic acid and aspartic acid. In another aspect, the invention provides a method of treating a pathological condition associated with a disordered regulation of HtrAl protein activity comprising administering an effective amount of the HtrAl PDZ domain ligand to an individual in need thereof. In another aspect, the invention provides a method of treating a pathological condition associated with abnormal regulation of HtrAl protein activity, comprising administering to an individual in need thereof an effective amount of a HtrAl PDZ domain-ligand modulator, wherein The regulator can adjust 128788.doc -14- 200846358

Interaction between the HtrAl PDZ domain and any of the above HtrAl PDZ domain binding polypeptides. In another aspect, the invention provides a method of treating a pathological condition associated with a disordered regulation of HtrA1 protein activity, comprising administering to an individual in need thereof an effective amount of the above variant HtrA1 pDZ domain polypeptide, wherein the variant HtrAl The PDZ domain polypeptide is capable of modulating the interaction between the HtrA1 pDZ domain and the mrAi PDZ domain ligand. In some aspects, the regulation is to inhibit the interaction between the HtrAl PDZ domain and the ligand. In some aspects, it is modulated to enhance the interaction between the HtrAl PDZ domain and the ligand. ♦ In some cases, the pathological condition is selected from malignant and benign tumors or cancers, non-leukemia and lymphoid malignancies, neurodegenerative disorders, inflammatory and immune disorders, and intraocular neovascular disorders. In some aspects, the pathological condition is selected from the group consisting of cancer (including but not limited to ovarian cancer and endometrial cancer), Alzheimer's disease, rheumatoid arthritis, and age-related (wet type) Macular degeneration. In another sorrow, the present invention provides a method of treating a pathological condition associated with a regulatory abnormality in Htr A丨 protein activity comprising administering an effective amount of any of the above HtrAl PDZ domain binding proteins to a desired one. With ^^ eight! An agonist for the interaction of the PDZ domain. In another aspect, the invention provides a method of treating a pathological condition associated with abnormal regulation of HtrAl protein activity, comprising administering to an individual in need thereof an effective amount of any of the above HtrAl PDZ domain binding proteins and HtrAl. An antagonist of the interaction of the pDZ domain. In some aspects, the pathological condition is selected from the group consisting of malignant and benign tumors or cancer, non-leukemia and lymphoid malignancies, neurodegenerative disorders, inflammatory and immune disorders, and intraocular neovascular disorders. In some cases, the disease 128788.doc -15- 200846358 is based on cancer (including but not limited to ovarian cancer and endometrial cancer), Alzheimer's disease, rheumatoid arthritis And age-related (wet) macular degeneration. In another embodiment, the invention provides methods of detecting the presence, amount or function of a HtrAl or HtrAl PDZ domain in a sample using one or more of the above polypeptides, polynucleotides and/or antibodies. In one embodiment, the invention provides an isolated polypeptide that specifically binds to the HtrA3 PDZ domain, wherein the polypeptide comprises a sequence having tryptophan at position -1 relative to the C-terminus. In one aspect, the polypeptide has an overall hydrophobic character. In another aspect, the polypeptide further comprises an amino acid selected from the group consisting of valine, isoleucine, and alanine at position 0. In another aspect, the polypeptide further comprises an amino acid selected from the group consisting of glycine and serine at position -3. In another aspect, the amino acid at position 0 is selected from the group consisting of lysine, isoleucine, and alanine; and wherein the amino acid at position -3 is selected from the group consisting of glycine and serine. In another aspect, the amino acid at position 0 is valine; and the amino acid at position -3 is selected from the group consisting of glycine and serine. In another embodiment, the invention provides an isolated polypeptide that specifically binds to the HtrA3 PDZ domain, wherein the polypeptide comprises a sequence of a sequence selected from the HtrA3 PDZ domain binding peptides listed in Tables 1 and 3. In one aspect, 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) 126788.doc -16- 200846358 FERWV (SEQ ID NO: 17) - FYRWV (SEQ ID NO: 18), FGAWV (SEQ ID NO: 19) and FARWV (SEQ ID NO: 20). In one aspect, the polypeptide comprises the sequence FGRWV (SEQ ID NO: 11). In another embodiment, the invention provides an isolated polypeptide that specifically binds to the Hti*A3 PDZ domain, wherein the polypeptide directly interacts with at least one specific HUA3-PDZ domain residue. In one aspect, the HtrA3 PDZ domain binds to the C-terminal carboxylate group of the polypeptide and is coordinated to at least one HtrA3 PDZ domain residue selected from the group consisting of Phe356, Ile357 and Gly35. In another aspect, the tryptophan at position-1 of the HtrA3 PDZ domain binding polypeptide interacts with at least one HtrA3 PDZ domain residue selected from the group consisting of Glu390 and Ala392. In another aspect, the amino acid at position -2 of the HtrA3 PDZ domain binding polypeptide interacts with the HtrA3 PDZ domain residue Gln423. In another aspect, the amino acid at position -3 of the HtrA3 PDZ domain binding polypeptide interacts with the HtrA3 PDZ domain residue Arg360. In another aspect, the HtrA3 PDZ domain binding polypeptide interacts and/or coordinates with at least one HtrA3 PDZ domain residue selected from the group consisting of Phe356, Ile357, Gly358, Glu390, Ala392, Gln423, and Arg3 60. In another embodiment, the invention provides an isolated polypeptide that specifically binds to a HtrA3 PDZ domain, wherein the polypeptide comprises a sequence having a conserved acidic residue following one or more hydrophobic residues. In one aspect, the polypeptide comprises the sequence WVL. In another aspect, 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). 128788.doc 17 200846358 In another embodiment, the invention provides an isolated polypeptide that specifically binds to a HtrA3 PDZ domain, wherein the polypeptide comprises a sequence selected from the HtrA3 PDZ domain binding sequences set forth in Table 2. In another embodiment, the invention provides an isolated polypeptide that specifically binds to a HtrA3 PDZ domain, and which comprises an amino acid sequence comprising a peptide selected from the amino acid sequences listed in Tables 1, 2 and 3. C-terminal, N-terminal or internal amino acid sequence. In one aspect, the C-terminal amino acid sequence is selected from the group consisting of 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). In another embodiment, the invention provides a separate polypeptide comprising an amino acid sequence that competes with any of the above HtrA3 PDZ domain binding polypeptides for binding to the Hti*A3 PDZ domain. In another embodiment, the invention provides an isolated polypeptide comprising an amino acid sequence that binds to the same epitope on the HtrA3 PDZ domain as any of the above HtrA3 PDZ domain binding polypeptides. In another embodiment, the invention provides the isolation of a variant HtrA3 PDZ domain polypeptide. In one aspect, the isolated polypeptide comprises a Htr A3 PDZ variant sequence, wherein at least one of the HtrA3 PDZ domain residues selected from the group consisting of Phe356, Ile357, Gly358, Glu390, Ala392, Gln423, and Airg360 is substituted with another amino acid. In another aspect, the variant polypeptide is isolated from Glu390 and/or 128788.doc -18- 200846358

Ala392 is substituted with another amino acid. In some aspects, the other amino acid is alanine. In another embodiment, the invention provides an isolated polypeptide comprising an amino acid sequence that competes with any of the above variant HtrA3 PDZ domain polypeptides for binding to a ligand of the HtrA3 PDZ domain. In another embodiment, the invention provides an isolated polypeptide that binds to the same epitope on a ligand of the HtrA3 PDZ domain with any of the above variant HtrA3 PDZ domain polypeptides. In another embodiment, the invention provides methods of using the HtrA3 PDZ domain variant polypeptides described above and the HtrA3 PDZ domain binding polypeptide. In one aspect, the invention provides a method of identifying a compound capable of modulating an HtrA3 PDZ domain-ligand interaction comprising comprising a fragment comprising a HtrA3 PDZ domain, a HtrA3 PDZ domain, and/or a functional equivalent thereof, and at least one The Hti:A3 PDZ domain binding polypeptide sample is contacted with the candidate compound, and the amount of the HtrA3 PDZ domain-ligand interaction is present in the presence of the candidate compound compared to the absence of the candidate compound; The change in the amount of HtrA3 PDZ domain-ligand interaction in the presence of the candidate compound compared to the amount in the absence of the candidate compound indicates that the candidate compound is a compound capable of modulating the HtrA3 PDZ domain-ligand interaction. In another aspect, the invention provides a method of rationally designing a modulator of HtrA3 PDZ domain-ligand interaction, comprising designing a modulator to comprise or mimic at a position -1 relative to the C-terminus or relative to The function of tryptophan at position-2 of leucine in the peptide, wherein the modulator is capable of specifically binding to the HtrA3 PDZ domain. In another aspect, the amino acid at position 缬 is selected from the group consisting of lysine, isoleucine, and alanine, and the amino acid at position -1 is color 128788.doc -19-200846358 aminic acid, and The amino acid at position -3 is selected from the group consisting of glycine and serine. In another aspect, the amino acid at position 0 is valine acid, the amino acid at position -1 is tryptophan, and the amino acid at position -3 is selected from the group consisting of glycine and silk. acid. In another aspect, the modulator comprises an amino acid sequence WVL. In another aspect, the invention provides a method of treating a pathological condition associated with a disordered regulation of Htr A3 protein activity comprising administering an effective amount of the HtrA3 PDZ domain ligand to an individual in need thereof. In another aspect, the invention provides a method of treating a pathological condition associated with a disordered regulation of Htr A3 protein activity, comprising administering to an individual in need thereof an effective amount of a HtrA3 PDZ domain-ligand modulator, Wherein the modulator is capable of modulating the interaction between the HtrA3 PDZ domain and any of the above HtrA3 PDZ domain binding polypeptides. In another aspect, the invention provides a method of treating a pathological condition associated with a regulatory abnormality in Htr A3 protein activity, comprising administering to an individual in need thereof an effective amount of the above variant HtrA3 PDZ domain polypeptide, wherein the variation The Htr A3 PDZ domain polypeptide regulates the interaction between the Htr A3 PDZ domain and the Htr A3 PDZ domain ligand. In some aspects, the regulation is to inhibit the interaction between the Htr A3 PDZ domain and the ligand. In some aspects, it is modulated to enhance the interaction between the HtrA3 PDZ domain and the ligand. In some aspects, the pathological condition is selected from the group consisting of malignant and benign tumors or cancer, non-leukemia and lymphoid malignancies, and placental dysfunction. In some aspects, the pathological condition is cancer (including but not limited to, endometrial cancer). In another aspect, the invention provides a method of treating a pathological condition associated with a regulatory abnormality in Htr A3 protein activity, comprising administering to the desired one of 128788.doc -20-200846358 an effective amount of any of the above The HtrA3 PDZ domain binds to an agonist of the interaction of the protein with the HtrA3 PDZ domain. In another aspect, the invention provides a method of treating a pathological condition associated with abnormal regulation of Htr A3 protein activity comprising administering to an individual in need thereof an effective amount of any of the above HtrA3 PDZ domain binding proteins and HtrA3 PDZ Antagonists of domain interactions. In some aspects, the pathological condition is selected from the group consisting of malignant and benign tumors or cancer, non-leukemia and lymphoid malignancies, and placental dysfunction. In some aspects, the pathological condition is cancer (including but not limited to, endometrial cancer). In another embodiment, the invention provides methods of detecting the presence, amount or function of a HtrA3 or HtrA3 PDZ domain in a sample using one or more of the above polypeptides, polynucleotides and/or antibodies. In another embodiment, the invention provides an isolated polynucleotide encoding the above HtrAl PDZ domain binding polypeptide, HtrA3 PDZ domain binding polypeptide, variant HtrAl PDZ domain or variant HtrA3 PDZ domain or a complement thereof. In another embodiment, the invention provides a vector comprising one or more of such polynucleotides. In another embodiment, the invention provides host cells comprising one or more of such vectors. In another embodiment, the invention provides a method of making a polypeptide comprising culturing the host cells under conditions in which the polynucleotide is expressed and optionally recovering and/or purifying the polypeptide. In another embodiment, the invention provides a non-human mammal that expresses one or more of the polynucleotides of the polynucleotide. In one aspect, the transgenic gene non-human mammal further has at least one inactivated HtrAl or HtrA3 gene. In another embodiment, the invention provides for the specific binding of one or more of the H.sub.2 PD. antibody. In another embodiment, the invention provides a kit comprising one or more molecules of the invention. In one aspect, the kit comprises at least one of the HtrAl PDZ domain binding polypeptide, the HtrA3 PDZ domain binding polypeptide, the variant HtrAl PDZ domain or the variant HtrA3 PDZ domain. In one aspect, the kit comprises at least one of the above-described HtrAl PDZ domain binding polypeptide, HtrA3 PDZ domain binding polypeptide, variant HtrAl PDZ domain or variant Hti*A3 PDZ domain. In another aspect, the kit comprises at least one antibody that specifically binds to at least one of the HtrAl PDZ domain binding polypeptide, the HtrA3 PDZ domain binding polypeptide, the variant HtrAl PDZ domain or the variant HtrA3 PDZ domain. [Embodiment] The present invention provides molecules capable of regulating the binding interaction between the PDZ domain of the HtrAl or HtrA3 protein and its cell binding partner, and methods for identifying and using such molecules. In one aspect, the molecules are produced by a combination of methods that allow the identification of a peptide binder that binds to the HtrAl PDZ or HtrA3 PDZ domain with different affinities. As described herein, the identification of these binder molecules and the structural kinetics of the binding interaction between the HtrAl PDZ domain or the HtrA3 PDZ domain and its respective binding partners further provide for the identification of other bindings to the HtrAl PDZ domain or the HtrA3 PDZ domain. The method of adjusting the agent. Given the importance of HtrAl and Htr A3 in a variety of cellular and physiological processes, such modulators will have significant utility in, for example, prophylactic, therapeutic, and/or diagnostic settings. 128788.doc -22- 200846358 General Techniques Unless otherwise indicated, the practice of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, such techniques. Within the technical scope of this technology. Such techniques are explained in detail in the following documents, such as ''Molecular Cloning: A Laboratory Manual', 2nd edition, (Sambrook et al., 1989); "Oligonucleotide Synthesis" (edited by J. Gait, 1984); Animal Cell Culture" (edited by R. I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (F·M· Ausubel et al., 1987, and Sexual update); "PCR: The Polymerase Chain Reaction", (Mullis 4 ed. '1994); "A Practical Guide to Molecular Cloning,, (Perbal Bernard V.? 1988) 募 used or described in the present invention Nucleotides, polynucleotides, peptides, polypeptides, and small molecules can be produced using standard techniques known in the art. Definitions When referring to a molecule, "separation" means having been identified from the components of its natural environment. Molecules that are isolated and/or recovered, one-to-Knife with a visible component, are components that interfere with diagnostic or therapeutic use. As used herein, the control sequence " To make the operability of the coding: to list the DNA sequence that is expressed in a particular host organism. The prokaryotic control sequence includes the promoter, the price of the longitudinal sequence and the ribosome binding site. The sequence includes a promoter, a face Φ person, a "polar control ^ ΛΚ gland" and a sputum signal and a fortifier. When the nucleic acid is made into a functional relationship with another nucleic acid and is in a functional relationship, it can be, 128788.doc -23- 200846358: linked ". For example, when the promoter or enhancer affects the sequence of the transfection 1 which is operatively linked to the coding sequence, or when the ribosome binding site is positioned to facilitate transfection, it is detachably linked to the coding sequence. . "Operative connection" usually means that the connected sequence is adjacent to the field, and in the secretory preamble amp&, 丨軎,, WT ^月J ¥斤幻之之哥, Read the phase ((4)峋 phase). However, the enhancers do not have to be adjacent.

The active peptide or fragment thereof retains the biological activity of the active or naturally occurring counterpart of the active polymorphism. Biological activity refers to the function mediated by the native or naturally occurring counterpart of the active polypeptide. For example, binding or protein-protein interactions constitute biological activity. The terms "," and "immunoglobulin" are used interchangeably and include monoclonal antibodies (eg, full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (eg, double Specific antibodies, as long as they exhibit the desired biological activity, and may also include certain antibody fragments (as described in more detail herein). The light chain of an antibody to any vertebrate species can be assigned to one of two distinct types known as kappa and lambda based on the amino acid sequence of its constant domain. The antibody (immunoglobulin) can be assigned to a different species depending on the amino acid sequence of its heavy chain. There are five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, and several of them can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgA-1, IgA-2. Wait. The heavy-chain constant domains corresponding to different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional configuration of different types of immunoglobulins are known to us and are generally described, for example, in Abbas et al., Cellular and Mol. 128788.doc -24 - 200846358

Immunology, 4th edition, (20〇〇). The antibody may be part of a more fused molecule formed by covalent or non-covalent association of the antibody with one or more other egg-mass or peptides. The antibody may be a chimeric antibody, a human antibody, a humanized antibody, and/or an affinity matured antibody. "antibody fragment" contains only a portion of an intact antibody, &> preferably retains at least one, preferably most or all of the functions normally associated with that portion when present in an intact antibody. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homologous antibodies, i.e., in addition to a naturally occurring mutation that may be present in minor amounts, the individual antibodies comprising the population are identical. Individual antibodies have a high specificity for a single antigen. Furthermore, unlike individual antibody preparations which typically include different antibodies directed against different determinants (antigenic determinants), each monoclonal antibody system is directed against a single determinant on the antigen. Monoclonal antibodies herein include, inter alia, "chimeric" antibodies in which the heavy chain and/or the light chain portion are homologous or homologous to the corresponding sequence in an antibody derived from a particular antibody (a) belonging to a particular antibody class or subclass. At the same time, the remainder of the strand or the strands are identical or homologous to the corresponding sequences derived from another species or antibodies belonging to another antibody class or subclass; and fragments of such antibodies, as long as they exhibit the desired biological activity, ie (U.S. Patent No. 4,816,567; and Morrison et al., ed. 81685, 6855 (1984)), a non-human (eg, murine) antibody "humanization, in the form of a non-human immunoglobulin derived from The smallest sequence of chimeric antibodies. To a large extent, human 128788.doc -25-200846358 antibody is a human immunoglobulin (recipient antibody) in which the residue of the hypervariable region of the recipient has the desired specificity, affinity and Replacement of residues in the hypervariable regions of non-human species (donor antibodies) such as mice, rats, rabbits or non-human primates. In some cases, the framework of human immunoglobulins The (FR) residue is replaced by a corresponding non-human residue. In addition, the humanized antibody may comprise a residue that is not present in the recipient antibody or in the donor antibody. Such modifications are made to further improve the potency of the antibody. A humanized antibody will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to a hypervariable loop of a non-human immunoglobulin, and all or substantially all of the FRs are human immunoglobulins The protein sequence of fr. The humanized antibody will, as appropriate, also comprise at least a portion of the immunoglobulin constant region (Fc), typically the constant region of human immunoglobulin. For more details, see J〇nes et al.' Nature 321:522_525 (1986); Riechmann et al, Nature 332: 323-329 (1988), and Presta, Curr Op. Struct. Biol 2: 593-596 (1992). See also the following review articles and references cited therein :Vaswani and Hamilton, Yimo j//er illusion;, 乂 (10) Qing Min, ImmunoL 1:105-1 15 (1998); Harris, Biochem, S〇〇, 23:1035-1038 (1995); Hurle and Gross, CWr,

Op. Biotech. 5: 428-433 (1994) ® n human antibodies" are antibodies having an amino-based sequence corresponding to the amino acid sequence of an antibody produced by humans and/or using any of the fabrications disclosed herein. Antibodies produced by the techniques of human antibodies. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen-binding residues. Methods for making human antibodies are well known in the art and include transgenic mice (xen〇m〇) Use) 128788.doc -26- 200846358 Technique as a non-limiting example (eg, as described in WO 96/33735). "Affinity maturation" antibodies have one or more variations in one or more of their CDRs. The antibodies, such mutations, increase the affinity of the antibody to the antigen compared to the parent antibody without the variants. Preferred affinity matured antibodies will have nemo or even picomolar affinity for the target antigen. Affinity Mature antibodies can be prepared by procedures known in the art. Marks et al, 10: 779-783 (1992) describe the recombination of the affinity of the VH and VL domains into a random mutation of CDRs and/or framework residues. Induction _ is described by the following literature: Barbas et al, 91: 3809-3813 (1994); Schier et al, G (9) e 169: 147-155 (1995), Yelton et al 'J. i55: i994_2 〇〇 4 (ip%) ·,

Jackson et al., 乂 (10) 〇 / 154(7): 331〇-9 (1995); and

Hawkins et al., J. Mo/. Price / 226: 889-896 (1992). A 'polypeptide-tagged" polypeptide refers to a chimeric polypeptide fused to a "tag polypeptide." The tags provide an epitope for which the antibody can be made or obtained but does not substantially interfere with the activity of the polypeptide. The reactivity of the target antibody with an endogenous epitope, the tag polypeptide is generally unique. Suitable tag polypeptides typically have at least 6 amino acid residues, typically about 8 and 50 amino acids. Between, preferably between 8 and 20 amino acids. Examples of epitope tag sequences include HA, GD and c_myc, poly-His and FLAG of influenza A virus, as used interchangeably herein, polynucleoside An acid, or, a nucleic acid, refers to a polymer of nucleotides of any length and includes, but is not limited to, DNA and RNA. The nuclear acid can be a deoxyribonucleotide, a ribonucleotide, a Modified nucleosides 128788.doc -27- 200846358 or a test group and/or an analog thereof, or any substrate which can be incorporated into a polymer by DNA or RNA polymerase or a synthetic reaction. The polynucleotide may comprise The core of the brocade is acid-like Glycosylates and their analogs. If present, the nucleotide structure can be modified before or after assembly of the polymer. The sequence of the nucleotide can be interrupted by non-nucleotide components. The polynucleotide can be further synthesized after synthesis. Modifications 'such as by binding to a label. Other types of modifications include, for example, a towel replacing one or more naturally occurring nucleotides with an analog; inter-nucleotide modification, such as having an uncharged bond (eg, Methyl phosphonate, di gadolinium phosphinate, ph〇Sph〇amidate, urethane vinegar, etc. and those with V-bonds (eg thiolates, dithiosity Ester, etc., δ has a hanging P knife (such as proteins (such as nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), with intercalators (such as acridine, bone) A chelating agent (such as ps〇raien), a chelating agent (for example, a metal, a radioactive metal, boron, an oxidizing metal, etc.), a substance containing a burning agent, and a modified binding group (for example, a α-helical heteromeric nucleic acid) And the unmodified form of the polynucleotide. In addition, in the sugar Any of the hydroxyl groups normally present may, for example, be replaced by phosphonate groups, phosphate groups, protected by standard protecting groups or activated to make other linkages with other nucleotides; or may be associated with solid or semi-solid supports Engaging. 5, and 3, the terminal oxime may be phosphorylated or partially substituted with an amine or an organic cap group having 1 to 20 carbon atoms. Other hydroxyl groups may also be derivatized to a standard protecting group. Similar forms containing ribose or deoxyribose sugars generally known in the art include, for example, 2,_〇-methyl-ribose, 2'-0-allyl-ribose, 2,-fluoro-ribose or 2, _ azido-ribose, a carbocyclic sugar analog, a helicotropic sugar, an epimeric sugar (such as 128788.doc •28-200846358 arabinose, xylose or lyx〇ses) Halose, furanose, sedoheptuloses, acyclic analogs and non-basic nuclear analogs, such as methylribose. One or more phosphate dimerization bonds may be replaced by an alternate linkage group. The alternative linking groups include, but are not limited to, the following examples wherein the phosphate group is via P(0)s("thioester"), /(Us("dithio Acidate "), (0) NR2 ("melamine" "), ρ(〇)Κ, P(〇)〇R|, CO or CH2("甲缩搭) replacement, each of which is independent a substituted or unsubstituted alkyl (1 〇 2 〇 C), aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl group which is an oxime or an ether bond (_〇_) as desired. Do 1). It is not necessary to have all of the linkages in the polynucleotides the same. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA. As used herein, "raised nucleotides" generally refers to short, usually single-stranded, usually synthetic, polynucleic acids, typically, but not necessarily, less than about 2 nucleotides in length. The terms "oligonucleotides," and ", polynucleotides, are not mutually exclusive. The above description of nucleating acid is equally and fully applicable to the recruitment of pupa. The term "peptide" generally refers to a peptide group. A continuous and relatively short amino acid sequence linked by a bond. The peptide typically, but not necessarily, has a length of from about 2 to 50 amino acids, 4 to 4 amino acids, or from 10 to 30 amino acids. Although the term "polypeptide" broadly refers to the longer form of the peptide, the two terms are used interchangeably and interchangeably in some instances herein. The terms "amino acid" and "residue" are used interchangeably herein. The "region" of a polypeptide is a contiguous sequence of two or more amino acids. In other embodiments, the zone has at least about any of about 3, 5 or 1 连续 of a continuous amino acid. The "C-terminal region" or variant thereof refers to a region of the polypeptide comprising the residues located at the end of the 〇128788.doc-29, 200846358, which are closest to the polypeptide. ''N-terminal region' or a variant thereof refers to a polypeptide region comprising 1-5 residues closest to the N-terminal position of the polypeptide. The "internal 11 region" of the polypeptide refers to neither the N-terminus nor the polypeptide. The polypeptide region of the C-terminus of the polypeptide. "PDZ domain", also known as DHR (DLG homology region) or GLGF repeat, originally described as a 95 kDa post-synaptic dense protein (PSD-95), Drosophila tumor suppressor disk-large and tight junction The protein domain of the conserved structural elements in the protein zonula occludens-1 (ZO-1), which can be found in a large and diverse proteome, including the HtrAl protein and the HtrA3 protein. The PDZ domain typically binds to a short carboxy terminal peptide sequence located at the carboxy terminus of the interacting protein. The PDZ domain typically contains two alpha helices and six beta sheets. "HtrAl PDZ domain", "HtrAl PDZ" and variations thereof refer to a sequence of partial or entire SEQ ID NO: 1 (KKYIGIRMMSLTSSKAKELK DRHRDFPDVISGAYIIEVIPDTPAEAGGLKENDVIISINGQ SVVSANDVSDVIKRESTLNMVVRRGNEDIMITVIPEEIDP Lu (SEQ ID NO: 1); see Figure 1), directly or indirectly Participates in cellular HtrAl PDZ-ligand interactions. "HtrA3 PDZ domain ", "HtrA3 PDZn and its variants refer to a partial or entire sequence of SEQ ID NO: 2 (KRFIGIRMRTITPSLVDELK ASNPDFPEVSSGIYVQEVAPNSPSQRGGIQDGDIIVKVNG RPLVDSSELQEAVLTESPLLLEVRRGNDDLLFSIAPEVVM (SEQ ID NO: 2); see Figure 1), which is directly or indirectly involved in the cell HtrA3 PDZ-ligand interaction. 128788.doc -30- 200846358 Ligand refers to a naturally occurring or synthetic molecule or moiety that is capable of interacting with a specific site on a protein or other molecule. The HtrAi PDZ domain ligand is a molecule or moiety that specifically interacts with the HtrA i PDZ domain. The HtrA3 PDZ domain ligand is a molecule or part that specifically interacts with the HtrA3 PDZ domain. Examples of ligands include proteins, peptides, and small organic and inorganic molecules. "Fu3 protein" refers to a polypeptide having two portions covalently linked together, wherein each portion is derived from a different protein. The two portions may be directly linked via a single peptide bond or via one or more amino acid residues. Peptide linker ligation. In general, the two moieties and linkers will be in a reading frame and produced using recombinant techniques. "Illness" or "pathological condition" is to benefit from the use of the present invention Any condition that is treated by substance/molecule or method. It includes both chronic and acute conditions or diseases, including those pathological conditions that predispose the mammal to the disorder in question. Non-limiting examples of HtrAl related disorders to be treated herein include malignant and benign tumors or cancer, non-leukemia and lymphoid malignancies, neurodegenerative disorders, inflammatory and immune disorders, and new intraocular diseases. Non-limiting examples of HUA3-related disorders include malignant and benign tumors or cancer, non-leukemia and lymphoid malignancies, and placental dysfunction. "Technology and cancer" refers to or describes in a mammal

. Examples of cancer, sarcoma and leukemia More specific examples of such cancers include squamous cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, small cell lung cancer, melanoma, peritoneal cancer, liver 128788. Doc -31 - 200846358 Cell carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer Or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, genital cancer, thyroid cancer, hepatic carcinoma, and various types of head and neck cancer. "Neurodegenerative disorders" as used herein includes, but is not limited to, diseases of the central and/or peripheral nervous system that are typically characterized by degradation of neural tissue or degradation of intercellular communication in neural tissue in a mammal or Illness. Examples of neurodegenerative disorders include, but are not limited to, neurodegenerative diseases including, but not limited to, Lewy body disease, postpoliomyelitis syndrome, and Shy-Draeger syndrome (Shy-Draeger) Syndrome), olivopontocerebellar atrophy, Parkins〇n, s disease, multiple system atrophy, striat〇nigral degeneration, tau proteinosis (including (but not limited to) Alzheimer disease and nuclear paralysis), prion diseases (including but not limited to bovine spongiform encephalopathy, scrapie (scrapie) ), Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease and lethal family Insomnia), bulbar palsy, motor neuron disease, and nervous system heterodegenerative disorders (including (but not limited to) Canavan disease, Huntington's disease, God 128788.doc -32- 200846358 Transmembrane lipofuscinosis (neur〇nal ceroid-lipofuscinosis) ), Alexander, s disease, Tourette, syn (jrome), Menkes kinky hair Syndrome, c〇ckayne syndrome, Halle Halerv〇rden-Spatz syndrome, laf〇ra disease, Rett syndrome, hepatolenticular degeneration, Leah-Niegen syndrome (Lesch_Nyhan syndrome) and Unverricht-Lundborg syndrome (Unverricht - Lundborg

Syndr〇me)), dementia (including but not limited to, Pick, s disease and spinocerebellar ataxia). "Intraocular neovascular disease" as used herein includes, but is not limited to, proliferative retinopathy (eg, diabetic retinopathy), macular degeneration (eg, age-related macular degeneration (AMD) or "dry", macular Denaturing), neovascular lunate eye and immune rejection of transplanted corneal tissue and other tissues Age-related macular degeneration (AMD) is the most common cause of severe irreversible loss of vision in elderly adults (Nati〇nal S〇ciety t0 prevent Blindness 1980). It is characterized by a large number of clinical and pathological findings, such as pale yellow spots called choroidal ridge, retinal pigment epithelium (RpE) rupture, choroidal neovascularization (CNV), and discoid macular degeneration. There are two forms: non-exudative (dry) and exudative (wet or new blood). The choroid is a characteristic form of the dry form, while neovascularization is characterized by a wet form. Discoid AMD The stage of fibrosis for neovascularization. 128788.doc •33- 200846358 The incidence of AMD increases significantly with age. See, for example, Leibo Witz et al, The Framingham Eye Study monograph: an ophthalmological and epidemiological study of cataract, glaucoma, diabetic retinopathy, macular degeneration, and visual acuity in a general population of 2631 adults, 1973-1975. Swrv 24 (suppl): 335-610 (1980); and Klein et al, Prevalence of age related maculopathy. The Beaver Dam Eye Study. Ophthalmology 99: 933-43 (1992). Although the wet form of the disease is rare, it causes 80%-90% Serious visual loss associated with AMD (Ferris et al., 102: 1640-2 (1984)). There are an estimated 100-1.2 million dominant cases of wet AMD. Although the cause of AMD is unknown, the risk of developing AMD is clearly Increased by age. Other known risk factors include family history and smoking. Presumed risk factors also include oxidative stress, diabetes, alcohol intake, and sun exposure (Damico DJ. Diseases of the retina. N Engl J Med 1994; 331:95 -106 (1994); and Christen WG et al., 276: 1147-51 (1996)). Dry AMD is characterized by changes in RPE and Bruch's membrane. It is believed that RPE damaged by age and other risk factors deposits lipofuscin and cell debris on Bruch's membrane. These changes can be seen as choroidal hernias, which are scattered throughout the macula and the posterior pole of the retina. There is also a variable degree of RPE atrophy and pigmentation. Dry AMD can be free of symptoms or accompanied by variable and often minimal loss of vision and is considered to develop a wet AMD prelude. 128788.doc 34· 200846358 Wet AMD is characterized by cNV in the macula. Choroidal capillary hyperplasia and penetration of Bruch's membrane reaches the RPE and can extend into the subretinal space. The increased permeability of the newly formed capillaries results in the accumulation of serum or blood in the RpE and/or sensory nerve subretinal or sensory nerve retina. When the litter becomes swollen or detached, it causes vision loss, which may follow fibrosis and structure, leading to an increase in subretinal mass called discoid scar, which constitutes terminal AMD and is associated with permanent visual loss. (D, Amic〇Dj. #心y ·/咖33 i :95] 〇6 (1994)). Elderly therapeutic physiotherapy for wet AMD includes Photodynamic therapy (PDT, for example with VISUDYNE). As used herein, "inflammatory and immune disorders" include, but are not limited to, by abnormal immune mechanisms and / Or a disorder caused by abnormal cytokine signaling. Examples of inflammatory and immune disorders include, but are not limited to, autoimmune diseases, immunodeficiency syndromes, and allergies. "autoimmune disease" as used herein is a non-malignant disease or condition produced by an individual's own tissues and directed against such tissues. The autoimmune diseases herein do not specifically include malignant or cancerous diseases or conditions, especially including B. Cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, and chronic myeloblastic leukemia. Examples of autoimmune diseases or conditions include, but are not limited to, inflammatory responses, Such as inflammatory skin diseases, including psoriasis and dermatitis (such as 'atopic dermatitis'); systemic scleroderma and sclerosis · reactions associated with inflammatory bowel disease (such as Crohn's disease (Cr〇hn, s and Ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome (ARDS)); dermatitis; meningitis; encephalitis; uveitis; colitis / I28788.doc •35· 200846358 spheroid nephritis; allergic disease, Such as eczema and asthma and other conditions involving tau cell infiltration and chronic inflammatory reactions; atherosclerosis; leukocyte adhesion defects; rheumatoid arthritis Systemic lupus erythematosus (including (but not limited to) lupus nephritis, cutaneous lupus); diabetes (eg, diabetes or insulin-dependent diabetes); multiple sclerosis; Reynaud's syndrome; Autoimmune verrumitis; Hashimoto's thyroiditis; allergic cerebrospinal inflammation; Sjogren's syndrome; juvenile onset diabetes; and commonly seen in tuberculosis, meat Immune response associated with acute and delayed allergies mediated by cytokines and T lymphocytes in oncology, polymyositis, granulomatosis, and vasculitis; pernicious anemia (Addison's disease) Diseases involving leukocyte oozing; central nervous system (CNS) inflammatory conditions; multiple organ injury syndrome; hemolytic poor (including but not limited to cryoglobulinemia or Cooms-positive anemia (c〇〇mbs p〇shive anemia)); myasthenia gravis; antigen-antibody complex-mediated disease; anti-glomerular basement membrane disease (anti-g Lomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenia gravis syndrome (Lamber

Eaton myasthenic syndrome); pemphig〇id bullous; pemphigus; autoimmune polyendocrine gland; Reiter s disease, stiff-man syndrome; Behcet disease; giant cell arteritis; immune-induced nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura 128788.doc -36- 200846358 癜 (ITP) or autoimmune platelets Reduced symptoms, etc. The HtrAl PDZ domain and HtrA3 PDZ domain modulatory compounds of the invention can be used in combination with one or more other pharmaceutical agents to treat or prevent one or more η&αι related disorders or HtrA3 associated disorders. For example, as is well known in the art, the polypeptide of the present invention can be used in combination with one or more cytotoxic agents, chemotherapeutic agents, growth inhibitors, anti-inflammatory agents, anti-angiogenic agents, and physical therapies to treat or prevent Or a variety of HtrAl related disorders or HtrA3 related disorders. • The term π cytotoxic agent as used herein refers to a substance that inhibits or prevents cell function and/or causes cell destruction. The term is intended to include radioisotopes (eg, At2] 1, I131, I125, γ9〇, Re186, Rei88,

Sm, 12, P32 and 1^ radioisotopes); chemotherapeutic agents, such as methotrexate, adriamicin, vinca alkaloids (vincristine), periwinkle (vinblastine), etoposide, doxorubicin, mephilan, mitomycin C, chlorambucil, darno Daunorubicin or other intervening agents, enzymes and fragments thereof (such as nucleases), antibiotics and toxins (such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments thereof and/or Variants, as well as various anti-tumor or anti-cancer agents disclosed below. Other cytotoxic agents are described below. Tumor killing agents cause destruction of tumor cells. "Chemotherapeutic agents" are compounds suitable for the treatment of cancer. Examples of chemotherapeutic agents include: a burning agent such as thiotepa (e.g., 0_hook and 128788.doc-37-200846358)

CYTOXAN® ring-filled amine; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; azepine, such as benzodopa, Carboquone, meturedopa and uredopa; ethyl imino and decyl melamine, including altretamine, triethylenemelamine , trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; aeetogenin (especially bulatacin) and cloth Deltatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOl®); beta-lapachone; lapitol (lapachol); colchicine; betulinic acid; camptothecin (including synthetic analogues topotecan (HYCAMTIN®), CPT-11 (ijiticon) Irinotecan), CAMPTOSAR®), acetylcamptothecin , scopolectin and 9-aminocamptothecin; bryostatin; calistastat; CC-1065 (including its adolescence) (adozelesin), carzelesin and bizelesin synthetic analogues; podophyllotoxin; podophyllinic acid; teniposide; candida Cyclopeptide (cryptophycin) (especially Candida cyclic peptide 1 and Nostoccal cyclic peptide 8); dolastatin; doocarmycin (including synthetic analogues, 128788.doc -38 - 200846358)

KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard, such as phenylbutyric acid Chlorambucil, chlornaphazine, cyclosporin, estramustine, ifosfamide, mechlorethamine, dichloropurine hydrochloride Mechlorethamine oxide hydrochloride, methadine, neomethane, phenesterine, prednimustine, trofosfamide, urinary feeding Nia urea mustard; nitrourea, such as carmustine, chlorozotocin, fotemustine, lomustine, sulphonate Nimustine and ranimnustine; antibiotics, such as dilute fast antibiotics (eg, calicheamicin, especially calicheamicin gammaΐΐ and calicheamicin coll (see, eg, ,Agnew,C/zem /W/·五乂A2g/·, 33:1 83-186 (1994)); dynemicin, including Damisin A; esperamicin; and neocarzinostatin chromophore and related chromoprotein diacetylene antibiotics Chromophore), aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, actinomycin C ( Cactinomycin), carabincin, carminomycin, carzinophilin, chromomycinis, actinomycin (<^〇1^11〇1117(^11) , daunorubicin, 128788.doc -39- 200846358

Detorubicin, 6-diazo-5-oxo-L-positive leucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanide) Cyanomorpholino-doxorubicin, 2-port specific lenyl-doxorubicin and deoxydoxorubicin, epirubicin, essobi Esorubicin, idarubicin, marcellomycin, mitomycin (such as mitomycin C), mycophenolic acid, nogamycin ( Nogalamycin), olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, chain Streptonigrin, streptozocin, tubercidin, ubenimex, netstatin (redundant 111〇31 壮1^11), zorubicin ( 2〇1^1^(^11); antimetabolites such as guanamine pterin and 5-fluorouracil (5-FU); folic acid analogues such as sino Denopterin, pteridopterin, pteropterin, trimetrexate, purine analogs, such as fludarabine, 6-mercaptopurine, °thiamiprine, thioguanine; 5 dense sigma analogs, such as ancitabine, azacitidine, 6-azapine (6- Azauridine), carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine Androgen, such as Karoo 128788.doc -40- 200846358 testosterone J (calusterone), dromostanolone propionate, epitiostanol, mepitiostane, testicular (testolactone); anti-adrenalin, such as aminoglutethimide, mitotane,

Trilostane; folic acid supplements, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid ); ililuracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; Demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; Gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoid, such as maytansine and ansamitocins; Mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; Pilarubicin; losoxantrone; 2-ethylhydr Azide); procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; (spirogermanium); tenuazonie acid; three 128788.doc -41 - 200846358

Triaziquone; 2,2^2^-trichlorotriethylamine; trichothecene (especially T-2 toxin, verracurin A, spores) Roridin A and anguidine; urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannitol (mannomustine); mitobronitol; mit〇lactol; pipobroman; gacytosine; cytarabine (丨'Ara-C "); thiotepa; taxoid, such as TAXOL® Pacific Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ·); ABRAXANETM without hexadecanol polyoxyethylene ether, albumin engineered Pacific Paclitaxel Nanoparticle Formulation (ABRAXANETM Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel, American Pharmaceutical Partners, Schaumberg, Illinois) and TAXOTERE® doxetaxel (Rh6ne-Poulenc Rorer, Antony, France); Chloranbucil ·, Geexi Gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; analogues of the class, such as cisplatin and carboplatin ); vinblastine (VELBAN®); initial; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®) ·, oxali Oxalplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; etadashasha 128788.doc -42- 200846358

(edatrexate); daunorubicin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoid, such as Retinoic acid; capecitabine (XELODA®); a pharmaceutically acceptable salt, acid or derivative of any of the above therapeutic agents; and a combination of two or more therapeutic agents, Such as CHOP (abbreviation of combination therapy with CHOP (cycloheximide, doxorubicin, vincristine and prednisolone) and FOLFOX (oxaliplatin) (ELOXATINTM) with 5-FU and Luka Abbreviation for the treatment plan of the Vervin combination). Other chemotherapeutic agents include cytotoxic agents suitable for use as antibody drug conjugates, such as maytansinoids (e.g., DM1) and auristatin (e.g., MMAE and MMAF). Also included in the definition are anti-hormonal agents that modulate, reduce, block or inhibit the effects of hormones that promote cancer growth and are typically in the form of systemic or systemic treatments. It can be a hormone itself. Examples include anti-estrogen and selective estrogen receptor modulators (SERMs) including, for example, tamoxifen (including NOLVADEX® tamoxifen), EVISTA® raloxifene, and Droloxifene, 4-hydroxytamoxifen, trioxifene, leroxifene hydrochloride, LY117018, onapristone and FARESTON® toremifene; anti-progesterone; estrogen receptor down-regulator (ERD); an agent that inhibits or stops the action of the ovaries, such as the lutein-releasing hormone (LHRH) agonist Such as LUPRON® & ELIGARD® leuprolide 128788.doc -43-200846358 acetate, goserelin acetate, buserelin acetate and triptorelin Tripterelin); other antiandrogens, such as flutamide, nilutamide, and bicalutamide; and aromatase inhibitors that inhibit the regulation of estrogen production by the estrogen in the adrenal gland, Such as 4(5)-imida, amine Glut 旅 amino amino amino amino (aminoglutethimide), MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® voltachlor Vorozole, FEMARA8 letrozole and ARIMIDEX® anastrozole. In addition, this definition of chemotherapeutic agent includes: bisphosphonates such as clodronate (eg, BONEFOS® or OSTAC®), DIDROCAL® etidronate, NE-58095, ZOMETA8 Zoledronic acid/zoledronate, FOSAMAX® alendronate, AREDIA pamidronate, SKELID® tiludronate or ACTONEL® risedronate Salt (risedronate); and troxacitabine (l,3-dioxolan nucleoside cytotoxin); antisense nucleotides, especially genes that inhibit gene involvement in abnormal cell proliferation Antisense oligonucleotides expressed in the transduction pathway, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as THERATOPE® vaccines and gene therapy vaccines, such as ALLOVECTIN® Vaccine, LEUVECTIN® vaccine and VAXID® vaccine; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; lapatinib ditosylate (ErbB-128788.doc-44-200846358 2 with EGFR double-depleted private acid kinase small molecule inhibitor, also known as GW572016); η λ non pharmaceutically acceptable salts of any more than one therapeutic agent, acids or derivatives. Growth Inhibitor "Spring A j Field, as used herein, refers to a compound or composition that inhibits cell growth in vitro or in vivo. In certain embodiments, the compounds or compositions inhibit tumor cell growth. An example of a certain growth inhibitor of growth of A, a growth inhibitor γ, which significantly reduces the percentage of cells (e.g., tumor cells), includes an agent that blocks cell cycle progression (in other periods than the s phase), such as An agent that induces (1) block and phase block. Typical sputum blockers include vinblastine (vincristine and vinblastine), taxanes (1&\& this), and topoisomerase inhibitors (such as doxorubicin, epirubicin, Daunorubicin, etoposide and bleomycin. The agents that block G1 also act on the 8-stage block, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine , mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and C. Additional information can be found in The Molecular Basis of Caneei, edited by Mendelsohn and Israel, Chapter 1, Murakami et al.

Cell cycle regulation, oncogenes, and antineoplastic drugsrf (WB Saunders: Philadelphia, 1995), especially page 13. Yew fever (pacific paclitaxel and docetaxel) are anticancer drugs derived from yew trees. TAXOTERE® (Rhone-Poulenc Rorer) is a semi-synthetic analog of paclitaxel (TAXOL®, Bristol-Myers Squibb). Pacific paclitaxel and docetaxel promote the assembly of microtubules by tubulin dimers and 12S788.doc-45-200846358 stabilizes microtubules by preventing depolymerization, which results in inhibition of cell mitosis. "Doxorubicin" is an onion cyclin antibiotic. The chemical name of doxorubicin is (8S-cis)-10-[(3-amino-2,3,6-trideoxy-α- L-lysyl-hexapipetanosyloxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetoxy)-1-methoxy -5,12 - 蔡二嗣. As used herein, the anti-angiogenic agent π is an agent that prevents or inhibits angiogenesis. Such anti-angiogenic agents include agents known in the art, such as antibodies to VEGF (eg, , anti-VEGF neutralizing antibodies or fragments (eg, humanized Α4·6·1, AVASTIN® (Genentech, South San Francisco, CA), Y0317, M4, G6, B20, 2C3, etc. and Lucentis 8). See, for example, US patents 6, 582, 959, 6, 884, 879, 6, 703, 020; WO 98/45332; WO 96/30046; WO 94/10202; EP 0666868B1; US Patent Application No. 20030206899, 20030190317,

20030203409 and 20050112126; Popkov et al., Jowrna/ 〇/ Immunological Methods 288:149-164 (2004); WO

2005 0123 59), antibodies to VEGF receptors, small molecules that block VEGF receptor signaling (eg PTK787/ZK2284, SU6668, SUTENT®/SU1 1248 (sunitinib malate), AMG706) And native angiogenesis inhibitors, such as angiostatin, endostatin, and the like. See, for example, Klagsbrun and D'Amore, corpse 53:217-39 (1991); Streit and Detmar, Oncogene, 22:3172-3179 (2003) (eg, anti-angiogenic therapy for malignant melanoma listed in Table 3) Ferrara and Alitalo, Nature Medicine 5(12): 1359-1364 128788.doc -46- 200846358 (1999); Tomm et al., 〇ncoge-like, 22:6549 6556 (2〇〇3-8, for example, Table 2 Listed as an anti-angiogenic factor); and Sat 〇〇//, 8:200-206 (2003) (for example, Table i lists the sputum producers used in clinical trials). "Treatment" as used herein refers to a clinical intervention that seeks to alter the natural processes of the individual or cell to which it is treated and which may be performed for prophylactic purposes or during clinical pathology. The desired therapeutic effect includes preventing the onset or recurrence of the disease, soothing the symptoms, reducing any direct or indirect pathological outcome of the disease, preventing metastasis, reducing the rate of disease progression, improving or reducing the condition of the disease, and improving or improving the prognosis. In some embodiments, the use of a modulatory compound of the invention delays the progression of a disease or condition. The π effective amount π is the amount effective to achieve the desired therapeutic or prophylactic result at the desired dose and for the time necessary. The "therapeutically effective amount of a substance/molecule, agonist or antagonist of the invention" may vary depending on factors such as the disease condition, age, sex and weight of the individual, and the substance/molecule, agonist or antagonist. The ability of an individual to elicit a desired response. A therapeutically effective amount is also one in which the therapeutically beneficial effect of the substance/molecule, agonist or antagonist exceeds any of its toxic or detrimental effects. "Preventive effective amount" The amount of the desired prophylactic result is effective at the dose and for a period of time necessary. Since the prophylactic dose is administered to the individual prior to or at an early stage of the disease, the prophylactic benefit is usually, but not necessarily, less than the therapeutically effective amount.

HtrAl PDZ domain·ligand or HtrA3 ρι> ζ domain-ligand interaction regulator The present invention provides a regulator and identification of HtrAl PDZ domain-ligand interaction bite HtrA3 PDZ domain ligand interaction in vivo These adjustments are called 128788.doc • 47, 200846358. One method of modulating the interaction between the HtrAl PDZ domain or the HtrA3 PDZ domain and its ligand is to inhibit this interaction. Any molecule that disrupts the HtrAl PDZ domain-ligand interaction or the HtrA3 PDZ domain-ligand interaction can be a candidate inhibitor. Screening techniques well known to those skilled in the art can identify such molecules. Examples of inhibitors include: (1) small organic and inorganic compounds, (2) small peptides, (3) antibodies and derivatives, and (4) peptides closely related to HtrAl PDZ domain ligands or HtrA3 PDZ domain ligands. (5) aptamers. ''HtrAl PDZ domain-ligand interaction inhibitor' includes any molecule that partially or completely blocks, inhibits or neutralizes the interaction between the HtrAl PDZ domain and its ligand. Molecules useful as such inhibitors Peptides that bind to the HtrAl PDZ domain are included, such as the peptide binders listed in Tables 1, 2, and 3 (eg, and in particular, the peptides DIETWLL (SEQ ID NO: 3), GWKTWIL (SEQ ID NO: 4), DSRIWWV (SEQ ID NO: 5) or WDKIWHV (SEQ ID NO: 6)), antibody (Ab) or antibody fragment, fragment or variant of endogenous HtrAl PDZ domain ligand, cognate containing HtrAl PDZ polypeptide; containing HtrAl A variant of a PDZ polypeptide (eg, wherein the HtrAl PDZ domain sequence comprises one or more substitutions at positions Ile383, Ile385, Gly384, Tyr382, Arg386, Ile418, Ala445, Met387, Gln446, Ile415, Arg386, Ser389, Lys380, Lys381, G1411 , Tyr413, Ile414, Val417, Thr421 and Pro422 (numbered according to human HtrAl protein amino acid sequence), for example substituted with an amino acid such as Ala or its functional equivalents), peptides, and small organic molecules. nHtrA3 PDZ domain-g Self-interacting inhibitor It includes any 128788.doc -48 · 200846358 partially or fully blocks, inhibits interaction between molecules and HtrA3 PDZ domain or its ligand. Molecules useful as such inhibitors include peptides that bind to the Htr A3 PDZ domain, such as the peptide binders of 歹 ij in Tables 1, 2 and 3 (e.g., and especially the 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), FGAWV (SEQ ID NO: 19), FARWV (SEQ ID NO: 20), GVVVDEWMLSLL (SEQ ID NO: 21), GVVVDEWVLSLL (SEQ ID NO: 22), ELLVDGYVLELL (SEQ ID NO: 23), or GVVVNEWVLSLL (SEQ ID NO) : 24)), an antibody (Ab) or antibody fragment, a fragment or variant of an endogenous HtrA3 PDZ domain ligand, a homologous Htr A3 PDZ polypeptide; a variant comprising a Htr A3 PDZ polypeptide (eg, wherein HtrA3 PDZ The domain sequence includes substitutions at one or more positions Phe356, Ile357, Gly358, Glu390, Ala392, Gln423, and Arg360 (numbered according to human HtrAl protein amino acid sequence numbering), such as with an amino acid such as Ala or a functional equivalent thereof Substituting), peptides and Organic molecule. Small Molecular Htr A1 PDZ Domain or Htr A3 PDZ Domain Modulator Small molecules can be suitable modulators of HtrAl PDZ domain-ligand interactions and/or HtrA3 PDZ domain-ligand interactions. Small molecules that inhibit any interaction are potentially useful inhibitors. Examples of small molecule modulators include small peptides, peptide-like molecules, preferably soluble and synthetic non-peptidyl organic or inorganic compounds 128788.doc-49-200846358. By "small molecule" is meant a composition having 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 library of chemical and/or biological mixtures, such as fungal, bacterial or seaweed extracts, is known in the art and can be screened by any assay. Examples of methods for synthesizing molecular libraries have been described (Carell et al.,

Angewandte Chemie International Edition. 33:2059-2061 (1994); Carell et al., Angewandte Chemie International Edition. 33:2061-2064 (1994); Cho et al., Sciewe. 261:1303-5 (1993); DeWitt et al. , Proe S, 90:6909-13 (1993); Gallop et al, Chm. 37:1233-51 (1994); Zuckermann et al, J Med Chem. 37:2678-85 (1994)) Each mode is presented in solution (Houghten et al., Biotechniques 13: 412-21 (1 992)), or on beads (Lam et al., (3) re 354: 82-84 (1991)), on the wafer (Fodor et al. Human, TVaiwre. 364:555-6 (1993)), bacteria, spores (Ladner et al., U.S. Patent No. 5,223,409, 1993), plastids (Cull et al., /Vocr dead Sc/i/SA 89:1865) -9 (1992)) or phage (Cwirla et al. AJ Proc Natl Acad Sci USA. 87: 6378-82 (1990); Devlin # K ^ Science. 249:404-6 (1990); Felicif A ^ J Mol Biol. 222:301-10 (1991); Ladner et al., U.S. Patent No. 5,223,409, 1993; Scott and Smith, Science. 249:386-90 (1990)). A cell-free assay comprises contacting a HtrAl PDZ domain or a HtrA3 PDZ domain with a known binding 128788.doc -50-200846358 sub-compound, such as one or more of the binder peptides described herein, to form a assay mixture, the assay mixture and test Compounds are contacted, and the ability of the test compound to interact with the HtrAl PDZ domain or the HtrA3 PDZ domain or the binder compound is determined, wherein the ability to determine the interaction of the test compound with the HtrAl PDZ domain or the HtrA3 PDZ domain or the binder compound comprises determining the HtrAl PDZ domain/ Whether the detectable characteristics of the binding partner complex or the HtrA3 PDZ domain/binding partner are regulated. For example, the interaction of the HtrAl PDZ domain with the binding of the binder compound, as determined by the amount of the complex formed, can indicate whether the test compound is capable of modulating the interaction between the HtrAl PDZ domain and the binder compound. Similarly, the binding interaction of the HtrA3 PDZ domain with the binder compound, as determined by the amount of complex formed, can indicate whether the test compound is capable of modulating the interaction between the HtrA3 PDZ domain and the binder compound. The amount of complex can be assessed by methods known in the art, some of which have been described herein, such as ELISA (including competitive binding ELISA), yeast two-hybrid, Biacore® assay, and proximity (glory resonance energy transfer, enzyme-substrate ) Verification. Polypeptide/Peptide and Antibody HtrAl PDZ Domain or HtrA3 PDZ Domain Modulator One aspect of the present invention is an isolated peptide/polypeptide regulation of the interaction between the HtrAl PDZ domain or the HtrA3 PDZ domain and its cellular and/or physiological binding partners. Agent. The binder peptides described herein and the peptide modulators obtained by the methods described herein are also suitable for use as immunogens for the production of antibody modulators of such interactions. In one embodiment, modulators (such as peptides and antibodies) can be isolated from a cell or tissue source by standard purification techniques using standard protein purification techniques. In another embodiment, the modulator is made by recombinant DNA technology. 128788.doc -51 - 200846358 Instead of recombinant performance, modulators can be chemically synthesized using standard peptide synthesis techniques. The HtrAl PDZ domain binder peptide of the present invention and the jjtrA3 PDZ domain binder peptide include the peptides described in Tables 1, 2 and 3. The invention also provides mutant or variant protein shells, any of which may be altered by the corresponding residues of the peptides while still encoding a peptide that maintains regulatory activity. In one embodiment, the sequence of the binding peptide/polypeptide/ligand and the reference binder peptide/polypeptide/ligand have at least 5 G%, 6 G%, 7 G%, 8 G%, 9 G%, 95%. , (iv), 99% amino acid sequence-induced. In general, based on industry-recognized binding assay quantitative units/metrics, the variant exhibits substantially the same or higher binding affinity than the reference binder peptide/polypeptide/ligand, eg, at least fold, 0.8 times , 0.9 fold, 1. fold, fold or 15 fold the binding affinity of the reference binder peptide/polypeptide/ligand. Variants of the invention typically include variants in which residues at specific positions in the sequence have been substituted with other amino acids, and further comprising inserting one or more other residues between the two residues of the parent protein/peptide The possibility of residues and the possibility of deletion from the parent sequence - or multiple residues or addition to the parent sequence - or multiple residues. The invention encompasses any amino acid substitution, insertion or deletion. In some circumstances, the substitution is a conservative substitution as described herein. The "amino acid sequence-to-percent (%)" is defined as the percentage of amino acid residues consistent with the amino acid residues in the reference (parent) polypeptide sequence when the two sequences are aligned. In order to determine the amino acid identity %, the sequence is aligned and 2 is introduced into the gap to achieve the maximum sequence identity %; conservative substitutions are not considered to be part of the column-specificity. Sequence alignment procedures are well known to those skilled in the art. Peptide sequences are typically aligned using publicly available electrons 128788.doc -52.200846358 brain software such as BLAST, BLAST2, AUGN2 or cardiac (4) such as (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment with the full length sequence being compared. When aligning amino acid sequences, a given amine group can be calculated as follows. % acid sequence identity of the acid sequence VIII with a given amino acid sequence B (or, may be expressed as a given amino acid sequence A having or occupying a given amino acid sequence specific amino acid sequence identity %) : amino acid sequence identity % = Χ / Υ · 1 〇 Wherein: X is the number of amino acid residues that are consistently matched by the sequence alignment program or algorithm of Α and 且 and γ is the total number of amino acid residues in B. If the amino acid sequence The length of A is not equal to the length of the amino acid sequence B, and the % identity of the amino acid sequence of A to B will not be equal to the % identity of the amino acid sequence of 8 to 8. The "separation" or "purification" The peptide, polypeptide, protein or biologically active fragment is isolated and/or recovered from components of its natural environment. Contaminant components include materials that would normally interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous materials. A formulation having less than 3% by weight, preferably less than 20%, 10% and preferably less than 5% by weight of the non-desired substance (contaminant) is considered to be substantially separated. Preferably, the recombinantly produced peptide/polypeptide or biologically active portion thereof is 128788.doc • 53- 200846358 is substantially free of medium, that is, the medium comprises preferably less than 20%, preferably less than about 10% by volume of the peptide/polypeptide preparation. % and preferably less than about 5%. Examples of contaminants include cell debris, culture media, and materials used and produced during the in vitro synthesis of peptides/polypeptides. Conservative substitutions are shown in Table A as "better substitutions". If such substitutions result in a change in biological activity, it may be introduced as shown in Table A as ''exemplary substitution'', or as further described below with reference to the amino acid class, more substantial changes and screening of the product.

• Table A. The original substitution of the original residue is preferably substituted for Ala(A) val, leu, ile val Arg(R) lys, gin, asn lys Asn(N) gin, his, asp, lys, arg gin Asp(D) Glu, asn glu Cys(C) ser, ala ser Gln(Q) asn, glu asn Glu(E) asp, gin asp Gly(G) ala ala His(H) asn, gin, lys, arg arg He(I) Leu, val, met, ala, phe, n-amine 酉 leu Leu (L) leucine, ile, val, met, ala, phe ile Lys (K) arg, gin, asn arg Met (M) leu ,phe,ile leu Phe(F) leu,val,ile,ala,tyr tyr Pro(P) ala ala Ser(S) thr ' cys cys Thr(T) ser ser Trp(W) tyr,phe tyr Tyr(Y) ) trp ^ phe ^ thr ^ ser phe Val(V) ile, leu, met, phe, ala, substantial modification of the biological properties of the leucine leu peptide/polypeptide by selecting for its effect on maintaining the following conditions Substantially different substitutions are achieved by: (a) the structure of the polypeptide backbone in the substitution region, such as a folded sheet or a helical configuration; (b) the charge or hydrophobicity at the target site in the molecule -54-128788.doc 200846358; (C) Side chain volume. The naturally occurring residues are classified into the following groups based on the properties of the common side chain: (1) 3⁄4丨l water-based, leucine, met, ala, val, leu, ile; (2) neutral hydrophilicity: cyS, Ser, thr; (3) acidity: asp, glu; (4) testability · asn, gin, his, lys, arg; (5) residues that affect chain orientation: gly, pr〇; and (6) · trp, tyr, phe. Non-conservative substitutions will require members of one of these categories to be exchanged for another category.

Variants of HtrAl PDZ domain-ligand interactions or antibody modulators of the interaction of the serotonin-3 ligand-ligand interaction may also be based on information known in the art without substantially affecting antibody activity. preparation. For example, an antibody variant can have at least one amino acid residue substituted with a different residue in the antibody molecule. In the case of antibodies, although the site of most interest for substitution mutation induction usually includes hypervariable regions, framework region (FR) variation is also contemplated. In the case of antibodies, one type of substitution variant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). The resulting variants selected for further development will generally have improved biological properties relative to the parent antibody from which they are produced. A convenient method for producing such substituted variants involves affinity maturation using phage presentation. Briefly, 'mutation of several hypervariable regions (e.g., 6-7 sites) is made to generate all possible amino acid substitutions at each point. When the antibody thus produced is fused to the gene III product of Ml3 encapsulated in each particle, it is presented by filamentous phage 128788.doc • 55- 200846358 body particles. Phage-presented variants are then screened for biological activity (e.g., binding affinity) of the antibodies disclosed herein. To identify candidate mutated region sites for modification, alanine scanning mutations can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Additionally or alternatively, it may be useful to analyze the crystal structure of the antigen-antibody complex to identify the point of contact between the antibody and the antigen. The contact residues and adjacent residues are candidate residues that are substituted according to the techniques detailed herein. Once such variants are produced, the panel of variants is subjected to screening as described herein, and antibodies having superior properties can be selected for further development in one or more relevant assays. Nucleic acid molecules encoding amino acid sequence variants of antibodies are prepared by a variety of methods known in the art. Such methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants), or mediated by nucleotides in the form of non-variant forms of previously prepared variants or antibodies Mutation induction (or site specificity), PCR mutation induction and sequence cassette mutation induction preparation. It may be desirable to introduce one or more amino acid modifications in the Fc region of an immunoglobulin polypeptide of the invention to produce an Fc region variant. An Fc region variant may comprise a human Fc region sequence comprising an amino acid modification (eg, a substitution) at one or more amino acid positions (including hinge cysteine positions) (eg, human IgG 1, IgG2, IgG3, or IgG4) Fc area). In one embodiment, the Fc region variant can exhibit altered neonatal Fc receptor (FcRn) binding affinity. The variant Fc regions may comprise amino acid modifications at any one or more of the following amino acid positions of the Fc region: 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305 307 , 128788.doc -56- 200846358 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, where? (The numbering of the residues in the region is the number of the EU index as in ^5&1. The Fc region variant with weakened binding to FcRn may comprise any one or more of the amino acid positions of the following amino acid positions of the Fc region. Modification: 252, 253, 254, 255, 288, 309, 386, 388, 400, 415, 433, 43 5, 436, 439 or 447, wherein the numbering of the residues in the Fc region is the number of the EU index as in Kabat. Alternatively, the Fc region variant Φ mentioned above may exhibit enhanced amino acid modification of FcRn binding and comprising any one or more of the following amino acid positions of the Fc region: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, wherein the number of residues in the Fc region is such as the EU index in Kabat The Fc region variant that is attenuated by binding to Fc (R may comprise an amino acid modification at any one or more of the following amino acid positions of the Fc region: 238, 239, 248, 249, 252 '254, 265 , 268, 269, 270, 272, 278, 289, • 292, 293, 294, 295, 296 298, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438 or 439, wherein Fc The numbering of the residues in the region is the number of the EU index as in Kabat. For example, the Fc region variant may exhibit any one or more of the following amino acid positions that are attenuated to bind to Fc (RI and include the Fc region) Amino acid modification: 23 8, 265, 269, 270, 327 or 329, wherein the numbering of the residues in the Fc region is the number of the EU index as in Kabat. 128788.doc -57- 200846358

The Fc region variant may exhibit amino acid modification at any one or more of the following amino acid positions that are attenuated to bind to Fc (RII and comprise an Fc region): 238, 265, 269, 270, 292, 294, 295 298 ' 303 , 324 , 327 , 329 , 333 , 335 , 338 , 373 , 376 , 414 , 416 , 419 , 435 , 438 or 439 , wherein the numbering of the residues in the Fc region is the number of the EU index as in Kabat. The Fc region variant of interest may exhibit amino acid modification at any one or more of the following amino acid positions that are attenuated to bind to Fc (RIII and comprise an Fc region): 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 , 43 4, 43 5 or 437, wherein the numbering of the residues in the Fc region is the number of the EU index as in Kabat. Fc region variants with altered (i.e., increased or decreased) Clq binding and/or complement dependent cytotoxicity (CDC) are described in WO 99/51642. The variants may comprise an amino acid substitution at any one or more of the following amino acid positions of the Fc region: 270, 322, 326, 327, 329, 331, 333 or 334. For Fc region variants, see also Duncan and Winter 322: 73 8-40 (1988); U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821 and WO 94/29351. Vector Construction Polynucleotide sequences encoding the peptides and polypeptides described herein can be obtained using standard synthetic and/or recombinant techniques. The desired nucleotide sequence can be isolated and sequenced from appropriate source cells. The source cells of the antibody will include antibody producing cells, such as fusion tumor cells. Alternatively, a polynucleotide can be synthesized using a nucleotide synthesizer or PCR technique 128788.doc • 58- 200846358. Once the sequence encoding the peptide or polypeptide is obtained, it can be inserted into a recombinant vector capable of replicating in a host cell and displaying the heterologous polynucleotide. For the purposes of the present invention, a variety of carriers available and known in the art can be used. The choice of a suitable vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed by the vector. Each vector will contain a plurality of components depending on its function (amplification or expression of the heterologous polynucleotide or both) and its compatibility with the particular host cell in which it resides. Vector components typically include, but are not limited to, an origin of replication (especially when the vector is inserted into a prokaryotic cell), a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, heterologous nucleic acid insertion and transcription. Termination sequence. A plastid vector containing a replicon and a control sequence derived from a species compatible with the host cell is typically used in conjunction with such host. The vector typically carries a replication site and a marker sequence capable of providing phenotypic selection in transformed cells. For example, 'the plastid pBR3 22 transformed from Escherichia coli (5·co/〇 species) is usually used. pBR322 contains a factor encoding ampicillin (Amp) and tetracycline (Tet) resistance, and It thus provides an easy way to identify transformed cells. pBR322, its derivatives or other microbial plastids or phage may also contain or have been modified to contain a promoter that can be used by microorganisms for the expression of endogenous proteins. A bacteriophage vector compatible with the replicon and control sequences can be used as a transformation vector in conjunction with such hosts. For example, phage preparations such as XGEM.TM-11 can be used to facilitate the transformation of e.g. Recombinant vectors for host cells. Constitutive or inducible promoters can be used in the context of the specific situation as determined by those skilled in the art, and are well known to be recognized by a variety of potential host cells. Promoter. The promoter can be removed from the source DNA by restriction enzyme digestion and the isolated promoter sequence inserted into the selection The operon ligation of a selected promoter with a cistron D encoding a polypeptide described herein is used to direct amplification and/or expression of the target gene using both a native promoter sequence and a plurality of heterologous promoters. Because of the ratio to the native target polypeptide promoter: a heterologous promoter usually allows for higher transcription and a higher yield of the target gene being expressed, so it is preferred.

Promoters suitable for use in prokaryotic hosts include the ph〇A promoter, β-galactosidase and lactose promoters (iv), the tryptophan (iv) promoter strand, and hybrid promoters (such as the promoter of (10) or). However, it is also suitable to have a promoter in bacteria (such as other known bacteriophage promoters). Its nuclear (four) sequence has been published so that skilled workers can use operators or adaptors to operably link to the cistrons encoding the light and heavy chains (Siebenlist et al. (198) (4) 2〇 2) to provide any desired restriction sites. a In some embodiments, each cistron in the recombinant vector comprises a secretion signal sequence component that directs the transmembrane migration of the expressed polypeptide. The signal sequence can generally be a component of the vector' or it can be part of the stem polypeptide DNA inserted into the vector. The signal sequence selected for the purposes of the present invention shall be a signal sequence which is recognized by the host cell and processed (i.e., cleaved by a signal peptidase). For prokaryotic host cells that do not recognize and process the native signal sequence of the heterologous polypeptide, the signal sequence is substituted, for example, with a prokaryotic signal sequence selected from the group consisting of: an (10) enzyme, a penicillinase, an ipp or Stable stability 128788.doc 200846358 Enterotoxin II (STII) leader sequence, LamB, PhoE, PelB, OmpA and MBP. Prokaryotic host cells suitable for expressing a polypeptide include, but are not limited to, Archaebacteria and Eubacteria, such as Gram-negative organisms or Gram-positive organisms. . Examples of suitable bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., Bacillus subtilis (Enterum bacteria), Pseudomonas species (e.g., Pseudomonas aeruginosa (Housin (6), Salmonella typhimurium, Salmonella typhimurium, Seiratia maixescans, Klebsie Ua, Proteus, Chi Shigella, Rh1Z〇bia, Vitre〇sciUa or Paracoccus ^ In some embodiments, Gram-negative cells are used. Host cells are preferred. The lowest amount of proteolytic enzyme should be secreted and other protease inhibitors may be required to be incorporated into the cell culture. Peptide or polypeptide preparation The host cell is transformed or transfected with the above expression vector and, as appropriate, modified for induction of the promoter, Choosing a transformant or culturing in a conventional nutrient medium that encodes a gene encoding the desired sequence. Turning wood refers to host cell pair performance that does or does not exhibit any coding sequence. .- absorbent member as known in the art that a variety of transfection methods, e.g.

CaPCU precipitation and electroporation. Transfection is generally considered successful when any indication of the operation of the vector occurs in the primary cell of the stomata. 128788.doc • 61 - 200846358 Transformation means the introduction of DNA into a prokaryotic host so that it can be replicated as a bicolor in vitro element or as a chromosomal component. Depending on the host cell used, transformation is performed using standard techniques appropriate to the cell. For bacterial cells containing a parenchymal cell wall barrier, calcium using calcium chloride is usually used. (4) Method (4) Polyethylene glycol/D. Another technique used is electroporation. Other techniques are known in the art. Prokaryotic cells useful for producing the polypeptides of the invention are known in the art and are suitable for growth in a culture medium in which the selected host cells are cultured. Examples of suitable media include LuHa brGth (LB) plus essential nutrient supplements. In a preferred embodiment, the medium also contains a selection agent selected based on the construction of the expression vector for selectively allowing the prokaryotic cell containing the expression vector to be exemplified by 5, and the female piracetin is added to the medium for performance. Cell growth of the ampicillin resistance gene. In addition to the carbon, nitrogen and inorganic phosphate sources, any necessary supplements may be included, either alone or in a suitable concentration in the form of a mixture with another supplement or medium, such as a complex nitrogen source. The medium may optionally contain one or more reducing agents selected from the group consisting of glutathione, cysteamine cysteamine, thioglycolate, dithioerythritol, and dithiothreitol. . Prokaryotic host cells are cultured at a suitable temperature. For E. coli growth, for example, a preferred temperature is in the range of about 2 (rc to about 39, more preferably about 251 to about 37 ° C, even more preferably about 3 (rc). The pH of the medium is predominant. Depending on the host organism, it may be any value in the range of from about 5 to about 9. For E. coli, the pH is preferably from about 6.8 to about 7, 4, and more preferably about 7 Å. Inducible promoters are used in the expression vector to induce protein expression under conditions suitable for activation initiation 128788.doc -62 · 200846358. For example, if the ph〇A promoter is used to control transcription, it can be in phosphate The transformed host cells are cultured in a restriction medium for induction. As is known in the art, a variety of other inducers can be used depending on the vector construct employed. The polypeptides described herein can be secreted in a host cell. It is recovered from and recovered from the periplasm. Protein recovery usually involves destroying the microorganisms usually by means such as osmotic shock, ultrasonic treatment or cell lysis. Once the cells are lysed, cell debris or whole cells can be removed by centrifugation or filtration. can For example, the protein is purified by affinity chromatography or the protein can be transported to and isolated from the medium. The cells can be removed from the culture and the culture supernatant filtered and concentrated for further purification. Produced protein. The polypeptides can be further separated and memorized using commonly known methods such as fractionation by immunoaffinity or ion exchange column; ethanol precipitation; reverse phase HPLC; after sulphur dioxide or such as DEAE Cation exchange resin chromatography; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using (for example) Sephadex G-75; hydrophobic affinity resin; using a suitable antigen immobilized on a substrate Ligand Affinity and Western blot analysis In addition to prokaryotic host cells, eukaryotic host cell systems are well established in this technology. Suitable hosts include, but are not limited to, mammalian cell lines such as CHO. And insect cells, such as the cells described below. Polypeptide/peptide purification purifies the resulting polypeptide/peptide to obtain Step verification and use. 128788.doc -63- 200846358 Substantially homogeneous preparations. Standard protein purification methods known in the art can be used. The following procedures are examples of suitable purification procedures but are not intended to be limited by this: immunoaffinity Separation by sex or ion exchange column; ethanol precipitation; reverse phase HPLC; chromatography with cerium oxide or cation exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; and use of, for example, Sephadex G-75 Gel filtration,.

Identification and Characterization of HtrAl PDZ Domains and HtrA3 PDZ Domain Modulators: General Methods • For example, candidate HtrAl PDZ domains and HtrA3 PDZ domain modulators for binding peptides can be identified by a number of methods known in the art. Modulatory characteristics of the modulator can be assessed by assaying the ability of the modulator to modulate the interaction between the HtrAl PDZ domain and its cellular binding partner or HtrA3 PDZ domain and its cellular binding partner. An important feature is the binding affinity. The binding characteristics of the candidate modulator (e. g., peptide) of interest can be assessed in any of a number of ways known in the art. The initial step in the method can include generating one or more candidate peptides comprising the sequence of interest, which are then presented under conditions suitable for determining the binding characteristics of their HtrAl PDZ domain or HtrA3 PDZ domain. For example, a protein fusion with a sheath protein such as p3 or p8 can be used to present the candidate peptide as a carboxy terminus (C-terminus) of the peptide on the surface of a phage or phagemid such as a filamentous phage (phagemid). library. C-terminal presentations are known in the art. See, for example, Jespers et al., Price Magic; (W Γ) 13:378-82 and WO 00/06717. These methods can be used to prepare the fusion gene, fusion protein, vector, recombinant phage particle, host cell of the present invention and its library 128788.doc-64-200846358. As described herein, in some embodiments, it can be adapted to present a candidate peptide on the surface of a phage or phagemid as an amine-terminal (N-terminal) presentation library of the peptide. Methods for the presentation of N-terminal phage (phagemids) include those described herein and those well known in the art, for example, as described in U.S. Patent No. 5,750,373 (the disclosure of which is incorporated herein by reference). Said. Methods for characterizing the binder molecules obtained by such methods are also known in the art, including those disclosed in the references cited above (&3?61^ et al., \¥0 00 /06717 and U.S. Patent No. 5,750,3,73) and methods as described herein. (i) Isolation of binding phage of HtrAl PDZ domain or Htr A3 PDZ domain phage display library with the presented candidate HtrAl PDZ domain binding peptide or HtrA3 PDZ domain binding peptide and HtrAl PDZ domain protein or fusion protein or HtrA3 PDZ domain protein Alternatively, the fusion protein is contacted in vitro to identify members of the library that bind to the HtrAl PDZ domain or the HtrA3 PDZ domain target. Ex vivo protein binding can be assayed using any method known to those skilled in the art. For example, one, two, three or four or more rounds of binding can be selected, after which individual phages are isolated and analyzed by phage ELISA as appropriate. Peptide ELISA assay peptides can be used to exhibit binding affinity of phage particles to immobilized PDZ|& proteins (Barrett et al, dna/Biochem. 204:357-64 (1992)) °

Appropriate binding competition conditions are provided in the case where the candidate competes with the known HtrAl PDZ domain binder or the HtrA3 PDZ domain binder for the ability to bind to the HtrAl PDZ domain or the HtrA3 PDZ domain, respectively. By way of example, in one embodiment, screening/selection/biopanning can be performed in the presence of one or more concentrations of known HtrAl PDZ 128788.doc -65-200846358 domain binder or HtrA3 PDZ domain binder. In another embodiment, candidate binders isolated from the library can then be assessed in the presence of a known HtrAl PDZ domain binder or HtrA3 PDZ domain binder in a competitive ELIS A assay. (ii) Preparation of HtrAl PDZ domain or HtrA3 PDZ domain The HtrAl PDZ domain or the HtrA3 PDZ domain can be conveniently prepared in the form of a protein fragment or fusion polypeptide containing the domain using conventional synthetic or recombinant techniques. The fusion polypeptide is suitable for phage (phagemid) presentation, wherein the HtrAl PDZ domain or the HtrA3 PDZ domain is a dry antigen in performance studies, cell localization, bioassay, ELISA (including binding competition assay) and the like. The HtrAl PDZ domain or HtrA3?0 domain "chimeric protein" or "fusion protein" comprises a HtrAl PDZ domain or a HtrA3 PDZ domain fused to a non-PDZ domain polypeptide. Non-PDZ domain polypeptides are not substantially homologous to the PDZ domain. HtrAl PDZ Domain Fusion Protein or HtrA3 PDZ Domain Fusion proteins can include any portion of the entire PDZ domain, including any number of biologically active portions. The fusion protein can then be purified using affinity chromatography and a capture reagent combined with a non-PDZ domain polypeptide according to known methods. The HtrAl PDZ domain or the HtrA3 PDZ domain can be fused to the C-terminus of the affinity sequence 歹 ij, such as the GST (glutathione S-transferase) sequence. The fusion proteins facilitate purification of the recombinant HtrAl PDZ domain using, for example, glutathione bound to a solid support and/or attached to a solid support (eg, a matrix for peptide screening/selection/biopanning) Or HtrA3 PDZ domain. Other exemplary fusions are presented in Table B, including some common uses of such fusions. Fusion proteins can be readily produced using recombinant methods. Nucleic acid 128788.doc-66.200846358 encoding the HtfAl PDZ domain (or a portion thereof) or the HtrA3 PDZ domain (or a portion thereof) can be fused in-frame to the non-PDZ domain-encoding nucleic acid at the N-terminus, end or internal of the PDZ domain. The fusion gene can also be synthesized by a conventional technique including a DNA synthesizer. PCR amplification is also applicable, which uses an anchor primer that produces a complementary overhang between two consecutive gene segments. These gene fragments can then be ligated and re-amplified to produce a conjugated gene sequence (Ausubel et al., Current protocols). In molecular biology. John Wiley & Sons, New York 1987). A number of vectors are commercially available which facilitate the incorporation of the HtrAi pdz domain or the HtrA3 PDZ domain into a fusion protein. Φ Table B for non-PDZ domain fusion peptide fusion partners in vitro - in vivo human growth hormone (hGH) radioimmunoassay without P-glucuronidase (GUS) colorimetric, fluorescent or chemiluminescent colorimetric (using X-gluc for histochemical staining) Green fluorescent protein (GFP) and related molecules (RFP, BFP, YFP domain, etc.) Fluorescent ' ~ Fluorescent luciferase (Firefly) Bioluminescent bioluminescence Chloramphenicol Enzyme (CAT) chromatography, differential extraction, fluorescence or immunoassay without p-galactosidase colorimetric, fluorescent, chemiluminescence colorimetric (histochemical staining with X-gal), bioluminescence secretion in living cells Alkaline phosphatase (SEAP) colorimetric, bioluminescent, chemical-free Tat-free regulation of transport to the cytoplasm and nucleus regulates transmission into the cytoplasm and nucleus as an example of a fusion of the HtrAi PDZ domain or the HtrA3 PDZ domain GST- The HtrAl PDZ fusion can be prepared from the gene of interest in the following manner. Using the full-length gene of interest as a template, PCR was used to amplify a DNA fragment encoding the PDZ domain using a restriction enzyme site introduced in favor of subcloning. Each amplified fragment is digested with an appropriate restriction enzyme and colonized into a similarly digested plastid such as pGEX6P-3 or pGEX-4T-3, which contains 128788.doc-67-200846358 with GST and is designed to The subcloned fragment will be ligated in frame with GST and operably linked to a promoter to produce a plastid encoding a GST-HtrAl PDZ fusion protein or a GST-HtrA3 PDZ fusion protein. To produce a fusion protein, E. coli cultures containing appropriately expressed plastids are typically grown in LB broth, for example at about 37 °C, to mid-log phase (Α_=1·0) and can be induced with IPTG. The bacteria were pelleted by centrifugation, resuspended in PBS and solubilized by ultrasonic treatment. The suspension was centrifuged and the GST-HtrAl PDZ domain fusion protein or the GST_HtrA3 PDZ domain fusion protein was purified by affinity chromatography using 0.5 ml of glutathione-agarose from the supernatant. It will be apparent to those skilled in the art that many variations will achieve the goal of isolating the HtrAl PDZ domain protein or the HtrA3 PDZ domain protein and can be used in the present invention. For example, a fusion of the HtrAl PDZ domain or the HtrA3 PDZ domain with an epitope tag can be constructed as described above and the HtrAl PDZ domain or the HtrA3 PDZ domain can be purified using the tag affinity. The HtrAl PDZ domain or the HtrA3 PDZ domain protein/peptide can also be prepared without any fusion; in addition, instead of using a microbial vector to produce a protein, in vitro chemical synthesis can be used instead. Other cells can be used to produce HtrAl PDZ domains or HtrA3 PDZ domain proteins/peptides, such as other bacteria, mammalian cells (such as COS) or baculovirus systems. A variety of polynucleotide vectors that produce a plurality of fusions can also be used. The final purification of the HtrAl PDZ domain or the HtrA3 PDZ domain fusion protein will generally depend on the fusion partner; for example, the polyhistidine tag fusion can be purified on a nickel column. (iii) determining the sequence of the presented peptide 128788.doc • 68 - 200846358 The phage (phagemid) that binds to the HtrAl PDZ domain or the HtrA3 PDZ domain with the desired characteristics (and optionally does not bind to an unrelated sequence) can be subjected to sequences analysis. The phage (phagemid) particles presenting the candidate binding peptide are amplified in the host cell, the DNA is isolated and the appropriate portion of the genome (coding candidate) is sequenced using any suitable known sequencing technique. Other methods for identifying HtrAl PDZ domain-ligand interactions or modulators of HtrA3 PDZ domain-coordination 馥 interactions Another identification of HtrAl PDZ domain-ligand binding or HtrA3 PDZ domain-ligand binding regulators The method is to incorporate rational drug design; that is, to understand and utilize the biology of PDZ interaction. In this method, key residues in the PDZ ligand are determined and the optimal peptide length is determined as appropriate. Subsequently, a small molecule with information is known; for example, if tryptophan is found to be a key residue that binds to the PDZ domain, a small molecule containing a tryptophan residue will be prepared and tested as an inhibitor. Typically 2, 3, 4 or 5 amino acid residues will be determined to be critical for binding and a candidate small molecule inhibitor containing such residues or residue side chains will be prepared. Subsequent inhibition of HtrAl PDZ domain-ligand or HtrA3 PDZ domain-ligand interaction capabilities The test compounds are screened using protocols well known in the art (e.g., competitive inhibition assays). Compounds that modulate HtrAl PDZ domain-ligand binding interactions or HtrA3卩02 domain-ligand binding interactions are useful for treating diseases and conditions associated with abnormal regulation of binding interactions with the HtrAl PDZ domain or the HtrA3 PDZ domain. Diseases and conditions associated with the regulation of interaction with the HtrAl PDZ domain include malignant and benign tumors or cancer, non-leukemia and lymphoid malignancies, neurodegenerative disorders, inflammatory and immune disorders, and intraocular neovascular disorders. 128788.doc -69- 200846358 The diseases and conditions associated with the regulation of the interaction of the HtrA3 PDZ domain include malignant and benign tumors or cancer, non-leukemia and lymphoid malignancies and placental dysfunction. Determining key residues in the HtrAl PDZ domain binding polypeptide or HtrA3 PDZ domain binding polypeptide (a) Alanine scanning can be determined using a tripping of the HtrAl PDZ domain binding peptide sequence or the HtrA3 PDZ domain binding peptide sequence to determine the coordination The relative contribution of each residue in the body to the PDZ junction. To identify key residues in the PDZ ligand, residues were replaced with a single amino acid, typically a propylamine residue, and the effect on PDZ domain binding was assessed. See US 5,580,723, US 5,834,250 and examples. (b) Truncated (missing series)

The truncation of the HtrAl PDZ domain binding peptide or the HtrA3 PDZ domain binding peptide not only clarifies the binding of key residues, but also determines the minimum peptide length at which binding is achieved. In some cases, truncation will reveal ligands that bind more tightly than the native ligand; the peptide is suitable for modulating the HtrAl PDZ domain · PDZ ligand phase interaction or HtrA3 PDZ domain: PDZ ligands are mutually effect. Preferably, a series of HtrAl PDZ domain binding peptide truncated forms or HtrA3 PDZ domain binding peptide truncated forms are prepared. One series will cut the amino terminal amino acid in turn; in another series, the truncation will begin at the carboxy terminus. As in the case of alanine scanning, the peptide can be synthesized in vitro or prepared by recombinant methods. (c) Rational modulator design based on information obtained from alanine scanning and truncation analysis, familiar with this technique 128788.doc -70- 200846358 The surgeon can design and synthesize small molecules or select small compounds rich in possible binding binding Molecular library. For example, based on the information as described in the Examples, the modulator peptide can be designed to include two suitably spaced hydrophobic moieties. (d) Binding assay The complex of the HtrAl PDZ domain binding peptide and the HtrAl PDZ domain or the complex of the HtrA3 PDZ domain binding peptide and the HtrA3 PDZ domain is facilitated to separate the complex form from its uncomplexed form and impurities. HtrAl PDZ domain: Binding Ligand complex or Htr A3 PDZ domain: The binding ligand complex can be formed in solution or one of the binding partners can be combined with an insoluble support. The complex can be isolated from the solution, for example, using column chromatography and, when combined with a solid support, can be isolated by filtration, centrifugation, etc. using well known techniques. Combining a polypeptide comprising a PDZ domain or a ligand thereof with a solid support facilitates high yield assays. The test compound can be screened for the ability to modulate (eg, inhibit) the interaction of the binder polypeptide with the HtrAl PDZ domain or the HtrA3 PDZ domain in the presence and absence of a candidate binding compound, and can be used in, for example, microtiter plates, test tubes, and micro Screening is achieved in any suitable container for the centrifuge tube. Fusion proteins can also be prepared to facilitate testing or isolation, wherein the fusion protein contains another domain that allows one or both of the proteins to bind to the matrix. For example, a GST-PDZ binding peptide fusion protein or a GST-PDZ domain fusion protein can be adsorbed to glutathione agarose beads (SIGMA Chemical, St. Louis, MO) or a trace amount derivatized with glutathione. The plate is then combined with the test compound or test compound and the unadsorbed HtrAl PDZ domain protein or HtrA3 PDZ domain protein or PDZ binding peptide, and the mixture 128788.doc-71 - 200846358 is allowed to form a complex (eg Cultivate under physiological conditions of salt and pH. After incubation, the beads or microtiter wells are washed to remove any unbound components, the matrix is immobilized in the case of beads, and the complex is assayed directly or indirectly. Alternatively, the complex can be dissociated from the matrix and the binding level or activity determined using standard techniques. Other fusion protein techniques for immobilizing proteins on substrates can also be used in screening assays. The HUAI PDZ binding peptide or HtrAl PDZ domain or the # HtrA3 PDZ binding peptide or HtrA3 PDZ domain can be immobilized using the biotin-avidin or biotin-anthreptin system. Biotinylation can be achieved using a number of reagents such as biotin-N-trans- succinate (NHS; PIERCE Chemicals, Rockford, IL) and immobilized on streptavidin coated 96-well plates (PIERCE) Chemical) in the hole. Alternatively, an antibody that reacts with the HtrAl PDZ domain binding peptide or the HtrAl PDZ domain or with the HtrA3 PDZ domain binding peptide or the HtrA3 PDZ domain but does not interfere with binding of the binding peptide to its target molecule can be derivatized in the pores of the plate and allowed to bind The HtrAl PDZ domain or the binder peptide or the unbound HtrA3 PDZ domain or the binder peptide is captured in the pore by antibody ligation. Methods of detecting such complexes, in addition to the methods described for GST-immobilized complexes, also include immunodetection using a complex of antibodies reactive with a binding peptide or HtrA1 PDZ domain or HtTA3 PDZ domain.

(e) Binding assay: competition ELISA To assess the binding affinity of peptides, proteins or other HtrAl PDZ domains or HtrA3 PDZ domain ligands, a competitive binding assay can be used in which the ligand binding to the HtrAl PDZ domain or the Htr A3 PDZ domain is assessed. The ability (and, if necessary, binding affinity), and its ability to bind to compounds that bind to the PDZ domain is compared to 128788.doc -72-200846358. A number of methods are known and can be used to identify binding affinities of binding molecules (e.g., peptides, proteins, small molecules, etc.); for example, binding affinities can be determined as IC5 〇 values using competition ELISA. The IC5G value is defined as the concentration of the binding block that blocks the binding of the HtrAl PDZ domain to the ligand or the binding of the HtrA3 PDZ domain to the ligand. For example, in a solid phase assay, a assay plate can be prepared by coating a microplate with neutral streptavidin, avidin or streptavidin, preferably treated to efficiently adsorb proteins. The non-specific binding site is then blocked by the addition of a solution of bovine serum albumin (BSA) or other protein (e.g., skim milk), and then preferably washed with a buffer containing a detergent such as Tween-20. A biotinylated known HtrAl PDZ binder or HtrA3 PDZ binder (e.g., a phage peptide fused to GST or another such molecule to facilitate purification and detection) is prepared and combined with a plate. Serial dilutions of the molecules to be tested with the HtrAl PDZ domain or the HtrA3 PDZ domain were prepared and brought into contact with the bound binder. The plate coated with the fixed binder was washed, and then each of the binding reactants was added to the wells and briefly cultured. After further washing, antibodies that recognize non-PDZ fusion partners and markers that recognize primary antibodies (such as horseradish peroxidase (HRP), alkaline phosphatase (AP), or fluorescent labels such as luciferin) are commonly used. Secondary antibodies detect binding reactions. The plate is then colored with the appropriate substrate (depending on the label) and the signal is quantized, for example, using a spectrophotometer plate reader. The least squares method can be used to fit the absorption signal to the binding curve. Thus, the ability of various molecules to inhibit binding of the PDZ domain to known PDZ domain binders can be measured. Many variations of the above assays will be apparent to the average technician. 128788.doc -73 - 200846358 See. For example, instead of the avidin-biotin based system, the PDZ domain binder can be chemically linked to the substrate or simply adsorbed. PDZ domain peptide ligand PDZ domain peptide ligands visible during phage display, even ligands that bind with relatively low affinity (eg, compared to consensus sequences), are HtrAl PDZ domain-ligands Potentially useful inhibitors of interactions or Htr A3 PDZ domain-ligand interactions, including those described in the Examples (and Tables 1, 2 and 3). Competitive binding ELISA is a suitable method for determining the efficacy of PDZ domain binding peptides exhibited by each phage. ? aptamer An aptamer is a short oligonucleotide sequence that can be used to recognize and specifically bind to almost any molecule. The Exponentially Enriched Ligand System Evolution (SELEX) method can be used (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, SckNce. 249:505-10 (1990)) looking for such aptamers. Aptamers have many diagnostic and clinical uses; aptamers can also be used for almost any clinical or diagnostic use of antibodies. In addition, aptamers, once identified, can be manufactured inexpensively and can be readily applied in a variety of formats, including pharmaceutical, bioassay, and diagnostic tests in the form of pharmaceutical compositions (Jayasena, C7h C/zem. 45:1628-50 (1999) )). In the above competitive ELISA binding assay, screening of candidate aptamers involves the incorporation of aptamers into assays and their ability to modulate HtrAl PDZ domain ligand binding or Htr A3 PDZ domain: ligand binding. 128788.doc -74- 200846358 I antibody (Ab) any regulatory (eg, inhibitory) ligand: HtrA1 PDZ domain binding or ligand: HtrA3 PDZ domain binding antibody can be divided into another HjAl PDZ domain-ligand Interaction or modulator of HtrA3 PDZ domain-ligand interaction (eg, inhibitor). Examples of suitable antibodies include multiple strains of Ab, single plant VIII>, single chain Ab, anti-idiotype Ab, chimeric Ab or humanized forms of such antibodies or fragments thereof. The antibody may be from any suitable source, including synthetic sources and any species that produce an immune response. Screening Methods The present invention encompasses methods for screening compounds to identify compounds that modulate HtrAl PDZ domain-ligand interactions or HtrA3 PDZ domain-ligand interactions. Screening assays are designed to identify compounds that bind to or complex with, or otherwise interfere with, the HtrAl PDZ domain and/or ligands of the HtrAl PDZ domain and/or ligands, or the HtrA3 PDZ domain interacts with cytokines. One method for determining the ability of a candidate compound to be a modulator is to evaluate a known HtrAl PDZ domain binder or a known HtrA3 PDZ, such as any of the binder peptides (eg, the high affinity binders described in the Examples) disclosed herein. The activity of the candidate compound in a competitive inhibition assay in the presence of a domain binder. Such screening assays will include assays suitable for high yield screening of chemical libraries to make them particularly suitable for identifying small molecule drug candidates. Verification 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 this technique. Common to all assays of modulators is the need to bind the drug candidate 12B788.doc -75- 200846358 to the HtrAl PDZ domain (or its equivalent) and/or to the binding interaction of the HtrAl PDZ domain with the binding ligand. The binding ligand, or a binding ligand that interacts with the HtrA3 PDZ domain (or its equivalent) and/or the HtrA3 PDZ domain and the binding ligand, is sufficient to allow interaction of the two components. The conditions of contact and duration are sufficient to allow the two components to interact. In the binding assay, the interaction is binding and the complex formed in the reaction mixture can be separated or detected. In a particular embodiment, the candidate substance or molecule is immobilized on a solid phase, such as a microtiter plate, by covalent or non-covalent attachment. Non-covalent attachment is typically achieved by coating the solid surface with a solution of matter/molecule and drying. Alternatively, a fixed affinity molecule, such as an antibody (e.g., a monoclonal antibody), specific for the substance/molecule to be immobilized, can be anchored to the solid surface. The assay is performed by adding an unfixed component that can be labeled by a detectable label to a fixed component (e.g., a coated surface containing the anchor component). When the reaction is complete, the unreacted components are removed, for example, by washing, and the complex anchored to the solid surface is detected. When the initially unfixed component carries a detectable label, detection of the label immobilized on the surface indicates recombination. When the initially unfixed component is not labeled, the complex can be detected, for example, by using a labeled antibody that specifically binds to the immobilized complex. If a candidate compound interacts with, but does not bind to, a binding partner of the HtrAl PDZ domain or the HtrA3 PDZ domain or the HtrAl PDZ domain or the HtrA3 PDZ domain, its interaction with the polypeptide can be determined by well-known methods of binding protein-protein interactions. Such assays include conventional methods such as the administration of 128788.doc-76-200846358, co-immunoprecipitation, and co-purification via a gradient or chromatography column. In addition, as described by Chevray^ Nathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793 (1991), protein-protein interactions can be monitored by using yeast-based genetic systems as described by Fields and colleagues. And Song, Nature (London), 340:245-246 (1989); Chien et al, Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)). Many transcriptional activators such as the yeast GAL4 consist of two physically discrete modular domains, one acting as a DNA binding domain and the other acting as a transcriptional activation domain. The yeast expression system described in the aforementioned publication (commonly referred to as "two-hybrid system") utilizes this property and employs two hybrid proteins in which the target protein is fused to the DNA binding domain of GAL4 and is candidate in another The activated protein is fused to the activation domain. The expression of the GAL1-lacZ reporter gene under the control of the GAL4 activating promoter is dependent on the remodeling of GAL4 activity via protein-protein interaction. The chromogenic receptor of β-galactosidase is used to detect communities containing interacting polypeptides. A complete set of protein-protein interactions between two specific proteins using a two-hybrid technique (matchmakerTM) is available from Clontech. The system can also be extended to locate protein domains involved in specific protein interactions and to pinpoint amino acid residues that are critical to such interactions. In any of the above screening methods, it is generally desirable to determine the candidate compound and the HtrAl PDZ domain and a known high affinity binder (such as one of the binders described herein) or the HtrA3 PDZ domain and a known high affinity binder (such as One of the binders described herein) combines the ability to assess its ability to regulate. I28788.doc -77- 200846358 may be based on the information described herein, particularly with respect to HtrAl PDZ domain-ligand binding interactions or individual residues and partial pairs in the ligand with individual residues and moieties in the ligand. The contribution and importance of the HtrA3 PDZ domain-ligand binding interaction or information relating to the HtrAl PDZ domain or the HtrA3 PDZ domain sequence itself, the candidate compounds are produced from combinatorial libraries and/or mutations of known binders. Compounds that interfere with the interaction of the HtrAl PDZ domain with the binding ligand or the HtrA3 PDZ domain and the binding ligand can be tested as follows: typically allowing the interaction and binding of the PDZ domain to the ligand and allowing for that interaction and The reaction mixture containing the HtrAl PDZ domain or the HtrA3 PDZ domain and the ligand was prepared at the time of binding. To test the ability of a candidate compound to inhibit binding interactions, the reaction is carried out in the absence and presence of a test compound. Alternatively, a placebo can be added to the third reaction mixture to serve as a positive control. The binding of the test compound to the HtrAl PDZ domain and/or the binding ligand present in the mixture or the HtrA3 PDZ domain and/or the binding ligand present in the mixture (complex formation) is monitored as described above. Formation of the complex in the control reaction but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the HtrAl PDZ domain with the binding ligand or the interaction of the HtrA3 PDZ domain with the binding ligand. As described herein, the substance/molecule of the invention can be a peptide. Methods for obtaining such peptides are well known in the art and include screening for binders of target antigens in a peptide library. In one embodiment, a suitable target antigen comprises a HtrAl PDZ domain or a HtrA3 PDZ domain (or a portion thereof comprising a binding site for a HtrAl PDZ domain ligand or a HtrA3 PDZ domain ligand), which is described in detail herein. 128788.doc -78 - 200846358 A library of peptides is well known in the art and can also be prepared according to methods known in the art. See, for example, Clark et al., U.S. Patent No. 6,121,416. Peptide libraries fused to heterologous protein components, such as phage sheath proteins, are well known in the art, for example, as described in Ciark et al., supra. In one embodiment, the peptide having the ability to block the HtrA1 pDZ domain protein-protein interaction or the HtrA3 PDZ domain protein-protein interaction comprises any of the amino acid sequences of the binder peptides disclosed herein. In another embodiment, the peptide having the ability to block the HtrAl PDZ domain protein-protein interaction or the HtrA3 PDZ domain protein-protein interaction comprises an amine of the binder peptide obtained by the modulator screening assay as described above. Base acid sequence. In one embodiment, the peptide has the ability to compete with one or more of the binder peptides disclosed herein (see examples) for binding to the Htr A1 PDZ domain or the Htr A3 PDZ domain. In one embodiment, the peptide binds to the same antigenic determinant on the HtrAl PDZ domain bound to one or more of the binder peptides (see examples) disclosed herein or to the same epitope on the HtrA3 PDZ domain. Mutants of the first peptide binder can be produced by screening mutants of the peptide to obtain characteristics of interest (e.g., enhanced binding affinity of the target, enhanced pharmacokinetics, reduced toxicity, therapeutic index elevation, etc.). Mutation induction techniques are well known in the art. In addition, scanning mutation inducing techniques, such as those based on alanine scanning, may be particularly helpful in assessing the structural and/or functional importance of individual amino acid residues within a peptide. The HTRAl PDZ domain activity can be modulated by a candidate substance/molecule of the invention (such as a peptide comprising an amino acid sequence comprising a binder peptide disclosed herein) by assaying the substance/molecule in an in vitro or in vivo assay. 128788.doc -79- 200846358

Determination of the ability of the HtrA3 PDZ domain to be active, the assay being well established in the art, for example, as Martins et al. (乂5M/·C/zem. 278(49):49417-49427 (2003)) and Faccio Et al. (丄 Biol Chem. 275(4): 2581-2588 (2000)).

Examples of the use of HtrAl PDZ domain binders and HtrAl PDZ domain-ligand interaction modulators or HtrA3 PDZ domain binders and HtrA3 PDZ domain-ligand interaction modulators are as described herein for HtrAl PDZ domain peptides Identification and characterization of a binder or HtrA3 PDZ domain peptide • a binder provides valuable insights into the cellular function of the HUA1 protein or HtrA3 protein, respectively, and provides for the in vivo interaction between these important cellular proteins and their binding partners. Composition and method of action. For example, such peptides and homologs thereof can be utilized to interfere with in vivo binding interactions involving the HtrAl PDZ domain or the HtrA3 PDZ domain. The homologues can be conveniently produced based on their binding and/or functional characteristics relative to the well characterized peptides provided herein. These peptides can be further utilized to elucidate cells and ® physiological polypeptides that constitute the in vivo complex of the HtrAl PDZ domain or the HtrA3 PDZ domain. Indeed, as shown by the unexpected results described herein, the binding partner of the HtrAl PDZ domain or the HtrA3 PDZ domain can be localized to the conventional C-terminal region of the polypeptide and to the previously unknown N-terminal region and/or internal region. As described herein (see, for example, the examples), well characterized high affinity peptide binders of the HtrAl PDZ domain or the Hti*A3 PDZ domain can be used to elucidate important structural features of the HtrAl PDZ domain or the HtrA3 PDZ domain itself. Knowledge associated with this provides for the development of modulators based on modifications of the HtrAl PDZ domain or the HtrA3 PDZ domain sequence itself. The present invention provides HtrAl PDZ domain variants and HtrA3 PDZ domain variants having the ability to enhance or decrease binding to a HtrAl PDZ domain or a HtrA3 PDZ domain binding partner as disclosed herein 128788.doc-80-200846358. Other variants can be similarly identified. The regulatory effects of interest can be achieved using HtrAl®0 redundancy domain-binding partner modulators or HtrA3 PDZ domain-binding partner modulators developed based on the ligand peptides described herein. For example, the manipulation can include inhibiting association between the HtrAl PDZ domain and its homologous binding protein or inhibiting association between the HtrA3 PDZ domain and its homologous binding protein. In another example, the manipulation may include, by, for example, inducing a fine function as a result of binding of the modulator to the HtrAl PDZ domain or the HtrA3 PDZ domain or via enhancing the HtrAl PDZ domain and its homologous binding protein by a modulator The association or enhancement of the association between the HtrA3 PDZ domain and its homologous binding protein is enhanced by modulators.

Other uses of modulators of the HtrAl PDZ domain or the HtrA3 PDZ domain include diagnostic assays for diseases associated with HtrAl or Hti*A3 and their associated collocations, HtrAl PDZ domain or HtrA3 PDZ domain and HtrAl PDZ domain or HtrA3 PDZ domain The ligand is used as a purification handle and anchor for the substrate, respectively, in the fusion protein. As described herein, a binder capable of binding to the HtrAl PDZ domain or the HtrA3 PDZ domain with different affinities provides a suitable pathway for in vivo regulation of biologically important protein-protein interactions. As is well established in the art, HtrAl proteins are involved in important biological processes, including, for example, processing amyloid precursor proteins, and HtrAl downregulation has been implicated in cancer development (ie, especially endometrial cancer, ovarian Cancer and melanoma), while HtrAl upregulation has been implicated in arthritis and age-related (wet) yellow 128788, doc-81 - 200846358 plaque degradation. As is well established in the art, Ship 3 proteins are involved in important biological processes, including placental development, and regulation of HtrA3 has been implicated in the development of cancer (i.e., especially endometrial cancer). The secret,] and HtrA3 proteins each contain a pDZ domain, which is reported to be in protein-protein, . The interaction of the mouth is a crucial domain. The identification of molecules that are capable of modulating such interactions indicates that the therapeutic and/or diagnostic application pathways and strategies in which such molecules and interactions are not available are not possible strategies. Regulatory compounds (eg, 'inhibitory or agonistic) can be delivered to living cells to serve as a ship-specific basis using a known route of administration known in the art, for example via microinjection, an antennal peptide or a lipofection reagent. Or HtrA3 PDZ domain specific regulators to modulate and in some cases validate the HtrA1 PDZ domain. Ligand interactions or Α3観 domains · Ligand interactions in specific tissues, cells, organs or pathological conditions The physiological importance of it. There is a suitable assay for monitoring the interaction of the ship's ligands with each other and regulating the physiological effects of the interaction. This does not require that the physiological ligand of the pDz domain or the HtrA3 PDZ domain be revealed by phage discovery that only the modulator is required to be specific for the PDZ domain and has sufficient affinity to disrupt the interaction of the ligand with the domain. Finally, # with any protein associated with the disease process, it must be determined how the drug will affect the protein to achieve therapeutic benefit. Regulatory compounds such as peptides/ligands can be delivered to live cells or animal models that are responsible for the disruption of HtrA1 PDZ domain-ligand interaction pDz domain-ligand interactions Whether to provide a disease model that is consistent with the expectations of therapeutic benefit (ie, 'simulating certain properties of the disease'). 128788.doc -82- 200846358 Methods for detecting protein-protein (or peptide) interactions in vivo are known in the art. For example, the HtrAl PDZ domain containing protein or the HtrA3 PDZ domain containing protein (including any of the ones described herein) can be analyzed using the methods described in U.S. Patent Nos. 6,270,964 B1 and 6,294,330 B1 to Michnick et al. Interaction of source ligands or synthetic peptides, including any of those described herein. In addition, such methods can be used to assess the ability of a molecule such as a synthetic peptide to modulate the binding interaction of a HtrAl PDZ domain protein with its cognate ligand or HtrA3 PDZ domain protein with its cognate ligand in vivo. Therapeutic/prophylactic applications Compounds having properties that increase or decrease the protein activity of the HtrAl PDZ domain or the HtrA3 PDZ domain are suitable. This increase in activity can occur in a variety of ways, such as by administering to an individual in need thereof an effective amount of one or more of the modulators described herein. "Antagonist" or "negative modulator" includes partial or complete blocking, inhibition or neutralization of the biological activity of the HtrAl PDZ domain and/or its endogenous ligand or HtrA3 PDZ domain and/or its endogenous ligand Any molecule. Similarly, an "agonist" or "positive modulator" includes any molecule that mimics or enhances the biological activity of the HtrAl PDZ domain and/or its endogenous ligand or HtrA3 PDZ domain and/or its endogenous ligand. Molecules that can act as agonists or antagonists include HtrAl PDZ domain-binding/ligand interactions as described herein or modulators of HtrA3 PDZ domain-binding/ligand interactions, including but not limited to a fragment of an antibody or antibody fragment, HtrAl PDZ domain/ligand/binding agent, peptide, small organic molecule or the like or HtrA3 PDZ domain/ligand/combin, peptide, small organic molecule, etc. 128788.doc -83 - 200846358 Or variants. Native to provide a binding molecule based on the discovery of specific interactions with the HtrA i pDz domain or the HtrA3 Z domain and between the HtrA 1 pDZ domain and the ligand binding peptide or Htl<A3 Various methods for the unique characteristics of the binding interaction between ligand bindings. Various substances or molecules (including peptides, etc.) can be used as therapeutic agents. These materials or knives can be formulated according to known methods to prepare medicine. Applicable composition: thus will The product is in the form of a mixture with a pharmaceutically acceptable carrier vehicle, and δ. By mixing the active ingredient of the desired purity with an optional physiologically acceptable carrier in the form of a lyophilized formulation or aqueous solution. , Excipients or Stabilizers (Remington, s Pharmaceutica I Edition, Osol, A. Ed. (1980)) Prepare therapeutic formulations for storage. Acceptable

The carrier, excipient or stabilizer is non-toxic to the recipient at the dosages and concentrations employed, and includes buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid; Low molecular weight (less than about 10 residues) more than a; protein, such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer, such as polyvinylpyrrolidine _; amino acid, such as glycine, glutamine Amine, aspartame, arginine or amidoxime, monosaccharide, disaccharide and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugar alcohols, such as mannitol: Ge Sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as tweenTM, PLURONICSTM or PEG. This is achieved easily in the frozen transition valley. Formulations to be administered in vivo must be placed in a container having a sterile access port, typically before or after sterilizing and rehydrating via a sterile membrane 128788.doc-84-200846358 An intravenous solution bag or vial of a stopper pierceable by a hypodermic needle. The route of administration is consistent with known methods, for example, by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, subcutaneous, intraarterial or intralesional route injection or infusion, topical administration or via a sustained release system. The dosage of the pharmaceutical composition of the present invention and the concentration of the desired drug can be expected

Change for a specific use. The determination of the appropriate dosage or route of administration is well within the skill of the general practitioner. Animal experiments provide a guilty guide to identifying effective agents for human therapy. The scaling of the effective dose between species can be based on J. ^ Chappell, W. ^ The use of interspecies scaling in toxicokinetics^ In Toxicokinetics and New DrBg Devel〇pment, Ya(3) bi#, ed., pergaman ρ_, n (4) 1989 'pp. 42-96 The principles laid down are carried out. When the substance or molecule of the present invention is administered in vivo, the normal dose may vary from the weight per kilogram of mammalian body weight per day to at most (10) mg or more (10) mg per kilogram of mammalian body per day, and (iv) h per kilogram. Called (four) mg. Regarding the dose and the delivery side: the:: is provided by, and is provided; see, for example, the US Patent No.... :"Luo’Μ4 or 5’225,212. It is expected that different formulations will be effective for different therapeutic compounds and different conditions, and techniques that target at least a few 遂〆, mouth ε or tissue (for example) may need to be transmitted differently. Or a method of organizing a disease or disease that requires a sustained release of a substance or molecule in a formulation having a release profile suitable for the treatment of any disease or substance requiring administration of a substance or molecule, The substance or molecule is microencapsulated. Microencapsulation of recombinant proteins for sustained release has been successfully carried out using human growth hormone (rhGH), interferon- (rhIFN-), interleukin-2 and MN rgpl20. Johnson et al., VIII. 71^(^, 2:795-799 (1996); Yasuda, Biomed. Ther^ 27:1221-1223 (1993); Hora et al., J. 8:755-758 (1990); Cleland, ''Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems/1 in Vaccine Design: The Subunit and Adjuvant Approach, ed. Powell and Newman, (Plenum Press: New York, 1995), pp. 439-462; WO WO 96/40072; WO 96/07399 and U.S. Patent No. 5,654,010. The use of the polymer due to the biocompatibility of polylactic acid-co-glycolic acid (PLGA) polymer and a wide range of biodegradability To develop a sustained-release formulation of these proteins, the degradation products of PLGA, lactic acid and glycolic acid, can be rapidly eliminated in the human body. In addition, the degradability of the polymer can be adjusted from several months to several years depending on its molecular weight and composition. Lewis, "Controlled release of bioactive agents from lactide/glycolide polymer,'' at M. Chasin and R. Langer (ed.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York 1990), pp. 1-41. Pharmaceutical Compositions The modulator molecules/substance of the invention may be incorporated into compositions suitable for medical use in some embodiments. The compositions typically comprise a nucleic acid molecule, peptide / 128788.doc -86 - 200846358 Proteins, small molecules and/or antibodies, and acceptable carriers, such as pharmaceutically acceptable carriers. "Pharmaceutically acceptable carrier" includes any and all pharmaceutical pharmaceuticals compatible Solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like (Gennar®, Remington: The science and practice of pharmacy. Lippin-cott, Williams & Wilkins, Philadelphia, PA (2000)). Examples of such carriers or diluents include, but are not limited to, water, physiological saline, Finger's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils (fixed 01ls) can also be used. These compositions are intended to be used in the absence of conventional media or agents which are incompatible with the active compound. Supplementary active compounds can also be incorporated into the compositions. It is generally contemplated that pharmaceutical compositions are formulated to be compatible with the desired route of administration, including intravenous, intradermal, subcutaneous 'oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal. Dosing. Solutions or suspensions for parenteral, intradermal or subcutaneous use may include: sterile diluents such as water for injection, physiological saline solution, fixed oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; Bacterial agents, such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (edta); buffers such as acetate, citric acid or A dish salt; and a tonicity modifier such as sodium chloride or dextrose. The pH can be adjusted with an acid or a base such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be sealed in ampules, disposable syringes or vials made of glass or plastic or in vials of 128788.doc -87 - 200846358. Injectable Formulations Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (in the case of water) or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, hexadecanol polyoxyethylene suture elTM (B ASF, Parsippany or sulphate buffered saline (pBs). In this case, the mouthpiece must be sterile and should be fluid for administration using a syringe. The compositions should be stable during the manufacturing and storage period and must be preserved against microbial contamination such as bacteria and sputum. The carrier can be contained (eg Water, ethanol, polyhydric alcohols (such as glycerol, propylene glycol and liquid polyethylene glycol) and suitable mixtures / hydrazine or knives. Suitable fluidity can be achieved, for example, by the use of a coating such as lecithin. In the case of a dispersion, it is maintained by maintaining the required particle size and by using a surfactant. Various antibacterial agents and anti-true, such as p-nonylbenzimidazole, chlorobutanol, benzene, ascorbic acid And thimerosal, which may contain microbial contamination. The composition may include an isotonic agent such as a sugar, a polyol such as mannitol, sorbitol, and sodium chloride. The composition which delays absorption includes, for example, aluminum monostearate and gelatin Injectable solution of innocent injectable solution can be hunted by the active compound (for example, any regulator substance/molecule of the invention) as needed, together with a component or a combination It is prepared by incorporation into a suitable solvent, followed by sterilization. The dispersion is usually made by injecting the active person into a sterile medium containing an alkaline dispersion medium and other ingredients. # ^ , /4+ ^ Preparation. The preparation method for preparing the sterile injectable lysate powder includes..., including vacuum drying and freeze-drying from the sputum-free liquid to obtain the active ingredient and the powder of any desired ingredient, 128788.doc -88 - 200846358 Ί>. Oral composition

Oral compositions generally comprise an inert diluent or an edible carrier in a gelatin capsule or compressed into a keying agent. For oral therapeutic rate =, the active compound can be combined with excipients and used in the form of tablets, lozenges; Oral compositions can also be prepared using a fluid carrier such that the compound in the fluid carrier is administered orally.彳 Includes academically compatible binders and or adjuvants. Tablets, pills, gels: Tablets and their analogues may contain the following ingredients: , Zhejiang >" People 1 4 has similar properties. "Do not adhesive, such as microcrystalline cellulose, tragacanth Or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, PRIMOGEL or corn starch; Run (4), ^ @(4)

STEROTES; a glidant such as a gelatinous silica dioxide; a sweetener, a saccharide or a saccharin; or a flavoring agent such as peppermint, methyl salicylate or an orange flavoring. 4 · Inhalation composition For inhalation administration, the compound may be self-contained with a suitable propellant (for example,

In a spray or pressurized container such as a gas of carbon dioxide, it is delivered in the form of an aerosol mist. I ^ systemic administration can also be administered systemically via mucosal or transdermal. For transmucosal or transdermal administration, a penetrant that is permeable to the target barrier is selected. Transmucosal penetrants include detergents, bile salts, and fusidic acid derivatives. For transmucosal administration, nasal sprays or suppositories can be used. For transdermal administration, the active compound can be formulated as an ointment, ointment, gel or cream by 128788.doc -89-200846358. The compounds can also be used in rectal delivery suppositories (for example, with a base such as cocoa butter and other glycerides) or in the form of retention enemas. 6. Carriers In one embodiment, the compound is protected from rapid elimination by the body. The carrier is used to prepare active compounds, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable or biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, % glycolic acid, collagen, polyacids, and polylactic acid. The f f can be commercially available from alza

Corporation (Mountain View, CA) ^N〇VA Pharmaceuticals, Ine. (Lake, CA) is available or prepared by those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. Such suspensions can be prepared according to methods known to those skilled in the art, such as in U.S. Patent No. 4,522,811, issued to 1985. Ί · Unit Dose An oral or parenteral composition can be produced in a unit dosage form to facilitate administration and dose uniformity. By unit dosage form is meant a physically discrete unit of k δ as a single dose of the subject to be treated, which comprises a therapeutically effective amount of the active compound and the required pharmaceutical carrier. The unit dosage form is determined by the following If A and is directly dependent on the following: the unique characteristics of the active compound and the desired therapeutic effect of the compound and the inherent limitations of the compounding compound. Gene therapy conjugates Nuclear knives can be inserted into vectors and used as gene therapy vectors. For example, intravenous injection, topical administration (Nabel and Nabel, U.S. Patent No. 128788.doc-90.200846358 5,328,470, 199 or stereo injection (stere〇tactic injeetiQn) (Chen et al., proc Natl Acad Sci) US A 913054 7 (i994)) The gene therapy vector is delivered to an individual. The pharmaceutical preparation of the gene therapy vector may comprise an acceptable diluent or may comprise a buffer release matrix in which the gene delivery vehicle is embedded. Alternatively, for example When the entire gene delivery vector of the retroviral vector can be produced intact by the recombinant cell, the pharmaceutical preparation can include one or more cells that produce a gene delivery system. The dosage pharmaceutical composition and method can further comprise treating HtrA1 protein-related or Htr A3 protein-related (specific Other therapeutically active compounds commonly used in the pathology of Htr A1 PDZ domain-related or Htr A3 PDZ domain correlations. Htr A1 PDZ domain · Ligand regulation or HtrA3 PDZ domain-ligands are required for / in therapy or prevention In the adjusted condition, the appropriate dose level will usually be about 〇·01 mg to 500 mg per kg kg of patient per day, which can be single or It should be administered in multiple doses. The dosage will preferably be from about 1 mg to about 250 mg per kg per day; more preferably from about 55 mg to about 1 mg per kg per day. Kg about 〇·〇1 mg to 250 mg, about 0.05 mg to 100 mg per kg per day or about mg to mg mg per kg per day. Within this range, the dose can range from 0.05 mg to 〇5 per kg per day. Mg, 〇·5 mg to 5 mg or 5 mg to 50 mg. For oral administration, the compositions are preferably provided in the form of a syrup which contains from 1 〇 mg to 〇〇〇 〇〇〇 mg The active ingredient 'especially 1·0, 5·0, 1〇, 〇, 15.0, 20, 0, 25.0, 50.0, 75.0, 1〇〇·〇, ι50·〇, 2〇〇·〇, 250.0, 300.0, 400 · 〇, 500.0, 600·0, 750.0, 800.0, 900·0 and 1000.0 mg of activity into 128788.doc -91 · 200846358 The dose to reach the patient to be treated can be 1 to 4-day owed per day. - administered with a compound, a person who is administered one or two times a day, ... and a specific dose for any particular patient... these -= degrees, age, ":: Metabolic stability and duration of long-term effects, weight, body health, gender, diet, medication, and interpreting 'excretion rate, drug combination...' Subjects of therapy. The severity of Xiao Xiao and the

The kit composition of the composition (for example, the medical Huaren 4 in the kit, the container ^^^ with the dosing instructions - including the group mnn # in the form of a kit, :: no / components can be packaged in a separate container The function of the component immediately before use. The monocoat can permit long-term storage without compromising inactivation.

The kit can also include reagents in separate containers that facilitate performing such tests. ϋ 4 ϋ MU Diagnostic test or organization classification specific

MrT tests on any type that allows the maintenance of different components and that: different components are not adsorbed or altered by the substance of the container::! In the supply. For example, a sealed glass ampule may contain a buffer that has been sealed with = _ neutral non-reactive gas. Ampoules can be made of any suitable material such as shell/molecular and/or polymer, such as polycarbonate, polystyrene, such as glass; organic, other materials commonly used to hold reagents. Other examples of metal or any σ k Gu, including 128788.doc -92- 200846358 can be made from a substance similar to ampoules and a coating such as the inscription or the end. Other containers include test tubes, vials; bottles, sparse, syringes or the like. Capacitance, ~ a, a, 匁, ... 圏 取, 1 1 has a stopper pierceable by a hypodermic needle. #(四) Two by the group allowed after removal - there is room. The amine can be made into glass, plastic, rubber, etc. by removing the membrane from the "easy to remove." Description material

Instructions can also be supplied to the kit. The instructions may be printed on paper or other kebab 'and' or may be supplied in electronically readable media such as a flexible disk, CD-ROM, DVD_R〇M, zip disk, video tape, laser disc, (laserdisc), Tapes, etc. The detailed instructions may not be physically related to the kit; instead, the user may be directed to an Internet site designated by the manufacturer or distributor of the kit or by email to the user. The following examples are included to illustrate preferred embodiments of the invention. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent a technique that the inventors have found to be effective in the practice of the present invention and thus can be considered as a relatively ambiguous mode for its implementation. However, it will be apparent to those skilled in the art <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Example Example 1 · HtAl and HtrA3 PDZ domain binding specificity profile

Comparison of the HtrAl, HtrA2 and HtrA3 amino acid sequences (Fig. 1) shows that these HtrA proteins have a high degree of sequence homology with a consensus within the Pdz domain of greater than 30°/. . When it is expected that the degree of conservation of the sequence is converted to structural conservation, 128788.doc-93-200846358 can be expected to differ from the key sequence differences of the ligand binding site such that the ligand specificity is different. Therefore, the phage-presenting peptide library was used to evaluate the binding specificity profiles of the PDZ domains of human HtrAl and HtrA3. The GST-HtrAl-PDZ, GST-HtrAl-(I415Q/I418A)-PDZ or GST-HtrA3-PDZ fusion protein was used alone to screen the random ten of the C-terminal fusion with the M13 gene-8 major sheath protein at a high price. Peptide library (libC) (Held, Η. and Sidhu, SS (2004) Journal of Mo/ecw/ar illus; 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 (libN) fused to the N-terminus of the M13 major sheath protein. The library was constructed with the simplification codons encoding all 20 natural amino acids (ATCGACAGCGCCCCCGGTGGCGGANNKNNKNNKNNK NNKNNKNNKNNKNNKNNKTGATAAACCGATACA (SEQ ID NO: 25)). Nucleotide nucleotides were designed as previously described using a simple molar design (Sidhu, S. S., Lowman, Η B., Cunningham, B. C. and Wells, JA (2000) Methods Enzymol 328, 333_ 363). The libraries further include a stop codon that allows for the presentation of a truncated peptide. After three rounds of selection, individual pure lines were grown in 96-well format in 500 pL 2 YT broth supplemented with carbencilin and M13-K07, and culture supernatants were directly used in phage ELISA to detect A peptide specifically bound by HtrAl or HtrA3 PDZ. Specific binding to each PDZ domain was obtained. The culture supernatant containing phage particles was used as a template for amplification of PCR containing DNA fragments of the peptides. DNA tablets as previously described 128788.doc -94- 200846358

Segmentation (Vajdos, F. F., Adams, C. W., Breece, T. N., Presta, L. G., de Vos, AM and Sidhu, SS (2002) Mo/ 扪〇/ 320 ( 2), 415-428). The unique sequences of each library were aligned and analyzed for homology (see Tables 1 and 2). 128788.doc 95- 200846358

^W3qil^鳑ϊ&lt; s NO Ph 00 a in Called saw〇Xfl 6 CN 00 m inch u〇v〇 00 On 00 OO OO 00 OO 00 00 ο — (N m inch Os 0\ Os Q\ Ct\ ID V〇卜〇\Q\ 0\ 00 〇\ 〇\ 〇\ O rH o HH &gt;&gt;&gt;&gt;&gt;&gt;&gt;&gt;&gt;&gt; 1 窆爹窆爹参参参爹爹爹参参爹爹爹参参参参&lt;s hH &gt;&gt; ω w W Ph Ph H &gt; HH H &gt;—'[Xt &gt; ffi H &gt; — Pi ro oo in cr 00 o 〇 m 〇m 〇GO om GO 〇b in 00 〇ffi pH o Pi &gt; in Q Pi a Ui in in zm οί H P4 &gt;&lt; 〇00 〇H ϋ Q vo 00 a U ffi 00 HH P-. H m Pt 00 rH fH tH 1—4 4 τ-H tH rH rH rH rH τ-H t—H r—H r-^ e ”OM 0 I· z— £_ s, 9-aia3s zadlrAIv is H ^lyulqn衾2 fl 63⁄4ai 卜CO 00 cn ΟΝ Ο 寸 m inch rH 00 Q\ (N m inch 1/^〇卜 yr\ \r\ ^r\ \r\ yx^ \r\ &gt;&gt;&gt; Η &gt &gt; H &gt; H &gt;&gt; H &gt; H &gt;&gt; Η &gt;&gt; HH &gt; HH &gt; 参参爹参参参参参参参参Pi Η (Jh H IX| HH (Xi H-) H ω Η Η Η HH (Jh ti. H |X| P&lt; Ο Ο '. 00 in m 〇in ϋ 0- o oo ϋ ϋ Ο 〇Ο 〇〇 〇ϋ C7 〇Uh Qh Q m pin &gt; m H 1-1 HQ CU Ο ιη in mom H CO Oh Ρ-ι PL, Q mm xn &gt; Z 〇〇rH (NJ rH rH rH i-H rH rH τ —H r-^ rH r ζρβ,,ϊνύΗ 〇一丨ζ- ε- ΙΛ

Ph ^ IX, fi, pL, MW WP^^PQ^^UhW^W&gt;^!^W ^ m HHG〇〇&gt;-iQii,pH^Q^Q^Q〇' O ^ QQPU|&gt;&gt;Hp^HHh*HI«(&gt;0^Q 128788.doc -96- 200846358

£ I sa T&quot;H! r*H ^ r··^ τ^Η r*H τ·Η T&quot;H ν*·Η τ — (Nr〇4^nvo oo〇n〇i~4(N m寸卜ooon ο r^H r-~H τ^Η τ-Η rH r*H ^Η ^Η τ—Η r^ ι—Η rTMH rH r*H τ-Η rH τ-Η ▼ 爹窆爹爹爹爹爹爹爹窆爹爹窆爹爹爹 爹爹爹爹爹爹 爹爹爹爹爹爹 &gt;&gt;p^fe&gt;Hpiip^p^〇P^&gt;&gt;^HfeHpip^HH-lHfJHC〇pci〇〇〇ζΛ〇ζΛ^^ζ&gt;〇ζ〇ζ&gt;οα}^ϋ, 〇〇οο 〇〇c/^ 〇m Ζ fe^PiPH^HPH〇IL,^00〇, 1-1 〇Η ζΛ ί^ ζΛ Pti Μ m Η c^i &lt; ^ 〇) go ^ W οο οο οο ι-ί WQ ί&gt; Q Η Pci ΪΖ; Q co ζ/) ΙΖ; Η &gt;-1 &lt;ί Ρ^

(N zslrAvm ^—L_Zad-Ivml^lwbal 衾oooONO — csjm inch mvoi&gt;oo〇N〇—(Nm inch tnvot^ooONO — 卜^卜卜卜γ〇〇爹窆爹爹窆爹〇〇爹窆爹爹窆爹参参参参参爹爹窆Participate in the ginseng and participate in the ginseng: Ο 声 • 声 裘 裘 》 》 》 》 》 》 》 》 》 》 》 碟 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13: .^:-·ν:?· %^.\ \:^:&gt;^i^:^y^&lt;:\:\^:f〇Ρ〇0〇〇^0&quot;〇Ρ〇〇〇〇 ^〇Α〇〇〇〇〇^〇〇Q 00 00 H^l hl 〇〇pu, pci W ffi d 幺^ 〇Hq PLh &gt;^ ^ Q Ph ¢^ &gt; ^ ^ Q &lt; 00 00 05 00 Pu, ih Ah ζΛ HH &gt; 00 ζΛ ^; PL, Ph HS &lt; ffi ^ .ψ^-^皭矣皭矣¥(5απ碱 W shouting 1.wfiWK^4. Interpretation &lt;Ν- , τ , 0^^^键^|^^楚^-3孤^怀务砘-^€^^^^气^^ wn^og^zad chu 440 scales. 验#9^^孝^€镏鍩世维叫伯^-4^4^14怀^^ grip* I28788.doc -97- 200846358 Table 2: Peptides derived from LibN~ from LibNw^~

htrAUPDZ -2 -3 -4 -5 6 5 4 3 2

No D i.e., I s n gvrgrr vnfrlvilfllvlllllilllrwgm lpwllfaallivpvre egserlpgelvavrsl llllvllllwlwvvlv veyvmvlvfvfvvgya eededeeeead-eew, dr gggg) gggggsssfllswwwwwwwwvaaatltv strstatttpllvmrv vvvyvvyvLLLVGSGL sgesagggaeeeeeed 12345678901234 2222222223333333 --llllllll-1111111-111 6 11 4 11116

11 lx 11 11 IX

HtrA3-PDZ -2 -3 -4 -5 -6 -7 2 3

14_L L L L L L L L Lllllll SSESNSNS L L L L L L LL i-v;:-;:':·········::,;&quot;-: Μ y V V V L V MWWYWWWWW EEGEGEEEDDDNDDDDvvvvvvvvVVLVVyvF VVLVLVVV GGEGEGGG

NoIDQI E s η 21222324137138139140 2573 2 11 lx IX Ί - * Hydrophobic residues are shown in bold text and acidic residues are shaded in shades of gray. Position the locations as described in the text. For C-terminal library selection, HtrAl-PDZ and HtrA3-PDZ are preferably ligands having overall hydrophobic character. Similarly, it has previously been shown that the HtrA2 PDZ domain is also preferably a ligand having hydrophobic characteristics (zhang et al. (2007), preparation section). For C-terminal library selection, the specific profile of HtrAi_pDz is defined by 4 residues at the C-terminus of the domain. The C. terminal residue (position 0) of this domain exhibits a slightly more preferred leucine, which is the next most common residue. At the position -128 of 128788.doc -98- 200846358, residues having a large volume of side chain (tryptophan, isoleucine and phenylalanine) are preferred. Almost only tryptophan was found at position-2. Preferably, the hydrophobic residue at position-2 is a marker of the type II PDZ domain (Sheng, M. and Sala, C. (2001) 〇 / 24(1), 1_29). Position -3 is threonine or isoleucine, and a charged residue (Giu, Lys or Arg) is preferred at position _4. No preference was observed at any other position in the obtained HtrAl-PDZ binding peptide.

The HtrA3-PDZ specificity profile is defined by the two residues at the C-terminus of the domain and at position -3 as determined by the C-terminal library selection results. The C-terminal residue (position 〇) of this domain is slightly more preferred for valine acid, and the isokinetic acid is the most common residue selected for the next. Position-1 is only for tryptophan. Although hydrophobic residues are preferred at position-2 in many π-type PDZ domains, HtrA3-PDZ exhibits no preference at position-2 and at this position a non-hydrophobic amino acid is typically selected. Preferred glycine or serine is observed at position -3. The HtrA3-PDZ-binding peptide obtained by Libc selection was not observed at position_4 or any other position. Binding selection using an N-terminal library was also successful for both mrA丨_pDz and A3_PDZ, and the sequence of the pure lines revealed μ and 8 unique sequences, respectively (Table 2). The alignment of the sequences is more difficult than the LibC selection result in terms of the L_ selection result, since the latter is facilitated by the c_terminal residue acting as an anchoring position. In the case of HtrAl-PDZ, a subset of the LibN selection sequences contain a conserved w[D/E] sequence (SEQ ID NO: 141). It is possible that the acidic residue in the conserved region acts as a surrogate c-terminus to effectively position the tryptophan residue at the front of the two residues of the acidic residue at positions in the sequence of 128788.doc-99 - 200846358 Tryptophan. Alignment of all sequences based on this motif (which maintains the acidic residue as position 〇) does not reveal a consensus sequence for all selected sequences, however, it is suggested that there may be more than one consensus sequence in the selection results. There was no significant similarity between the sequences selected from the LibN and LibC library screens. For HtrA3-PDZ, most of the N-terminal selection ligands contained the W-V-L peptide (see Table 2). The sequences were aligned based on the homology, and tryptophan was used as position-1 to number the residues arbitrarily. Conserved acidic residues after hydrophobic residues were observed in multiple selections of HtrA3-PDZ. Example 2··Synthetic Peptide Binding To further improve the binding specificity of the HtrAl-PDZ domain and the HtrA3 PDZ domain, a set of synthetic peptides was generated based on the information obtained in Example 1. The peptide was synthesized using the standard 9-fluorenylmethoxycarbonyl (Fmoc) protocol for 2.5% triisopropyldecane and 2.5% H20 scavenging resin in trifluoroacetic acid (TFA) with reverse phase Purification by high performance liquid chromatography. The purity and quality of each peptide were verified by liquid chromatography/mass spectrometry (LC/MS).

The binding affinities of the peptides of HtrAl-PDZ or HtrA3-PDZ were determined as IC5G values using a solution phase competition ELISA. Immobilized with NeutrAvidinTM (Pierce) (Rockford, IL) by N-terminal biotinylated peptide (biotin-GWKTWIL for HUA1-PDZ and biotin-RSWWV for HtrA3-PDZ) The assay plates were prepared by BSA (Sigma) blocked MaxisorpTM plates (Nalge NUNC International) (Naperville, IL). HtrAl-PDZ and HtrA3-PDZ constructs with glutathione S-transferase (GST) fused to the N-terminus were prepared as described in the following literature (Laura, R. P., Witt, A. S., Held) ,Η·A., Gerstner, R., Deshayes, K., Koehler, Μ·128788.doc -100- 200846358 F·, Kosik, K. S., Sidhu, SS and Lasky, L, A. (2002)

Ckm 277 (15), 12906-12914). Pre-incubation of a fixed concentration of GST-HtTAl-PDZ (200 nM) or GST-HtrA3-PDZ (200 nM) in PBS, 0.5% BSA, 0.1% Tween 20 (PBT buffer) with serial dilutions of the peptide 1 hour and then transferred to the prepared assay plate. After an additional 1 hour of incubation, the plates were washed with PBS containing 0.5% Tween 20, incubated with HRP/anti-GST antibody (Amersham Pharmacia Biotech) diluted 1:10,000 in PBT buffer for 30 minutes, washed again, and Detection was carried out with 3,3',5,5'-tetradecyl-benzidine/oxime 202 (ΤΜΒ) peroxidase substrate (Kirkegaard and Perry Laboratories). The IC50 value is defined as the concentration of peptide that blocks 50% of the binding of the PDZ domain to the immobilized peptide. The IC50 values obtained for some of the synthetic peptides are shown in Table 3.

128788.doc 101 - 200846358 Table 3: IC50 value of the binding of synthetic peptide to HtrAl-PDZ and HtrA3-PDZ peptide ID position ί ~ ~~ (SEQIDNO) -10 -9 -8 -7 -6~^Λ~~5 ~~0~~Γ 1&lt;:50

UtrAl-ΡΏΖ coordination vessel Hl_cl (3) Hl_c2 (4) Hl-c3 (5) HI-c4 (6) Hl_c5 (142) HI-c3a(143) Hl_c3b (144) Hl_c3c(145) Hl_c3d (146) Hl_c3e(147) HI a c3f(148) Hl_c3g(149) Hl_c3h(150) GM130(151) CoBal (152)

D dgdwlddddddade

LLVVFAVVVVVVVVL LIWHVWAWWWWWWVF WWWWDWWWWWWWMC T T I n R I I n A T1 Tx I I I V ekrkdrrrrarrrkp IWSDWSSSSSASSVG

Hl-nl (153) Hl-n2(154)

S E

G Gw w s R V V

G R L V Lw E s L L V Y ED

HtrA3-PDZ Ligand H3-cl (11) H3_c2 (12) H3_c3 (13) H3_c4 (14) H3_cla(155) H3_clb(15) H3-clc H3_cld H3_cle H3~clf H3_clg(16) H3_clh(17) H3_cli( 156) H3_clj (18) H3_clk(157) H3_cll(158) H3_clm(159) H3_cln(19) H3_clo (20)

R w R R R R R G s G G G G F R F F rrrrrrrar lerygggga fffffffff

vvilvvvvagvvvvfavvvWWWWWWWWWWWWWWWWW H3_nl (22) H3_n2 (160)

G V V V D E L V V D

LLLL s NLLV Vww EG μΜ 23·3 士2.9 7·7 士0.6 0.9±0.1 2.8 士0.3 64·1 士13.6 3.5 士0.9 6·3土1 · 1 40.0 士5.1 13.0 士0.9 2.5 士0.4 1.3 士 0.1 2.8士 0.3 &gt;500 24.1 士 8.4 15.7 士 3.3 &gt;500 241.6 土169.10.6 土 0.1 0.6士0·1 1.0 土 0.1 2.9 ± 0.3 72 土 11 1.0 士 0·1 1.3 士 0.1 4.7 士 0.4 14.1 土1· 2 21·6 Soil 2.7 1.7 ± 0.2 0.6 0.1 2.6 ± 0.2 0.8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 The end is acetylated. An asterisk (*) indicates that the C-terminus is blocked by guanidine. The peptides used in the structural studies are shown in bold text and are substituted with a single alanine at each position within the peptides. 128788.doc -102- 200846358 For HtrAl-PDZ, peptide HI-c3 (DSRIWWV) (SEQ ID NO: 5) and biotin-0\¥1^ bound 11^(8£(5 10&gt;^0: 4) Competitive binding to GST-HtrAl-PDZ, where IC50 is 0·9 ± 0·1 μΜ, which is the lowest IC50 obtained for the synthetic peptide tested for binding to HtrAl-PDZ. Isothermal titration calorimetry is also used. The dissociation constant of the Hl_c3-HtrAl-PDZ interaction was measured. Briefly, ITC measurements were performed using a VP-ITC titration calorimeter (MicroCal) at 28 ° C. PBS pairs with 1 mM sodium azide Extensive dialysis was performed. Each titration experiment consisted of injecting 5 pL of peptide injection (284 μΜ) (unit volume of 1.4 mL) into 5.5 μΜ HtrAl-PDZ for 25 times. The concentration of peptide and protein was determined by amino acid analysis. The dilution heat was measured by injecting 10 pL of peptide into the buffer in a blank titration experiment. The nonlinear binding site model (MicroCal) was used to assume the nonlinearity of the single binding site model from the experimental measurements (after subtracting the dilution heat). Squared fit to determine binding heat and dissociation constants. 1.1 Soil 0.1 μΜ yields good agreement with competitive ELISA data (see Figure 2). Another peptide identified in phage selection (Hl_c2) binds to HtrAl-PDZ with a lower affinity than IC-c3 (IC5〇7·7 μΜ), although it has a higher selection during phage panning. Frequency. Blocking of the C-terminus by guanidination (see, eg, peptide Hl_c3h in Table 3) eliminates the competition of the peptide with biotin-GWKTWIL (SEQ ID NO: 4) for binding to HtrAl-PDZ at concentrations up to 500 μΜ Capability, indicating that the terminal carboxylate group is required for binding. Substituting alanine for tryptophan at position _1 results in a 7-fold decrease in binding (see, for example, peptides H1 to c3b in Table 3), while alanine substitution position - 2 tryptophan acids or iso-leucine at position -3 resulted in a 44-fold and 14-fold decrease in binding, respectively (peptides HI-c3c and HI-c3d). The best peptides were in position 〇, -4, -5 or - 6 (peptide 128788.doc -103- 200846358

Alanine substitution at a single position at H1-c3a, HI-C3e, Hl_c3f, and HI-c3g) has little effect on binding. Comparison of the peptides Hl_cl and Hl_c5 confirms the modest importance of tryptophan at position 1 and suggests that basic residues at position -4 (eg, lysine and arginine) provide superior acidity (eg, bran) The energy advantage of residues of aminic acid or aspartic acid. In the case of HtrA3, PDZ, peptide Η3-cl competes with biotin-RSWWV (SEQ ID NO: 12) for binding to GST-HtrA3-PDZ with IC50 of 0·6 +/- 0·1 μΜ. Similar to the results for HtrAl-PDZ, position 0 allows for any aliphatic side chains (ie, valine, isoleucine, leucine, and alanine), with lysine being preferred (comparative peptide H3-C1) , the result of H3—c2, H3_c3, H3_c4, and H3_cla), while the larger volume of the amphetamine side chain (peptide H3_c5) at position 使 reduced the binding by 14-fold. Blocking the carboxylate group (peptide H3_cle) by guanidation sharply reduces the binding affinity. Consistent with phage display data, position-1 is strongly preferred for tryptophan, and mutation to alanine results in a decrease in affinity over 400-fold (see, for example, peptide H3_clb). Significantly, even the tryptophan dipeptide at the first position binds to HUA3-PDZ with moderate affinity (see, peptides H3-cld (WV), H3-cle (WA) and H3-clf (WG)) . The alanine substitution at the -2 or -3 position has little effect on ligand binding to no effect (see, peptides H3_clc and H3_cld). The peptide H3_c6-9 further confirmed the lack of importance of the amino acid identity at the -3 and -4 positions. Previous reports have suggested that mouse HtrAl binds to the C-terminus of fibrous procollagen proteins such as type III collagen a3 C-propeptide (Col3al) and to Golgi matrix protein (GM130) (Murwantoko, Yano, M., Ueta) , Y., Murasaki, A., Kanda, H., Oka, C. and Kawaichi, Μ 128788.doc -104- 200846358 (2004) 381 (Part 3), 895-904). Synthesis corresponds to

C-terminus of Col3al and peptide of GM130. As described above, the C-terminus of Col3al and the binding of GM130 to HtrAl-PDZ were compared to the biotin _GWKTWIL (SEQ ID NO: 4) in a competition binding assay. The Col3al and GM130 peptides competed with biotin-GWKTWIL (SEQ ID NO: 4) for binding to GST-HtrAl-PDZ with IC50 values of 15.7 ± 3. 3 μΜ and 24.1 ± 8.4 μΜ, respectively (Table 3). This value is significantly higher than the IC50 value observed for GWKTWIL (SEQ ID NO: 4) binding (7·7 +/- 0,6 μΜ), indicating that the binding of Col3al and GM13 0 peptide to GST-HtrAl-PDZ is significant. It is weaker than the observed binding to the optimized peptide ligand. Although the results show that HtrAl-PDZ does bind to this optimized peptide ligand, the affinity measured here is significantly weaker than that reported by Murwantoko et al. (Col3al is 0.3 μΜ and GM130 is 6.0 nM). It is worth noting that the prior affinity is determined using assays using ligands immobilized on a solid surface, which leads to an overestimation of affinity due to the affinity effect induced by multivalent binding (Harris et al. (2001) Biochemistry 40: 5921 -5930; Harris and Lim (2001) J. Cell Sci. 114: 3219_323 1 ; Laura and Witt (2002) J, Biol Chem. 277(15): 12906-14). The solution phase competition assay described herein more accurately estimates monomer binding affinity. To further investigate the identification of internal peptide motifs, C-terminal amidated peptides derived from the N-terminal library of HtrAl-PDZ and HtrA3-PDZ were synthesized and tested for their ability to compete with biotinylated C-terminal peptides. In the case of HtrAl-PDZ, binding was detected only for one of the two peptides tested (peptide HI-n2), and the affinity of peptide Hl_n2 was significantly weaker than that observed for the C-terminal peptide 128788.doc-105- 200846358 Affinity (see Table 3). For Htr A3-PDZ, the two peptides tested (H3-nl and H3-n2) detected binding, but the two peptides showed significantly lower affinity than the peptide with free C-terminus. Combined (Table 3). Although the peptide H3-nl was derived from the sequence having the highest selection frequency in the N-terminal peptide phage selection, the affinity of HtrA3-PDZ was several hundred times weaker than the optimized C-terminal peptide (Table 3). Peptide H3-n2 binds to an affinity comparable to the non-optimized C-terminal dipeptide (compared to peptides H3-cle and H3_clf) (Table 3). In summary, these data suggest that although the HtrAl-PDZ and HtrA3-PDZ domains are capable of binding to internal peptide motifs in the ligand recognition groove (and therefore, the binding may be biologically relevant), C-terminal coordination is preferred. body. Example 3: Structure of the PDZ domain of HtrAl and HtrA3 a. NMR analysis To better understand the binding interaction between the HtrAl-PDZ domain and the peptide ligand, HtrAl_PDZ and peptide Hl_c3 (DSRIWWV) (SEQ ID NO: 5 The complex between the two was subjected to NMR analysis. A DNA fragment encoding human HtrAl residue 3 80-480 (corresponding to the PDZ domain) was cloned into the Ndel/BamHI site of the pET15b expression vector (Novagen) to generate a N-terminal His-tag and thrombin cleavage. A fusion of loci. BLR(DE3) pLysS cells containing plastids were supplemented with 15N-ammonium halide (&gt;99%, Spectra Stable Isotopes) and 12C- and/or 13Cc6-D-glucose (&gt;99%, Spectra Stable Isotopes) ) grown in M9 minimal medium. The cells were grown at 37 ° C to mid-log phase (〇D6〇〇=0.8). Protein expression was induced with 1 mM IPTG and the culture was incubated for an additional 12 hours at room temperature. Cells were harvested by centrifugation at 4,000 xg for 15 minutes. The resulting cell pellet was resuspended in Buffer A with 1 128788.doc -106 - 200846358 mM PMSF and the cells were solubilized by a high shear fluid processor. The cell lysate was clarified by centrifugation at 21,000 xg for 45 minutes, filtered through a 0.45 μηι filter and loaded onto a Nickel-NTA Superflow column (QIAGEN). The column was washed with 10 mM imidazole in 50 mM Tris-HCl (pH 8.0), 500 mM NaCl (buffer A), and used to dissolve 500 mM imidazole in buffer a. The fractions were pooled, thrombin (1 unit / house protein protein) was added and the sample was dialyzed overnight at 4 C with 50 mM Tris HCl (pH 7.5) with 2 mM CaCL, 100 mM NaCl (buffer B). The protein sample was concentrated and purified by Superdex-75 column (Pharmacia) a25 Tris HCl (pH 7.5), 300 mM NaCl. The sample was further purified via a MonoQ (Pharmacia) anion exchange column with Tris (pH 7.5) having a gradient of 〇u 〇 μ NaCl. The protein sample was concentrated to approximately 2 mM in 25 mM sodium phosphate (pH 6.00) containing 10% yttrium oxide (仏7) and mM sodium azide by freeze-drying the 10% D20 sample and dissolving it in Prepared in 99.996% D2〇 (Cambridge Isotope Labs, Inc.), 100%, 'D20 sample. Obtained on a Bruker DRX600 MHz or DRX800 MHz spectrometer equipped with a triple resonance triaxial active shielding gradient probe at 25 °C NMR spectroscopy. All NMR data were processed using NMRPipe (Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J. iBax, A. (1995) 6(3), 277 -293) and using Sparky program analysis (version 3.11, Goddard and Kneller, UCSF). Direct monitoring of 15N-HSQC 15N-13C-labeled HtrAl-PDZ by stepwise titration of peptide Hl_c3 The formation of the new peaks and some corresponding 128788.doc •107·200846358 The disappearance of the peak at the free HtrAl-PDZ is consistent with the slow exchange on the NMR time scale. Uses from 3D HNCA, HNCOCA, CBCACONH, CBCANH, HNCO and HNCACO experiment (Cavanagh, J., Fairbrother, W. J., Palmer, A. G. and Skelton, N. J. (1995)

Protein NMR Spectroscopy, Principles and Practice, Academic Press, New York) by Monte (Hitchens, T. K., Lukin, J. A., Zhan, Y., McCallum, S. Α· and Rule, G· S. (2003) / (10) rna / iVMi? 25 (1), 1-9) Auxiliary 1Hn, 15N, 13Ca, 130β and 13C, belonging. The side chain assignment was obtained by manually analyzing 3D-HCCH-TOCSY with D20. Analysis of 2D NOESY and 2D TOCSY peptide resonances by 13C and 15N filters in FI (Zwahlen, C., Legault, P., Vincent, S. J. F., Greenblatt, J., Konrat, R. and Kay? LE (1997) J. Am. Chem. Soc, 119(29), 6711-6721; Iwahara, J., Wojciak, J. M. and Clubb, R. T. (2001) 〇 / JVMi? V19 ( 3), 231-241). Analysis of 3D NOESY_15N by using the automatic NOE designation using the program CYANA (version 2.0, Herrmann, T., Guntert, Ρ· and Wuthrich, Κ (2002) J Mo/ Price·〇/ 319(1), 209-227) -HSQC, 3D NOESY-13C-HSQC and 3D 13C F1 Filter F3-Edit-NOESY-HSQC spectra to obtain initial structure and distance limits. The Φ, Ψ and χΐ dihedral angle limits are obtained by analyzing the HNHA, HNHB and TOCSY_15N-HSQC (35 ms mixing time) experiments based on the established Karplus relationship. Other loose backbone dihedral angle limits were obtained by analyzing the chemical shifts in the backbone using the program TALOS (Cornilescu, G_, Delaglio, F. Anti-A, {, 999, Journal of Biomolecular NMR 2-9-302). 128788.doc -108· 200846358 Applying a dihedral angle limit with a good fit as a chemical shift (as defined by the program), where the allowable range is the greater than the average of ±30° determined by TALOS or the standard calculated by TALOS Three times the deviation. The main chain dynamics were studied by analyzing the steady state iH-^N-NOE as described (Farrow, Ν·A·, Zhang, 0·, Forman-Kay, J. D. and Kay, L·Ε· (1994) σ/ Price TVMi? V4(5), 727-734). Using a simulated adhesion program CNX (Accelrys, Inc., 2002) used a distance, dihedral angle, and hydrogen bond restriction to calculate 100 structures starting from random protein and peptide configurations. The structure of the structure of the composite was determined by selecting 20 structures with a minimum energy limit violation. The NMR data of the Htr A1 -PDZ/peptide complex is sufficient to clearly determine the structure of the complex (Fig. 3, Table 4). Table 4 · NMR overall input limitation of HtrAl-PDZ in combination with Hl_c3 peptide and structural statistical poor material overview Parameter overall input limit: Total NOE 1352 Residue 179 Continuous 340 Medium range 252 Remote 504 Intermolecular 77 Total two sides Angle 178 Φ 77 Ψ 76 χΐ 21 Violation: RMSD of experimental limit: ΝΟΕ-distance (Α) 0.0041 ±0.0003 Dihedral angle (.) 0.056 ± 0.016 128788.doc •109- 200846358

NOE distance violation: Value &gt;0.01 A 34.1 ±4.8 Value &gt; 0.1 person 0.0 Average maximum violation (A) 0.07 ±0.01 Dihedral violation: Value &gt; 0.1. 6.6 ± 1.9 Average maximum violation (.) 0.5 ± 0.19 RMSD of idealized geometry 0.0009 ± 0.0001 bond (A) 0.27 ± 0.01 angle (.) 0.12 ± 0.01 瑕 (improper) (〇) Energy: capable part (kcal .mol/1) NOE 1.24±0.18 CDIH 0.04 ± 0.02 Key 1.5 ± 0.2 Angle 35.1 ± 1.4 瑕 2.1 ± 0.37 Van der Waals 16, 3 Earth 2 · 4 Stereochemistry: Rama Chandran ( Ramachandran) (%) favorable 74.39 allowed 24.18 generous 0.97a forbidden 0.46a structural accuracy: average structure average RMSD main chain (N, Ca, C) 0.52 ± 0.12b heavy chain (N*, C*, Ο*, S* 0.71 ± 0.35ba The residues belonging to the generous and forbidden regions are located at the ambiguous N-terminus of HtrAl-PDZ; no more than 3 members of the population have any of these residues. The RMSD of the ordered region of HtrAl-PDZ at residues 378-389, 411-463 and 468-475 is covered. 128788.doc -110- 200846358 bX-ray crystallography enables the HtrA3-PDZ domain (residues 354-453) in a complex with a high affinity pentapeptide sequence (FGRWVC00H) (SEQ ID NO: 11) identified by phage display crystallization. The PDZ_ligand complex was crystallized (herein referred to as HtrA3-PDZext) using the previously described strategy, including fusion of the 5-residue peptide sequence to the C-terminus of the PDZ domain via a triglycine linker (Appleton, Β · A·, Zhang, Y·, Wu, P·, Yin, J. P., Hunziker, W., Skelton, N. J., Sidhu, S. S. and Wiesmann, C. (2006) J. # Price·〇/. Chm. 281(31), 22312-22320). A DNA fragment encoding residues 354-453 of human HtrA3-PDZ was cloned into the Ndel/BamHI site of the pET22d expression vector, and this resulted in a HtrA3-PDZ encoding the N-terminal His-tag and thrombin cleavage site. Open reading frame. In addition, a 1 〇 residue extension was fused to the C-terminus of Htr A3-PDZ using standard molecular biology techniques to generate an open reading frame encoding HtrA3-PDZ-ext (extension, GGGFGRWV) (SEQ ID NO: 161). The E. coli BL21 (DE3) (Stratagene) culture containing the expression plasmid was grown to a mid-log phase (Α600=0·8) at 37 °C. Protein expression was induced with 0.4 M mM IPTG and the culture was grown for 16 hours at 16 °C. The bacteria were granulated 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 passed through a Microfluidizer® treatment facility (Model 110Y, Microfluidics Corp., Newton, MA, USA). ) dissolve the bacteria. The cell lysate was loaded onto a nickel-nitrotriacetic acid-agarose tube (Qiagen). The column was washed with buffer 128788.doc -111 - 200846358, flush A plus 20 mM imidazole, and the protein was used to dissolve the 25 mM imidazole in buffer a. The fractions containing the protein of interest were pooled, thrombin was added (1 unit / 3⁄4 gram of protein), and the sample was dialyzed overnight at 4 °C in PBS. The protein samples were hanked and further purified by Superdex-75 column with 50 mM Tris-HCl (pH 8.0), 300 mM NaCl and 5 mM β-Wilylethanol. By placing an equal volume of protein (10 mg/mL) with 0.1 M Bis-Tris (pH 6.5), 0.2 M MgCl 2 and 25% PEG 3350 (suitable solution) via a sinking vapor at 19 C Diffusion yields crystals. It is rapidly quenched in liquid nitrogen; before the east, the crystals are transferred to a low temperature buffer containing a suitable solution. Collect the complete data set under the beam line 5.0.1 of the Advanced Light Source (Berkeley, CA). All of the poor materials were treated with Denzo and Scalepack (Otwinowski, Z. a. W. M. (1997) Methods in Enzymology 276, 307-326) from HKL Suite. PDZ domain using HtrA2/Omi crystal structure (pdb accession number lLCY(Li, W·, Srinivasula, S, M,, Chai, J., Li, P·, Wu, J. W., Zhang, Z., Alnemri) , E·S· and Shi, Y· (2002) iVai Βζ·ο/ 9(6), 436-441)), using AMoRe (Navaza, J. (1994) Acta Cry st alio graphic a Section A 50 (2) ), 1 57-163) and the search model generated by SWISS-MODEL (Schwede, T., Kopp, J) Guex, N. and Peitsch, M. C. (2003) NucL Acids Res. 31(13), 3381 -3385) Solving the HtrA3-PDZext structure by molecular replacement. HtrA3-PDZext crystallizes in the space group P4JJ, where each asymmetric unit has two molecules. Each PDZ domain in the asymmetric unit is symmetric with crystallographic phase 128788 .doc •112- 200846358 The relative molecular ligand forms a crystalline dimer. This packing arrangement produces two non-equivalent PDZ-peptide dimers (Fig. 1B) with an RMSD of 0.3 A above the 102 Ca atom. It is proved that it is very similar in structure. The atomic model is established by Coot (Emsley, Ρ· and Cowtan, Κ· (2004) 60 (12 part 1), 2126-2132) and Refmac (Murshudov, G. N·, Vagin, A A. and Dodson, EJ (1997) Acta Crystallographica Section D 53(3), 240-255) ^: Into obtain a highly accurate structure of the HtrA3-PDZ/peptide complex structure (Fig. 4, Table 5). : HtrA3-PDZext crystallization data collection and improvement statistics overview data collection resolution (A) a 50-1.7 space group P4A2 unit cell parameters (A) a ' 6=73.0 &gt; c unique reflection 24,442 redundancy a 7.6 ( 5.6) Integrity C%)a 99.9(99.6) Rsym(%)ab 0.049(0.461) &lt;I&gt;/a(I)&gt;a Improved 39.2(3.3) Resolution 30-1.7 ^cryst J RfreeC 0·186 , 0.221 Reflection number 23,159 Non-hydrogen atom number 1849 Residue number 416 Number of water 202 RMSD bond (A) 0.012 RMSD angle (. ) 1·4 Ramachandran (Ramachandran 95.3, 4·7, 0 ^l〇tX%) d = 80.1

128788.doc -113· 200846358 &amp; The value in parentheses refers to the data b Rsym4|/-&lt;/&gt;|E/ in the highest resolution shell, where &lt;1&gt; is the symmetry correlation observation of unique reflection The average intensity of the results is C RcrysFRfreedl/Vi^l/Ei^, where Rfree represents 5% of the data selected by the P machine. The d values represent the percentage of residues in the most favorable zone, additional allowable zone, generous allowable zone, and prohibited zone of Lamarckrande, respectively (Laskowski, R. A., MacArthur, M. W., Moss, D. S) · and Thornton, J. Μ· (1993) Journal of Applied

Crystallography 26(2), 283-291) e. Structural Analysis In both structures, as seen in other PDZ domain structures, PDZ folding (Figures 3B and 4B) is enclosed by two alpha-helices (αΐ, α3) Composition of the five-chain β-layer (βΐ-β5) at the end (Sheng, Μ· and Sala, C· (2001) 〇/

Neuroscience 24(1), 1-29). Further, short β chains (βΝ and PC) were observed at the N-terminus and the C-terminus. Such as the PDZ domain of the HtrA2 protein (Zhang et al. Lu 2007, preparation section), and typical PDZ folding (for example, by the PDZ domain of Erbin (Skelton, NJ, Koehler, M. F., Zobel, K., Wong, W. L., Yeh, S., Pisabarro, Μ·T·, Yin, J·P·, Lasky, L· Α· and

Sidhu, SS (2003) J Biol Chem 278(9), 7645-7654)) ^ tb ? HtfAl-PDZ and HUA3-PDZ have a cyclic exchange fold, in which the first β chain of a typical fold corresponds to β5 of HtrAl-PDZ (See Figures 5C and 5D). The β1-β2 loop of the PDZ domain of all three HtrA family members forms a definitive α-spiral. However, the orientation of the helix relative to the rest of the domain varies, this hint 128788.doc -114- 200846358 The helix may be dynamic in configuration. In fact, for the HtrAl-PDZ complex, the heteronuclear NOE measurement is consistent with the presence of sub-nanosecond-scale kinetics in this region (Figure 6). (To facilitate the comparison of residues within each pdZ domain, reference to residues at each secondary structural element or position within the ring (Aasland, R., Abrams, C., Ampe, C., Ball, L. J) ·, Bedford, Μ·T·, Cesareni, G,, Gimona, M., Hurley, J. H., Jarchau, T. and Lehto, V.-P. (2002) LWiers 513(1), 141-144 Therefore, βΐ-ΐ is the first residue in the chain βΐ, and βΐ: β2-1 is the first residue in the ring between the chains βΐ and β2.

In the Pdz domain-peptide complex of all three members of the HtrA family, the peptide binds in an extended configuration to the gap between the chain βΐ and the helix α3, extending the antiparallel β-sheet formed by the chains βΐ and β2. Chain (Figures 3, 5 and 5C). The C-terminal carboxylate group is coordinated by three main chain guanamines just before the chain β 1 . In the case of HtrAl-PDZ, as determined by a hydrogen/gas exchange NMR experiment, the product is protected from solvent exchange in the complex. Although the residues βΝ: pl-1 (Ile383) and pl-1 (Ile385) are both directly directed to the Wei group, the distance is generally considered to be slightly longer than the distance determined by the hydrogen bond (the heavy atom distance is 3.9. .4 people and 3.5 bandits. 6 people). The guanamine hydrogen of 0&gt;1:01-2 (〇 384) does not directly point to the carboxylate group, and it may participate in the hydrogen bond of water transfer. Although the hydrogen/deuterium exchange experiments and low field chemical shifts observed for the two indole hydrogens suggest hydrogen bonding, the inherent lack of NOE limitations prevents the precise determination of the ligand carboxylate and carboxylate binding rings or The use of specific hydrogen bonds is limited in structural calculations. In the case of HtrA3-PDZ, all three guanamine groups directly point to the carboxylate oxygen atom (βΝ: βΐ-l, βΝ: β1-2 and βΐ-ΐ heavy atoms) at a typical distance from the hydrogen bond visible in the protein. The distances are 2·9 A, 2 8 A 128788.doc -115 - 200846358 and 3·2 A). Another similarity between the two structures is the recognition of the leucine side chain at position 0, which is oriented within the shallow hydrophobic pocket of the PDZ ligand binding groove. In both configurations, the hydrophobic binding pocket presents an aliphatic side chain that provides a complementary surface to which the proline side chain can be placed, however there is clearly enough in the pocket to accommodate multiple aliphatic peptide side chains at position 0. space. Although two rotationally isomeric configurations of the VaP side chain were observed in the NMR whole of the HtrAl-PDZ complex, the side chain forming the PDZ ligand binding pocket took a single configuration among all members of the whole.

The remaining protein-ligand interactions between HtrAl-PDZ and the HtrA3-PDZ domain are significantly different. In particular, the most striking difference between the two structures includes the recognition of tryptophan at position-1. In the case of HtrA3-PDZ, Trp·1 takes up almost other PDZ domains of preferred tryptophan at position-1 (eg, HtrA2 (Zhang et al. 2007, preparation)) and Erbin (Skelton, N. J., Koehler, Μ·F., Zobel, K., Wong, W. L., Yeh, S., Pisabarro, Μ. T., Yin, J. P., Lasky, LA and Sidhu, SS (2003) 5/ A conformal configuration visible in o/C/zem 278(9), 7645-7654). The indole ring of the tryptophan extends through the backbone of the chain βΐ and is interposed between the side chains of P2-5 (Glu390) and β2: cx2-l (Ala392) (Fig. 5C). The orientation of the Trp·1 side chain relative to the chain β2 reminiscent of the recent study of peptide β-hairpin stability (Cochran, Α·G·, Tong, R. T., Starovasnik, Μ. A·, Park, E. J., McDowell, R. S., Theaker, J. Ε and Skelton, N. J. (2001) Ckm 123(4), 625-632) Inter-strand tryptophanate contact observed. Thus, the preferred tryptophanic acid at position -1 suggests a general and slightly non-selective contribution to the high affinity binding mediated by the interaction of the aromatic and chain βΐ protein backbone phases 128788.doc -116-200846358. In the case of HtrAl-PDZ, Trp 1 also extends through the backbone of the bond βΐ, however, the orientation of the anthracene ring is significantly different from the orientation of HtrA3-PDZ (Fig. 7A). TYp·1 is located between the side chain of pN-3 (Tyr382) and pi-2 (Arg386) and is adjacent to the position β2: the isoleucine side chain (Ile418) at α2-1. The orientation of the ankle ring does not take a unique configuration across the whole (Figure 3 A). The lack of NOE limits (the three intermolecular NOEs of aromatic protons) is consistent with the unconformity of the configuration and the dynamic structure of the ring. The comparison of the measured chemical shift with the calculated chemical shift does not support the single, well-defined configuration of the ankle ring.

HtrAl-PDZ does show a strong preference for tryptophan at position-2. Trp·2 is specifically defined in its entirety by 20 unique intermolecular NOEs assigned to its aromatic protons. The anthracene ring package abuts the helix α3, producing a favorable hydrophobic interaction with α3-l (Ala445), pi-3 (Met387) and α3-2 methylene (Gln446) (Fig. 5A). The Bowbow ring is directly positioned above the alpha proton of Gln446, which is induced by the ring current effect to induce large (-1 ppm) chemical shift perturbations. Although the binding profile of HtrA3-PDZ and the synthetic peptide binding assay suggest that position-2 is not important for ligand binding, Arg.2 in the crystal structure display peptide H3_cl is localized to glutamine in position α3-4 The amine side chain (Gin 423) forms a hydrogen bond (Fig. 5C). Almost consistent interactions have been observed in the structure of the human CASK PDZ domain (Daniels, D. L., Cohen, A. R., Anderson, JM^Brunger, AT (1998) Nat Struct Mol Biol 5(4), 317-325).

The ligand binding profile of HtrA 1 -PDZ and the synthetic peptide binding assay suggest that it is preferably an isoleucine residue at position-3. The NMR structure shows 11 packaged into a hydrophobic patch block produced by 128788.doc-117-200846358 p2-3 (Ile415) and pl-2 (Arg386) (Fig. 5A). Peptide and protein β2-3 lie·3 packaging against the side chain methylene group of β1-2 (Αα386) may cause the Arg386-Glu416 salt bridge to affect the accessibility of position-1. In the case of the HtrA3-PDZ complex structure, Gly·3 of the peptide H3_cl does not directly contact HtrA3-PDZ, but adopts a positive Φ angle to locate the carbonyl oxygen of Phe-4 to form hydrogen with the side chain at position p2-2 (Arg360). The bond and the side chain of Phe-4 interact with the Arg360 side chain via a π-π stack (Fig. 5C). Although this is a favorable configuration in terms of energy and phage selection shows that glycine or serine is preferred at position -3 (Table 1), the synthetic peptide binding assay of HtrA3-PDZ does not suggest a position -3 glycine The energy advantage of the acid and the aromatic residue at position -4 of the ligand (see Table 3). Residues 4 to -7 did not appear to participate in the interaction with HtrAl-PDZ and were not sufficiently determined in the NMR whole. In HtrA3-PDZ, there is a hydrogen bond-stable type I inversion between Gly·6 carbonyl oxygen and Gly·3 decylamine, however, the synthetic peptide binding assay does not suggest that this configuration is important for ligand binding. . Genomic extensive search with potential extracellular ligands that match the C-terminus of HtrAl-PDZ specific profiles identified an amine thiol/cystamine amine with a C-terminal peptide KNLKSLTWWL (SEQ ID NO: 162) Peptidase isoform 1 (GenBank NP_005566) and leucine thiol/cystamine aminopeptidase isoform 2 with C-terminal peptide KNLKSLTWWL (SEQ ID NO: 163) (GenBank NP_787116). The suggestion that HtrAl specifically interacts with members of the TGF-β family (Murwantoko et al. (2004) Biochem J. 381 (Part 3): 895-904) is not supported by these results. Although the results confirm that HtrAl-PDZ does bind to the C-terminus 128788.doc-118-200846358 (such as the C-terminus of Col3al) and thus can play a role in the metabolism of fibrillar collagen C-propeptide, the analysis also shows a variety of other The C-terminal sequences are capable of binding with comparable or even higher affinity. HtrAl-PDZ appears to recognize a variety of targets with exposed hydrophobic C-termini rather than only a few selected targets. In the case of HtrA3_PDZ, a surprising preference for ligands containing tryptophan at position-1 suggests that HtrA3 binds preferentially to ligands containing tryptophan at this position, although the determinants of specificity are limited. Many 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 having the C-terminal sequence YYKRPVQEWV (SEQ ID NO: 166) (GenBank NP-006681) number). Melanoma-associated chondroitin sulfate (GenBank NP-001888) having the C-terminal sequence PALKNGQYWV (SEQ ID NO: 1 67) and keratinocyte-associated protein having the C-terminal sequence EIRASQRSWV (SEQ ID NO: 168) 3 (GenBank No. NP_776252) also has a conserved tryptophan acid at the penultimate position and thus may be a ligand for the HtrA3-PDZ domain. Example 4 · HtrAl_PDZ Ukrainian scan The contribution of individual residues of the Htr A1-PDZ domain to ligand binding was evaluated by a combined alanine scan (see, e.g., WO 2001/044463). Three libraries were constructed in which 64 positions in and around the peptide binding site were encoded by wild-type amino acids or alanine (or, when the wild-type amino acid was alanine, 128788.doc-119-200846358) , a trinucleotide representation of a non-alanine mutant). The library is constructed as follows. HtrAlPDZ was presented on the surface of M13 phage by modifying the previously described granules (pS2202b) (Skelton, N.J., Koehler, M. F., Zobel, K., Wong, W. L., Yeh, S., Pisabarro, Μ·T, 5 Yin, J. P., Lasky, L. A. and Sidhu, S. S. (2003) 278(9), 7645-7654). A fragment encoding pS2202b encoding human Erbin PDZ was replaced with a DNA fragment encoding HtrAl PDZ using standard molecular biology techniques. The resulting phagemid (p8HtrAl) contains an open reading frame encoding a secretion signal for maltose binding protein followed by an epitope tag (amino acid sequence: SMADPNRFRGKDLGS (SEQ ID NO: 169)) followed by HtrAl PDZ and M13 gene-8 The C-terminal domain of the secondary sheath protein ends. Escherichia coli containing p8HtrAl was co-infected with M13-K07 helper phage and allowed to grow at 37 °C without IPTG induction, thereby producing phage particles which encapsulated p8HtrAl DNA and presented HtrAl PDZ in a monovalent format. Use the previously described method (sidhu, s_S·, Lowman, Η·B·, Cunningham, BC and Wells, J. Α· (2000) Mei/zoA 328, 333-363) with appropriately designed p8HtrAl &quot "Terminal template" format construction library. For each library, we used a termination template containing a TAA stop codon in each region to be mutated. Using a termination template as a Kunkel mutation induction method (Kunkel, Τ·A·, Roberts, J. D. &amp; Zakour, RA (1987) A template for Mei/zoA &lt;9/154,367-382) in which the mutagenized nucleotides are designed to simultaneously repair the stop codon and Mutations are introduced at the desired loci. For bird grab scans, use Vajdos et al. (Vajdos, F. F., 128788.doc -120-200846358

Adams, C. W., Breece, Τ·N·, Presta, L. G., de Vos, A. Μ, and Sidhu, S. S. (2002) J Mo/320(2), 415-428) The corresponding simplification codons shown in Table 1 replace the wild type codon. Three libraries were constructed and each library mutated the discrete regions of HtrAlPDZ as follows: library 1, position 380-400; library 2, positions 401-422; library 3, positions 440-460. Libraries 1, 2, and 3 contain 3·0χ101(), 2.5xl01G, and 2.3xl01G unique members, respectively. The bacillus from the above library was propagated in E. coli XL1-blue by the addition of Μ13-K07 helper phage. As previously described (Sidhu, S. S., Lowman, Η B., Cimningham, Β·C· and Wells, J. A. (2000) 328, 333-363), grown overnight at 37 ° C The phage were concentrated by precipitation with PEG/NaCl and resuspended in PBS, 0·5°/〇BSA, 0.1% Tween 20. A sputum bacterial solution (1 〇 12 sputum bacteria/ml) was added to a 96-well MaxisorpTM immunoplate that had been coated with a capture target and blocked by BSA. Two different standardes are used: in terms of presentation options, the target is a fixed antibody that recognizes the epitope tag fused to the N-terminus of HtrAl PDZ, and in terms of function selection, will be high affinity with HtrAl-PDZ Binding biotinylated peptide (Biotin-GWKTWIL (SEQ ID NO: 26) or Biotin-DSRIWWV (SEQ ID NO: 5)) (Laura, R. P., Witt, A. S., Held, Η. A·, Gerstner, R., Deshayes, K., Koehler, M. F., Kosik, K. S., Sidhu, SS and Lasky, LA (2002) J 5ζ·ο/ C/zem 277(15), 12906-12914) was attached to a NeutrAvidinTM coated plate. After incubation for two hours to allow the cells to bind, the plates were washed 10 times with PBS, 0.05% Tween 20. The cells were combined with ·128788.doc -121 - 200846358 for 10 minutes with 0. 1 M HC1 and the lysing agent was neutralized with ΐ·〇 M Tris base. The lysing bacterium was expanded in E. coli XL 1 -Blue (Stratagene) and used in other rounds of selection. Individual pure lines from the second round of selection were grown in 96-well format in 500 gL 2YT broth supplemented with carbencillin and M13-K07, and the culture supernatant was used directly in the sputum ELISA (Sidhu, SS, Lowman, Η. C., Cunningham, BC and Wells, JA (2000) Mei/zoA 328, 333-363) for the detection of phage binding to biotin-GWKTWIL, biotin-DSRIWWV or anti-tag antibodies HtrAlPDZ variant. More than 50% of the pure lines exhibited a positive phage ELISA signal that was at least twice greater than the signal observed on the control plate coated with BSA. These positive lines are subjected to DNA sequence analysis. The sequence was analyzed using the program SGCOUNT (Weiss, G. A., Watanabe, C, K., Zhong, A., Goddard, Α· and Sidhu, SS (2000) 97(16), 8950-8954) as previously described. . SGCOUNT translates each aligned sequence of acceptable quality by using the Needleman-Wunch pairwise alignment algorithm relative to the wild-type DNA sequence and tabulates the appearance of each native amino acid at each position. For functional selection, the number of pure lines analyzed is indicated in parentheses following the name of each library: in terms of functional selection by biotin-GWKTWIL (SEQ ID NO: 4), Ll(80), L2 (91), L3 (89); in terms of functional selection by biotin-DSRIWWV (SEQ ID NO: 5), L1 (82), L2 (89), L3 (93); For the purpose, analyze the following number of pure lines: Ll (69), L2 (78), L3 (91). I28788.doc -122- 200846358 These libraries were subsequently selected for binding to peptide H1-c3 or peptide HI-c2 and will be sequenced positive for binding after two rounds of selection. The number of pure lines with wild type residues at each position is compared to the number with alanine (or mutant) to give a preferred indication that the wild type residue is superior to alanine (or mutant). To control changes in the expression or presentation content of different library members, libraries are also selected for binding to immobilized antibodies that recognize antigen epitopes present at the N-terminus of all library members. The ratio of wild type to _ mutant in the peptide selection was then determined by the ratio of wild type to mutant observed in antibody selection to give the frequency of normalization (F; Table 4). The substitution at each position is divided into three categories using the frequency of occurrence of this normalization: substitutions that reduce binding to the peptide (F &gt;5); substitutions that do not affect binding are about 〇; and substitutions that increase binding to the peptide (F ^ 〇, 5). The effect of the substitution of the alanine substitution in HtrAl-PDZ with the peptides H1 - c3 and H1 - c2 in Figure 7A and 7B, respectively, is reflected in the HtrA1_pDz^ structure. In the case of the peptide HI-c3, some of the substitution sites φ which have a significant influence on the binding (F &gt; 5) are in direct contact with the ligand (see, for example, Y3 82, 13 83 G384, M387 and S389) 'Others are located more than 4.5 A from the peptide ligand (see, for example, K38〇, K381, G411, 1414, V417, Τ421, and Ρ422). Although their residues do not directly contact the peptide ligand, it is possible to introduce a partial perturbation of the pDZ domain structure by replacing its side chain with alanine. For example, the isoleucine at position 414 (β2-2) is packed into the hydrophobic interior of the domain, and replacement of the bulk aliphatic side chain with a single thiol can perturb the hydrophobic inner packaging arrangement and may affect the β2_β3 inverse Parallel folds the sheet, thus disturbing the ligand binding site. Many residues are preferably substituted with alanine 128788.doc -123- 200846358 (see, for example, D400, 1418, V442, N446, D447, V448, S449 and L458). Of these residues, only residues 1418, N446 and S449 are within 4.5 A of the peptide of the complex. The effect of alanine substitution on H1-c2 peptide binding was very similar to H1-C3 (Figure 4A' Table 5), but there were some significant differences. The most striking difference between the alanine scan results for the two peptides can be found in residue 1418. In the case of the peptides H1-c2, the residue 1418 is advantageous (F = 17.7), and in the case of H1-c3, the alanine (F = 0.5) is slightly more preferable at this position. This may be related to the non-optimal interaction of Trp·1 inferred from structural analysis, and this can be improved by substitution of alanine at position 141 8 . Another significant difference between the two peptides can be found in residue S389 (pi-5). Wild type serine acid is advantageous at this position for binding to the peptide hi_c3 (F &gt; 46), but the substitution at this position does not bind to the HI-c2 (F = 2). Although this residue does not appear to be in direct contact with the peptide in the NMR structure, it is located at the periphery of the binding site and the side chain hydroxyl group of S389 is directed to the peptide in all members of the whole. In the NMr population, the side chain of the three N-terminal peptide residues was not fixed due to the lack of n〇e restriction, and it was not clear that S3 89 (or its lack) was directly involved in the &amp; Arg-4 side. chain. 128788.doc -124- 200846358 Table 6: Information on shotgun alanine scanning of HtrAl-PDZ using biotin-GWKTWIL * Ligand: biotin-GWKTWIL___

___wt/mutation_F residue' &amp; can choose rendering choice K380 K381 Y382 1383 G384 1385 R386 M3 87 M388 S389 L390 T391 5392 5393 K394 A395 K396 E397 L398 K399 D400 R401 H402 R403 D404 F405 P406 D407 V408 1409 S410 G411 A412 Y413 1414 1415 E416 V417 1418 1 8 3 4 8 9 0 7 7 3 /mlo.&gt;6&gt;7&gt;5&gt;273.&gt;1·&gt;4 wt A 4 2 5 80724772268604494476 t/5418&gt;5&gt;89120037311010021010 w 2 95 ❹ 6 6 3 12 rm 4·ί1.ί7.2.0.&gt;70.a wt/A wt/ml wt/m2 .0.3.9.3.0.8 11 1X 1A 11 ov .49 2.3.8.9.0.2 2 o 34 ool 1 5 9 6 5 6 1·&gt;8&gt;310.&gt;8 1000560116 13.90.144:&gt;32L1&gt;9&gt;8 3 3 4 .6.8.64.3.9.0.5 ο o 1- 2 1- o 1_ 2 5 0. 9 7 3 &gt;80.a .2 128788.doc 49.5.8.2.5.0.5.4.7.9.6.4.8.5.0.0.4.3.9.7.7.1.7.5.0.6.2.8.5.4.7.0.9.4. 2J.7.2· Αν ο 11 IX 11 Λν 11 ο CU11 ΙΑ Αν 11 ο ο f-Η ο ο 03 11 11 1i ο 11 0XW 1χ 11 11 ΙΑ 11 5 .3.7.0 0.0.1. 7 0. 4 2 4 7 0.1.0.ο* 0 2 5 8 ···* 8 2 6 1 6 2. .2.3.4.0.1.7.4 11 1i 11 11 11 11 .6 0. 0 5 0 5.2· 1· 8 5. 3 2 2 7 11 11 &gt; 1X 1 J 7*0·9·7·9·6 i l 11 11 tl 11 3 0. 5 6 0. 8 0. 7 ο· a4 7 0 169135253122049376564968843120357045 1Μ » · · mry. ····························· ······· » 7f. A15437&gt;2&gt;33600225121100210201200002233115&gt;2171&gt;3&gt;7

Ml m2 38.6 E 11.9 T &gt;87 E 25.6 T &gt;74 D 103.6 S &gt;82 T 3.7 V 3.6 T 0.4 V 33.9 G 1.0 P &gt;1.1 T 0.1 V 0.9 T 0.2 V &gt;17 P 1.3 V

2.4 E 0.5 T 0·7 E 0.5 T 2.5 P 1.6V 3.0 E 2.9 T 0.8 G 0.7 P 0.8 D LOP 1.5 G 1.8 P 0.8 S 1.4 V

2.5 T 1.6V

&gt;18 D &gt;61 S &gt;14 T 1.2 V 11.1 T 0.4 V

&gt;15 T 23.2 V 200846358

P419 21.8 D420 2.3 T421 &gt;91 P422 &gt;91 Q440 1.9 S441 3.2 V442 1.5 V443 1·6 S444 43.5 A445 1.0 N446 0.1 D447 1.4 V448 13.8 S449 0.5 D450 0.4 V451 0.4 1452 18.0 K453 0·2 R454 1.6 E455 0.2 S456 3.7 T457 1,1 L458 6.8 N459 1.4 M460 &gt;83 1.7 1.0 27.0 0.6 1.1 &gt;68 0.7 83·0 &gt;42 0.6 1.8 0.5 17.0 6.2 3.116.6 2.5 1.7 2.3 2.5 1.7 2.4 45.0 2.5 44.5 1.0 12.0 1.0 0.2 0.2 0.3 2.5 7.3 2.3 0,8 0.5 6.0 12.0 1.5 0.4 0.5 0.9 1.5 2.7 14.3 0.5 3.1 1.2 31·0 62.0 2.3 1.6 1.1 2.0 32·0 64.0 2.7

8·7 1,4 &gt;40 &gt;36 0.7 Ε &gt;0.9 Ρ 1.3 0·0 1.6 3.6 1.0 0·7 5.8 D 1.9Τ 0.6 1.9 0.2 0.6 0.8 3·〇2.3 Τ 1.2 V 0·5 1.0 Ε 0.6 Τ !·1 0.4 G 1.2Ρ 0.4 1.2 0.9 0·2 &gt;1.1 Τ 2,7 V °·9 0.6 D 1,6 Τ ^3 1.3 Τ 6.2 V * with high affinity peptide ligand (function selection) After selection by binding to an anti-gD_tag antibody (presentation selection), the Wt/mutation ratio is determined from the sequence of the isolated binding line. By dividing the function selection wr/mutation ratio by the rendering option

The frequency of occurrence of normalization is obtained by selecting the Wt/mutation ratio. In the case where no specific mutation is observed in the functional selection sequence, only the wt/mutation ratio and the lower limit of the F value (indicated by greater than the sign) can be determined. The F value of the alanine substitution and the two other substitutions (m2 and m3) were determined in the case where the alanine scan requires a tetranomial codon. The consistency of the non-alanine substitutions is shown in parentheses to the right of each F value. Bold numbers indicate mutations with more than 4 times the effect. (Continued on Table 7 on the next page) 128788.doc •126- 200846358 Table 7: HtrAl-PDZ Birds with Biotin-DSRIWWV Capture Alanine Acid*

Ligand: biotin-DSRIWWV wt/mutation J residue function selection _ ' 瓦 now choose K380 K381 Y382 1383 G384 1385 R386 M387 M388 S389 L390 T391 5392 5393 K394 A395 K396 E397 L398 K399 D400 R40! H402 R403 D404 F405 P406 D407 V408 1409 S410 G411 A412 Y413 1414 1415 E416 V417 1418 wt/A wt/ml wt/m2 4.2 8.3 3.6 5.5 66.0 22.0 8.4 74.0 3.9 61.0 61.0 2.9 WL 4 9 5 8 /Αο· ο. ο· 2· wt Ell wt/ M2 0.3 0.4 0.7 1.2 1.0 0.4 0.7 0.7

01080 00.4.9.4.3.6.6.6.0·0.0.5 41.1.31.7.18.18.39.3.41101 ο8528196 ο 6 632067963084923.7.0.8.8.8.3.6.9.6.6.0.0.3.0.8.5.0.6 2,4.1.0.5 .0.3.2.1.111102.10.110.0210301ί91Σ82841890 8 18 8 2 2 CC 3 3 7· 8· 1· 11 Q ο ο 11 3 ο 2 6 〇〇 1.6.5 2.2.3.2.2.2.0.1 5 0. 0 5 9 5.0.C5 8 7 25 0547964850043.9.7.71·.7.5.ο.62·8.54.7ο.94217 2· 2· 2* 1.1.1· 2· ο· 1·0.ο· 1· 1· OLOO.0.1 ο ο 3 1 1 1 ο 1 2 1 2 1 1 1 2 8.0 1·7 2.2 73.0 6.5 L2 1.8 1.2 2.6 1.1 o.2.34 ο· 1· 7· 4· 1- 1 1- 1 1- 11 ο 11 6 0 17.0.9.7.9.68· 11 Ίχ 1-Η ο ο ο 11 0. 2 .0.5.0.8 52.1 5 5 6 8 1.0.0. 7 0. la1.96.26.9&gt;22&gt;37L90.86.83,8sL24.5L42 .02.51.01.80.92.3L40:52.40.91.21.70.90.80.52.21.2u&gt;331.09.720:a4.01.4&gt;330.5 A 1 1 &gt;&gt; 4 2

Ml m2 29·8Ε 8.7 Τ 91.0 Ε 17.9 Τ &gt;74 D 11.0 S &gt;86 Τ 4.093 V &gt;5Τ 0.761 V 0.5 G 0.4 Ρ 4.8 Τ 0.6 V 42 Τ 0.7 V 7.0 Ρ 1.0 V

18.0 Ε 2.1 Τ 33.2 Ε 1.2 Τ 2.7 Ρ 3.2 V 3.4 Ε 3,1 Τ 1.4G 1.6 Ρ 1.2 D 2.3 Ρ 0.8 G 1.0 Ρ 1.1 S 0.9 V LOT 1.5 V

&gt;17 D 58.0 S &gt;11 Τ 0.8 V 20.0 Τ 1.1 V 1.1 Τ 2.3 V 128788.doc -127- 200846358 P419 D420 T421 P422 Q440 S441 V442 V443 S444 A445 N446 D447 V448 S449 D450 V451 1452 K453 R454 E455 S456 T457 L458 N459 M460 3.2 2.36.4 21.3 2.0 22 5.2 1.7 22.3 1.0 0.1 0.5 2.8 0.3 0.5 1.018.0 0.8 1·9 0.5 2.9 0.8 8·8 0.712.0 2Λ 23.5 36.0 3·3 3.7 0.7 1.08.0 1.5 2.5 1.7 2.3 2.5 1.7 2.5 44.5 1.012.0 1.0 0.2 2.5 73 2.3 0.8 0.5 6.0 0.4 1.5 0.5 3.1 1.231.0 1.632.0 24 45,0 〇·2 0.3 12.0 0.5 2.7 1.5 0.914.3 2.7

1.3 1.4 2.98.5 U 1.0 Ε 0.5 Ρ 0.9 0.1 1.7 1.9 1.0 0.5 20.4 D 2.0 Τ 0.2 0.4 0.1 0.6 1.8 3.0 1.9 1.3 0.9 0.9 0.6 0.3 &gt; 0.9 Τ 0.7 V 0.4 0.3 D 0.7 Τ 038 LIT L9V 3.0 Τ 0.4 V 6.0 Ε 1.2Τ 1.4 G 0,6 Ρ *With high-affinity peptide ligands (work U @, 择) or anti-tag antibodies (presenting „selection, the sequence of the isolated pure line isolated is used to measure 疋wt/m The :b rate 1 is normalized by dividing the function selection (10) mutation ratio by the selection of the wt/mutation ratio. The rate of occurrence is (F). In the case where no specific mutation is observed in the function selection sequence, only And the lower limit of the F value (indicated by greater than the symbol). In the case where the alanine scan requires a quadruple codon, the arginine substitution and the two other substitutions (5) and the value are called. The non-alanine substitution is shown in The bold number in the right parenthesis indicates a mutation with a more than 4-fold effect. Example 5: Binding specificity profile of the HtrAl-PDZ mutant The above structure and mutation analysis suggest a relationship between 1^1_1&gt;1)2 and HtrA3_pDZ The difference in specificity is that the adjacent peptide binding site is predominantly at position p 2 _ 3 , β 2: 128788.doc -128- 200846358 2_1 and the results of a limited number of sequence differences at α3-5. The effect of ligand specificity on the mutation in the case of HtrAl-PDZ was evaluated. Peptide library was presented using phage The binding specificity profiles of point mutations (I415Q, I418A and S449Q) and double mutants (I415Q/I418A) of HtrAl-PDZ were determined, wherein the residues of HtrA-PDZ were substituted by the corresponding residues of HtrA3-PDZ. Mutants were classified by a library of random heptapeptides at the C-terminus of the M13 p8 sheath protein. Only the double mutant HtrAl(I415Q/I418A)-PDZ was used to obtain a specific binding to the pure line. The mutant was specifically designed to mimic it. Binding properties of HtrA3-PDZ (see Table 1), and its specificity profile is substantially consistent with the specificity profile of HtrA3-PDZ. At position 0, HtrAl(I415Q/I418A)-PDZ exhibits proline and amine Preferably, the acid residue is selected, although other hydrophobic amino acids are also selected. Although the position 0 of HtrAl(I415Q/I418A)-PDZ is preferably slightly different from the preference of HtrA3-PDZ, the preference and the position of the remaining ligand positions are preferred. The preference observed for HtrA3-PDZ binding is indistinguishable Preferably, tryptophan is selected at position -1, there is no preference at position -2, position -3 shows preference for glycine and serine residues, and any other ligand position does not exhibit any preference. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts a sequence alignment of human Htf A family proteins in which the sequences are numbered to the left of each sequence. The elements of the secondary structure are indicated above the sequence. Residues that are identical across all four members are highlighted by a dashed box, and similar residues in all four members are framed by solid lines. Direct participation in ligand recognition is indicated by a solid star (site 0), a solid triangle (site-1 and site-3) or a solid circle (site - 128788.doc -129-200846358 2) below the sequence and A residue that is important for ligand specificity. The lower right panel of the figure summarizes the ligand binding specificity of HtrAl, HtrA2 and HtrA3 as determined by the steroidal and synthetic affinity assays, where X indicates no preference and Φ indicates that hydrophobic amino acids are preferred. Figure 2 depicts experimental calorimetry data for the combination of peptide Hl_c3 and HtrAl-PDZ. The top panel shows the raw heat data obtained during a single titration experiment in which 10 μ!&quot;peptide (284 μΜ) injections were titrated into the HtrAl-PDZ domain in PBS buffer with 1 mM sodium azide. (5·5 μΜ). The integrated thermal signal of the data in the above figure produces the binding curve shown in the figure below. The solid line represents the nonlinear least squares fit of the data after subtracting the dilution heat based on a single binding site model. Figure 3 depicts the structure of HtrAl-PDZ bound to a phage-derived peptide ligand as described in Example 3(a). Figure 3A shows the structural integrity of HtrAl-PDZ complexed with peptide Hl_3. The main chain Ν, Ca and C atoms are shown only by the lines. The HtrAl-PDZ structure is shown in light gray, while the peptide Hl_3 is shown in dark gray. Includes the selected peptide side chain heavy atom. The root mean square dispersion of the heavy chain of the main chain of residues 378-3 89, 411-463 and 468-475 is 0.52 ± 0.1 A. The distance or dihedral angle limits are not exceeded by 0.1 A or 1 °, respectively (Table 4). Figure 3B shows a banded view of HtrAl-PDZ bound to the Hl__3 peptide (peptides are shown in bar representation). The elements of the regular secondary structure and the peptide residues are labeled. Figure 4 depicts a dimer of the 11-8-8?026\1 structure as described in Example 3 (13). The PDZ domain is shaded dark gray and the pentapeptide is shown in light gray. The peptide is linked to the C-terminus of the PDZ domain via three glycine residues. HtrA3-PDZext structure Each asymmetric unit contains two molecules, and the two copies are well defined in terms of electron density except for the disorder between the chain βΐ and β2. 128788.doc -130- 200846358. Figure 4B shows a ribbon view of HtrA3-PDZext in which the peptides are shown in bar representation. Rules The components and remnants of the secondary structure are labeled. Figure 5 depicts the interaction of certain PDZ domains as described in Example 3 (C) with specific peptide ligands. Figure 5A shows HtrAl-PDZ in combination with peptide Hl_c3. Figure 5B shows HtrA2-PDZ in combination with peptide H2-cl (WTMFWVCOOH) (SEQ ID NO: 170). Figure 5C shows HtrA3-PDZ in combination with peptide H3_cl. Figures 5D-5F show DegP-PDZl (Figure 5D, pdb Accession No. 1KY9) without ligand binding, DegS-PDZ without ligand binding (Figure 5E, pdb Accession No. 1SOT) and binding to OmpC peptide DegS_PDZ (Figure 5F, pdb login number 1SOZ). In each of the figures, the structures are shown in the same relative orientation. The peptides shown in Figures A, B, C and F appear at the center of each figure. The side chain nitrogen atoms are shown in dark gray. The side chain of the peptide ligand is marked in gray with a three-letter amino acid code and the superordinated ligand position number, while the side chain of the PDZ domain is marked with black with a one-letter amino acid code and residue number. Figure 6 shows the heteronuclear NOE measurements of the HtrAl-PDZ complex as described in Example 3(c). Figure 7 depicts the localization of peptide Hl_c3 (Figure 7A) and peptide Hl_c2 (Figure 7B) on the HtrAl-PDZ structure (as shown by the surface) in a shotgun alanine scan as described in Example 4. Both peptides are shown as sticks. The PDZ domain residues that vary with the library are colored black (F &gt; 16), dark gray (16 &gt; F &gt; 5), medium gray (5 &gt; F &gt; 0.5), or light gray (F &lt; / = 0.5). The residues that do not change with the selection are added. The structural model of HtrAl-PDZ in combination with peptide HI-c2 was generated using a homologous modeling program Modeler (Accelrys, Inc.) using NMR of HtrAl-PDZ in combination with peptide Hl_c3. 128788.doc -131 -

Claims (1)

200846358 X. Patent Application Range: 1. An isolated polypeptide that specifically binds to the HtrAl PDZ domain, wherein the polypeptide comprises a sequence having tryptophan at position 2 relative to the C-terminus. 2. The isolated polypeptide of claim 1, wherein the polypeptide further comprises a small amino acid at position 0. 3. The isolated polypeptide of claim 2 wherein the amino acid at the position is selected from the group consisting of leucine and valine. 4. The isolated polypeptide of claim 1, wherein the polypeptide further comprises an amino acid comprising a bulky side chain at position _1. 5. The isolated polypeptide of claim 4, wherein the amino acid of the position is selected from the group consisting of tryptophan, isoleucine and phenylalanine. 6. The isolated polypeptide of claim 5, wherein the amino acid of the position is "tryptophan." The polypeptide of claim 1, wherein the polypeptide further comprises threonine or isokinetic acid at a position 3. The isolated polypeptide of claim 1, wherein the amino acid of position _4 is charged. 9. The isolated polypeptide of claim 8 wherein the amino acid of position _4 is selected from the group consisting of glutamic acid And an isolated amino acid of claim 9, wherein the amino acid at position -4 is selected from the group consisting of lysine and arginine. An isolated polypeptide, wherein the amino acid at the position is selected from the group consisting of leucine and valine; wherein the amino acid at position 4 is selected from the group consisting of tryptophan, isoleucine and phenylalanine; wherein the position -3 amino acid system selected 128788.doc 200846358 from threonine and isoleucine; and wherein the amino acid at position -4 is selected from the group consisting of glutamic acid, lysine, arginine and aspartame 12. The isolated polypeptide of claim 1, wherein the amino acid at the position is leucine; wherein the amino acid at position -1 is tryptophan; The amino acid at position -3 is selected from the group consisting of threonine and isoleucine; and wherein the amino acid at position -4 is selected from the group consisting of lysine and arginine. The isolated polypeptide, wherein the polypeptide comprises a sequence selected from the sequence of the HtrAl PDZ domain binding peptide set forth in Table 1. The isolated polypeptide of claim 1, wherein 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) o 15. The isolated polypeptide of claim 1, wherein the polypeptide comprises the sequence DSRIWWV (SEQ ID NO: 5). The isolated polypeptide of claim 1, wherein the polypeptide directly interacts with at least one specific HtrAl-PDZ domain residue. 17. The isolated polypeptide of claim 16, wherein C The terminal carboxylate group is coordinated to at least one HtrAl PDZ domain residue selected from the group consisting of Ile383, Ile385 and Gly384. 18. The polypeptide of claim 16, wherein the amino acid at position -1 is at least one selected from the group consisting of Tyr3 82. Dynamic interaction of the HtrA1 PDZ domain residues of Arg386 and Ile418. 19. The isolated polypeptide of claim 16, wherein The tryptophan acid at position -2 interacts with at least one HtrAl PDZ domain residue 128788.doc 200846358 base selected from Ala445, Met387 and Gln446. 20. The isolated polypeptide of claim 16, wherein the amino group at position -3 The acid interacts with at least one HtrAl PDZ domain residue selected from the group consisting of Ile415 and Arg386. The isolated polypeptide of claim 16, wherein the polypeptide interacts and/or coordinates with at least one HtrAl PDZ domain residue selected from the group consisting of Tyr382, Ile383, Gly384, Met387 and S389. An isolated polypeptide which specifically binds to a HtrAl PDZ domain, wherein the polypeptide comprises a sequence having a tryptophan acid at position -2 relative to the acidic amino acid. The isolated polypeptide of claim 22, wherein the polypeptide comprises a sequence of the formula XI-X2-W-X3-X4, wherein the XI is selected from the group consisting of lysine and leucine; wherein the X2 is selected from the group consisting of leucine , sulphate, arginine, alanine and valine; wherein X3 is selected from the group consisting of glycine, serine, phenylalanine and leucine; and wherein X4 is an acidic amino acid. 24. The isolated polypeptide of claim 23, wherein X3 is glycine and X4 is selected from the group consisting of glutamic acid and aspartic acid. An isolated polypeptide which specifically binds to the HtrAl PDZ domain and which comprises a C-terminus, N comprising an amino acid sequence of a peptide selected from the amino acid sequences listed in Tables 1, 2 and 3. End or internal amino acid sequence. 26. The polypeptide of claim 25, wherein the C-terminal amino acid sequence is selected from the group consisting of DIETWLL (SEQ ID NO: 3) ^ GWKTWIL (SEQ ID NO: 4) &gt; DSRIWWV (SEQ ID NO: 5) and WDKIWHV ( SEQ ID NO: 6) o 128788.doc 200846358 27. An isolated polypeptide comprising an amino acid sequence which competes with the polypeptide of any one of claims 1 to 26 for binding to the HtrAl PDZ domain. 28. An isolated polypeptide which binds to the same epitope on the HtrA1 PDZ domain as the polypeptide of any one of claims 1 to 26. 29. An isolated polypeptide comprising a HtrAl PDZ variant, wherein at least one HtrAl PDZ domain residue selected from the group consisting of the following amino acid substitutions: Ile383, Ile385, Gly384, Tyr382, Arg386, Ile418, Ala445 Met 387, Gln446, Ile415, Arg386, Ser389, Lys380, Lys381, G1411, Tyr413, Ile414, Val417, Thr421 and Pro422. The polypeptide of claim 29, wherein the Ile4 18 is substituted with another amino acid. 31. The isolated polypeptide of claim 29 or 30, wherein the other amino acid is alanine. An isolated amino acid comprising an amino acid sequence which competes with a polypeptide of any one of claims 29 to 31 for binding to a ligand of the HtrAl PDZ domain. 3. An isolated polypeptide which binds to the same epitope on the ligand of the HtrAl PDZ domain as the polypeptide of any one of claims 29 to 31. 34. A method of identifying a compound that modulates an HtrAl PDZ domain-ligand interaction, comprising: comprising a fragment comprising a HtrAl PDZ domain, a HtrAl PDZ domain, and/or a functional equivalent thereof, and at least one such as claim 1- A sample of the polypeptide of any one of 27 is contacted with a candidate compound, and the HtrAl PDZ domain-ligand interaction 1287δ8 is determined in the presence of the candidate compound. (1οο -4- 200846358 曰/, the compound is not Compared with the case of existence; the change in the amount of HtrAl PDZ domain-ligand interaction in the presence of the candidate substance, and the amount in the absence of the candidate compound indicates that the candidate is The chelate is a compound that modulates the HtrAl PDZ domain-ligand interaction. • The HtrAl PDZ domain-coordinator interaction regulator is designed to contain or modify a The tryptophan acid is located at a position _2 relative to the C-terminus or relative to the position of the acidic residue in the peptide _2 where the modulator can specifically bind to the HtrAl PDZ domain. 36. Method wherein the peptide 37. The method of claim 35, wherein the position lanthanide is selected from the group consisting of leucine and amidoxime, wherein position-1 is selected from the group consisting of tryptophan, isoleucine, and phenylalanine; 3 is selected from the group consisting of sulphate and isoleucine; and wherein the position 4 is selected from the group consisting of sulphate, aspartic acid, lysine, and arginine. 38. The method of claim 35, wherein the position is 〇 Is selected from the group consisting of leucine and valine; wherein position-1 is tryptophan; wherein position-3 is selected from the group consisting of threonine and isoleucine; and wherein position-4 is selected from the group consisting of glutamic acid and aspartame Acid. 39. Use of a HtrAl PDZ domain-ligand modulator for the manufacture of a medicament for the treatment of pathological conditions associated with abnormal regulation of HtrAl protein activity, wherein the modulator modulates the HtrAl PDZ domain The interaction with the polypeptide of any one of claims 1 to 26. 40. The use of claim 39, wherein the pathological condition is selected from the group consisting of malignant and benign tumors or cancer, non-leukemia and lymphoid malignancy Diseases, neurodegenerative disorders, inflammatory and immune disorders, and intraocular neovascular diseases. 128788.doc 200846358 41. 42. 43. The use of claim 39, wherein the steroidal T-Terminal inhibits the interaction between the HtrAl PDZ domain and the compound of the ceramide, as in the use of the ninth aspect, wherein The gentleman ^ Y $ modulator enhances the interaction between the HtrAl PDZ domain and the 5H polypeptide. For example, the HtrAl pDz domain ligand of any of the items 1 to 26 is used: 'It is used for manufacturing A drug that treats pathological conditions associated with abnormal regulation of the activity of this phantom protein. The term "the term" is selected from the group consisting of malignant and benign tumors or cancer, non-leukemia and lymphoid malignant diseases, neurodegenerative diseases, inflammatory and immune disorders, and intraocular neovascular diseases. 45. A drug according to the pathological condition associated with the abnormal regulation of the A1 protein shell activity of the polypeptide of any one of the claims α26. 46. The use of claim 45 wherein the pathological condition is selected from the group consisting of malignant and benign tumors or cancer, non-leukemia and lymphoid malignancies, neurodegenerative disorders, inflammatory and immune disorders, and intraocular neovascular disorders. 47. Use of an antagonist of the interaction of a HtrAl PDZ domain with one or more HtrA1 coffee domain ligands for the manufacture of a medicament for the treatment of pathological conditions associated with abnormal regulation of mrAi protein shell/tongue . 48. The use of claim 47, wherein the pathological condition is selected from the group consisting of malignant and benign tumors or cancer, (4) blood diseases and lymphoid malignancies, neurodegenerative disorders, inflammatory and immune disorders, and intraocular neovascular disorders. 49. An isolated polypeptide that specifically binds to the mrA3 pDZ domain, wherein 128788.doc 200846358 the polypeptide comprises a sequence having tryptophan at position -1 relative to the C-terminus. 50. The isolated polypeptide of claim 49, wherein the polypeptide has an overall hydrophobic character. The polypeptide according to claim 49, wherein the polypeptide further comprises an amino acid selected from the group consisting of lysine, isoleucine and alanine at a position 〇. 52. The isolated polypeptide of claim 49, wherein the polypeptide further comprises an amino acid selected from the group consisting of glycine and serine at position -3. 5. The isolated polypeptide of claim 49, wherein the amino acid at the position is selected from the group consisting of lysine, isoleucine, and alanine; and wherein the amino acid at position -3 is selected from the group consisting of glycine Acid and serine. 5. The isolated polypeptide of claim 49, wherein the amino acid at the position is guanamic acid and the amino acid at position -3 is selected from the group consisting of glycine and serine. 55. The isolated polypeptide of claim 49, wherein the polypeptide comprises a sequence selected from the group consisting of the HtrA3 PDZ domain binding peptides set forth in Tables 1 and 3. 56. The isolated polymorphism of claim 49, wherein the polymorphic sequence comprises: 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) 〇 128788.doc 200846358 5 7. The isolated polypeptide of claim 49, wherein the polypeptide comprises the sequence FGRWV (SEQ ID NO: 11). 58. The isolated polypeptide of claim 49, wherein the polypeptide interacts directly with at least one specific HtrA3-PDZ domain residue. 59. The isolated polypeptide of claim 58, wherein the C-terminal carboxylate group is coordinated to at least one HtrA3 PDZ domain residue selected from the group consisting of Phe356, Ile357, and Gly358. 60. The isolated polypeptide of claim 58, wherein the tryptophan acid at position 1 interacts with at least one HtrA3 PDZ domain residue selected from the group consisting of Glu390 and Ala392. 61. The isolated polypeptide of claim 58, wherein the amino acid at position-2 interacts with the HtrA3 PDZ domain residue Gln423. 62. The isolated polypeptide of claim 58, wherein the amino acid at position -3 interacts with the HtrA3PDZ domain residue Arg360. 63. The isolated polypeptide of claim 58, wherein the polypeptide interacts and/or coordinates with at least one HtrA3 PDZ domain residue selected from the group consisting of Phe356, Ile357, Gly358, Glu390, Ala392, Gln423, and ^Arg360. 64. An isolated polypeptide that specifically binds to a HtrA3 PDZ domain, wherein the polypeptide comprises a sequence having one acidic residue after one or more hydrophobic residues. 65. The isolated polypeptide of claim 64, wherein the polypeptide comprises the sequence WVL. 66. The isolated polypeptide of claim 64, wherein the polypeptide comprises the sequence GVVVDEWMLSLL (SEQ ID NO: 21), GVVVDEWVLSLL (SEQ ID NO: 22), ELLVDGYVLELL (SEQ ID NO: 23), or 128788.doc 200846358 GVVVNEWVLSLL (SEQ ID NO: 24). 67. The isolated polypeptide of claim 64, wherein the polypeptide comprises a sequence selected from the HtrA3 PDZ domain binding sequences set forth in Table 2. 68. An isolated polypeptide that specifically binds to the HtrA3 PDZ domain and comprises a C-terminus, N comprising an amino acid sequence comprising a peptide selected from the amino acid sequences set forth in Tables 1, 2 and 3. End or internal amino acid sequence. 69. The polypeptide of claim 68, wherein the C-terminal amino acid sequence is selected from the group consisting of PGRWV (SEQ ID NO: 7), SGKGIWV (SEQ ID NO: 8), GFWV (SEQ ID NO: 9) &gt; IFDGRWI ( SEQ ID NO: 10), FGRWV (SEQ ID NO: 11), RSWWV (SEQ ID NO: 12), FGRWI (SEQ ID NO: 13) &gt; 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) 70. An isolated polypeptide comprising an amino acid sequence which competes with the polypeptide of any one of claims 49 to 69 for binding to the HtrA3 PDZ domain. 7. An isolated polypeptide which binds to the same epitope on the HtrA3 PDZ domain as the polypeptide of any one of claims 49 to 69. 72. An isolated polypeptide comprising a HtrA3 PDZ variant sequence, at least one of which is selected from the group consisting of βΝ:β1-1, βΝ:β1-2 and βΐ-ΐ, Glu390, Ala392, Gln423 and Arg360 HtrA3 PDZ domain residues Substituted by another amino acid. 73. The isolated polypeptide of claim 72, wherein Glu390 and/or Ala392 is substituted with an amino acid of another 128788.doc -9 - 200846358. 74. The isolated polypeptide of claim 72 or 73, wherein the other amino acid is alanine. 75. An isolated polypeptide comprising an amino acid sequence which competes with a polypeptide of any one of claims 72 to 74 for binding to a ligand of the HtrA3 PDZ domain. 76. An isolated polypeptide which binds to the same epitope on a ligand of the Htr A3 PDZ domain as the polypeptide of any one of claims 72 to 74. ® 77. A method for identifying a compound that modulates an HtrA3 PDZ domain-ligand interaction, comprising a fragment comprising a 11忖8-3?02 domain, a 11忖8-3?02 domain, and/or its function And a sample of at least one polypeptide according to any one of claims 49 to 69 in contact with a candidate compound, and determining the amount of HtrA3 PDZ domain-ligand interaction in the presence of the candidate compound, and the candidate compound In the absence of the case; the change in the amount of HtrA3 PDZ domain-ligand interaction in the presence of the candidate compound compared to the amount in the absence of the candidate compound indicates that the candidate compound is A compound that modulates the HtrA3 PDZ domain-ligand interaction. 78. A method of rationally designing a modulator of HtrA3 PDZ domain-ligand interaction, comprising designing the modulator to comprise or mimic a tryptophan at a position relative to the C-terminus-1 or relative to the peptide The function of position -2 of the amino acid, wherein the modulator specifically binds to the Htr A3 PDZ domain. 79. The method of claim 78, wherein position 0 is selected from the group consisting of lysine, isoleucine, and alanine; wherein position-1 is tryptophan; and wherein position-3 is selected 128788.doc -10- 200846358 From glycine and serine. 80. The method of claim 78, wherein position 0 is proline; wherein position -1 is tryptophan; and wherein position -3 is selected from the group consisting of glycine and serine. The method of claim 78, wherein the modulator comprises an amino acid sequence tryptophan- lysine-leucine. 82. Use of a HtrA3 PDZ domain-ligand modulator for the manufacture of a medicament for the treatment of a pathological condition associated with abnormal regulation of HtrA3 protein activity, wherein the modulator modulates the HtrA3 PDZ domain and The interaction between the polypeptides of any of the items 49-69 is claimed. 83. The use of claim 82, wherein the pathological condition is selected from the group consisting of a malignant and benign tumor or cancer, a non-leukemia and a lymphoid malignancy, and a placental dysfunction. 84. The use of claim 82, wherein the modulator inhibits the interaction between the HtrA3 PDZ domain and the polypeptide. 85. The use of claim 82, wherein the modulator enhances the interaction between the Hti*A3 PDZ domain and the polypeptide. The use of the HtrA3 PDZ domain ligand according to any one of claims 49 to 69 for the manufacture of a medicament for the treatment of a pathological condition associated with abnormal regulation of HtrA3 protein activity. 87. The use of claim 82, wherein the pathological condition is selected from the group consisting of malignant and benign tumors or cancer, non-leukemia and lymphoid malignancies, and placental dysfunction. 8. The use of an agonist for interacting with a Hti:A3 PDZ domain of any one of claims 49 to 69 for use in the manufacture of a protein for treatment with HtrA3 128788.doc -11 - 200846358 A drug that modulates pathological conditions associated with abnormalities. 89. The use of claim 88, wherein the pathological condition is selected from the group consisting of malignant and benign tumors or cancer, non-leukemia and lymphoid malignancies, and placental dysfunction. 90. Use of an antagonist of the interaction of a HtrA3 PDZ domain with one or more HtrA3 PDZ domain ligands for the manufacture of a medicament for the treatment of pathological conditions associated with the regulation of Α3 protein activity. 91. The use of claim 88, wherein the pathological condition is selected from the group consisting of malignant and benign tumors or cancer, non-leukemia and lymphoid malignancies, and placental dysfunction. 92. The polypeptide of any one of claims 1 to 33 and 49 to 76, or a complement thereof. 93. A vector comprising the polynucleotide of claim 92. 94. A host cell comprising A vector according to claim 93. A method for producing a polypeptide comprising the host cell of claim 94 cultured under conditions in which the polynucleotide is expressed. 96. A mammal, which exhibits a polynucleotide of the claim %. 97. A non-human mammal of the gene of claim 96, wherein at least one of the original HtrAl or HtrA3 genes has not been activated. With request item 1 The specific binding of the polypeptide of any of 26 and 49 to 69, or a fragment thereof. 99. An antibody which specifically binds to the polypeptide of any one of claims 27 to 33 and 70 to 76, or a fragment thereof. 128788.doc -12-200846358 100, a kit comprising at least one polypeptide according to any one of claims 1 to 33 or 49 to 76 and a selective hybridization with the polynucleotide of claim 92 Compound. 128788.doc -13-