US2003186863A1PendingUtilityA1
Nck SH3 binding peptides
Priority: Jul 22, 1994Filed: May 31, 2002Published: Oct 2, 2003
Est. expiryJul 22, 2014(expired)· nominal 20-yr term from priority
Inventors:Andrew SparksBrian K. KayJudith M. ThornLawrence A. QuilliamChanning J. DerDana M. FowlkesJames E Rider
G01N 33/5041C07K 14/435A61K 38/00C07K 7/08G01N 33/68G01N 33/5011C07K 7/06G01N 33/5008A61K 47/62C07K 14/001C07K 2319/00C40B 30/04G01N 33/573A61P 35/00G01N 33/6845
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Claims
Abstract
Peptides having general and specific binding affinities for the Src homology region 3 (SH3) domains of proteins are disclosed in the present invention. In particular, SH3 binding peptides have been isolated from phage-displayed random peptide libraries which had been screened for isolates that bind to bacterial fusion proteins comprising SH3 and glutathione S-transferase (GST). Preferred peptides are disclosed which comprise a core 7-mer sequence (preferably, a consensus motif) and two or more, preferably at least six, additional amino acid residues flanking the core sequence, for a total length of 9, preferably at least 13, amino acid residues and no more than about 45 amino acid residues. Such peptides manifest preferential binding affinities for certain SH3 domains. The preferred peptides exhibit specific binding affinities for the Src-family of proteins. In vitro and in vivo results are presented which demonstrate the biochemical activity of such peptides.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A peptide having at least nine and up to forty-five amino acid residues, including an amino acid sequence of the formula, R-2-L-P-5-6-P-8-9 (SEQ ID NO:10), positioned anywhere along the peptide, in which each number represents an amino acid residue, such that 2 represents any amino acid residue except cysteine, 5 and 6 each represents a hydrophobic amino acid residue, 8 represents any amino acid residue except cysteine, and 9 represents a hydrophilic amino acid residue except cysteine, each letter being the standard one-letter symbol for the corresponding amino acid, said peptide exhibiting a binding affinity for the SH3 domain of Src, provided that said peptide is not R-P-L-P-P-L-P-T-S (SEQ ID NO:11).
2 . The peptide of claim 1 in which 2 is a P, R, A, L, Q, E or S.
3 . The peptide of claim 1 in which 5 is a P, M, I or L.
4 . The peptide of claim 1 in which 6 is a P, L, I or V.
5 . The peptide of claim 1 in which 8 is a T, R, P, I, N, E, V, S, A, G or L.
6 . The peptide of claim 1 in which 9 is a T, R, S, H or D.
7 . The peptide of claim 1 which further comprises a C-terminal-flanking amino acid sequence of the formula 10, 10-11, 10-11-12, 10-11-12-13 (SEQ ID NO:12) or 10-11-12-13-14 (SEQ ID NO:13), in which each number represents any amino acid residue except cysteine, such that 10 is bound to 9 by a peptide bond.
8 . The peptide of claim 7 in which 10 is T, R, L, S, D, P, A or N.
9 . The peptide of claim 7 in which 11 is R, P, A, Q, S or T.
10 . The peptide of claim 7 in which 12 is P, S, R or T.
11 . The peptide of claim 7 in which 13 is P, S, R, F, H or T.
12 . The peptide of claim 7 in which 14 is S, R, G or T.
13 . The peptide of claim 1 which further comprises an N-terminal-flanking amino acid sequence of the formula 1′, 2′-1′, 3′-2′-1′ or 4′-3′-2′-1′ (SEQ ID NO:14) in which each number represents any amino acid residue except cysteine, such that 1′ is bound to R by a peptide bond.
14 . The peptide of claim 13 in which 1′ is T. P, S, N, F, W, K, H, Q or G.
15 . The peptide of claim 13 in which 2′ is S, T, G, P, R, Q, L, A or H.
16 . The peptide of claim 13 in which 3′ is R, S, P, G, A, V, Y or L.
17 . The peptide of claim 13 in which 4′ is R, S, V, T, G, L or F.
18 . A peptide having at least thirteen and up to forty-five amino acid residues, including an amino acid sequence of the formula, 3′-2′-1′-R-2-L-P-5-6-P-8-9-10 (SEQ ID NO:15), positioned anywhere along the peptide, in which each number represents an amino acid residue, such that 3′, 2′, 1′, 2, 8, and 10 each represents any amino acid residue except cysteine, 5 and 6 each represents a hydrophobic amino acid residue, and 9 represents a hydrophilic amino acid residue except cysteine, each letter being the standard one-letter symbol for the corresponding amino acid, said peptide exhibiting a binding affinity for the SH3 domain of Src.
19 . The peptide of claim 18 in which 5 is a P or M.
20 . The peptide of claim 18 in which 1′ is T, P, S or N.
21 . The peptide of claim 18 in which 2′ is S or T.
22 . The peptide of claim 18 in which 3′ is R or S.
23 . The peptide of claim 18 in which 10 is T or R.
24 . The peptide of claim 1 the binding affinity of which is at least three-fold greater than that exhibited by the peptide RPLPPLP for the SH3 domain of Src.
25 . The peptide of claim 18 the binding affinity of which is at least three-fold greater than that exhibited by the peptide RPLPPLP (SEQ ID NO:9) for the SH3 domain of Src.
26 . The peptide of claim 1 the binding affinity of which is at least four-fold greater than that exhibited by the peptide RPLPPLP (SEQ ID NO:9) for the SH3 domain of Src.
27 . The peptide of claim 18 the binding affinity of which is at least four-fold greater than that exhibited by the peptide RPLPPLP (SEQ ID NO:9) for the SH3 domain of Src.
28 . The peptide of claim 1 which further exhibits a general binding affinity for the SH3 domain of Abl, Grb2, PLC- , PLC- , Ras GAP, Nck, p85 PI-3′Kinase, and proteins related thereto.
29 . The peptide of claim 1 which exhibits a selective binding affinity for the SH3 domain of Src and Src-related proteins.
30 . The peptide of claim 18 which further exhibits a general binding affinity for the SH3 domain of Abl, Grb2, PLC- , PLC- , Ras GAP, Nck, p85 PI-3′Kinase, and proteins related thereto.
31 . The peptide of claim 18 which exhibits a selective binding affinity for the SH3 domain of Src and Src-related proteins.
32 . A peptide having the amino acid sequence RSTPRPLPMLPTTR (SEQ ID NO:62).
33 . A peptide having the amino acid sequence RSTPRPLPPLPTTR (SEQ ID NO:67).
34 . A peptide having the amino acid sequence GILAPPVPPRNTR (SEQ ID NO:63).
35 . A peptide having the amino acid sequence VLKRPLPIPPVTR (SEQ ID NO:64).
36 . A peptide having the amino acid sequence GPHRRLPPTPATR (SEQ ID NO:65).
37 . A peptide having the amino acid sequence ANPSPATRPLPTR (SEQ ID NO:66).
38 . A peptide having an amino acid sequence selected from the group consisting of RSTRPLPILPRTT, STPRPLPMLPTTR, STNRPLPMIPTTR, RSTRPLPSLPITT, STSRPLPSLPTTR, RSTRSLPPLPPTT, RSTRQLPIPPTTT, STPRPLPLIPTTP, RSTRPLPPTPLTT, and RSTRPQPPPPITT (SEQ ID NOS:85-94).
39 . A peptide having the amino acid sequence selected from the group consisting of VLKRPLPIPPVTR (SEQ ID NO:64), YSTRPVPPITRPS (SEQ ID NO:76), SHKSRLPPLPTRP (SEQ ID NO:77), GPHRRLPPTPATR (SEQ ID NO:65), PATRPLPTRPSRT (SEQ ID NO:81), and SGGILAPPVPPRN (SEQ ID NO:84).
40 . A peptide having the amino acid sequence selected from the group consisting of PPNSPLPPLPTHL (SEQ ID NO:72), TGRGPLPPLPNDS (SEQ ID NO:74), YRFRALPSPPSAS, LAQRQLPPTPGRD ALQRRLPRTPPPA (SEQ ID NOS:78-80), YSTRPLPSRPSRT, and XPGRILLLPSEPR (SEQ ID NOS:82-83).
41 . A construct comprising a nucleic acid encoding a peptide of claim 1 or its complement.
42 . The construct of claim 41 which is a DNA polynucleotide.
43 . The construct of claim 41 which is a RNA polynucleotide.
44 . A construct comprising a nucleic acid encoding a peptide of claim 18 or its complement.
45 . The construct of claim 44 which is a DNA polynucleotide.
46 . The construct of claim 44 which is a RNA polynucleotide.
47 . The construct of claim 41 which is a transforming vector.
48 . The construct of claim 44 which is a transforming vector.
49 . A host cell transformed with the vector of claim 47 .
50 . A host cell transformed with the vector of claim 48 .
51 . A conjugate comprising a peptide of claim 1 and a second molecule.
52 . The conjugate of claim 51 in which said second molecule is selected from the group consisting of an amino acid, a peptide, a protein, a nucleic acid, a nucleoside, a glycosidic residue, a label, a drug or a small molecule.
53 . A diagnostic kit for the detection of SH3 domains comprising an SH3 domain-binding peptide and a detectable label conjugated to said peptide directly, indirectly or by complexation, said peptide comprising: (i) a core sequence motif of the formula RXLPφφP (SEQ ID NO:71), wherein X represents any amino acid except cysteine and φ represents a hydrophobic amino acid residue, each letter representing the standard one-letter designation for the corresponding amino acid residue; and (ii) two or more additional amino acid residues flanking said core sequence at its C-terminal end, N-terminal end or both.
54 . A drug delivery system comprising an SH3 domain-binding peptide and a drug conjugated to said peptide directly, indirectly or by complexation, said peptide comprising: (i) a core sequence motif of the formula RXLPφφP (SEQ ID NO:71), wherein X represents any amino acid except cysteine and φ represents a hydrophobic amino acid residue, each letter representing the standard one-letter designation for the corresponding amino acid residue; and (ii) two or more additional amino acid residues flanking said core sequence at its C-terminal end, N-terminal end or both.
55 . The drug delivery system of claim 54 which may be administered parenterally, orally, enterally, topically or by inhalation.
56 . The drug delivery system of claim 54 which may be administered intranasally, opthalmically or intravaginally.
57 . The drug delivery system of claim 54 which is in the form of a solid, gel, liquid or aerosol.
58 . A method of modulating the activity of Src or Src-related proteins comprising administering a composition comprising an effective amount of a peptide of claim 1 and a carrier.
59 . The method of claim 58 which inhibits the activity of Src or Src-related proteins.
60 . The method of claim 58 which activates Src or Src-related proteins.
61 . A method of identifying a peptide having a region that binds to an SH3 domain comprising:
(a) providing an immobilized target protein comprising an SH3 domain; (b) incubating said immobilized target protein with an aliquot taken from a random peptide library; (c) washing unbound peptide from said immobilized target protein; (d) recovering the peptide bound to said immobilized target protein; and (e) determining the primary sequence of the SH3 domain-binding peptide.
62 . The method of claim 61 in which said library is a displayed random peptide library.
63 . The method of claim 62 in which said library is a phage-displayed random peptide library.
64 . The method of claim 62 in which said library is a phagemid-displayed random peptide library.
65 . The method of claim 61 in which step (c) includes washing unbound phage from said immobilized target protein; step (d) includes recovering the phage bound to said immobilized target protein; and step (e) includes determining the relevant nucleotide sequence of said binding phage nucleic acid, from which the primary sequence corresponding to the SH3 domain-binding peptide is deduced.
66 . A method of identifying a peptide having a region that binds to an SH3 domain comprising:
(a) providing an immobilized target protein comprising an SH3 domain; (b) incubating said immobilized target protein with an aliquot taken from a phage-displayed random peptide library, which library includes peptides having a random sequence of 8 amino acid residues; (c) washing unbound phage from said immobilized target protein; (d) recovering the phage bound to said immobilized target protein; and (e) determining the relevant nucleotide sequence of said binding phage nucleic acid and deducing the primary sequence corresponding to the SH3 domain-binding peptide.
67 . The method of claim 66 which further comprises amplifying the titer of the recovered phage.
68 . The method of claim 66 which further comprises repeating the steps of incubation, washing and recovery to provide SH3 domain-binding peptide-enriched phage.
69 . A pharmaceutical composition comprising an SH3 domain-binding peptide and a pharmaceutically acceptable carrier, said peptide comprising: (i) a 9-mer sequence motif of the formula RXLPφφPXψ (SEQ ID NO:10), wherein X represents any amino acid except cysteine, φ represents a hydrophobic amino acid residue, and wherein ψ is a hydrophilic amino acid residue except cysteine, each letter representing the standard one-letter designation for the corresponding amino acid residue; and, optionally, (ii) additional amino acid residues flanking said 9-mer sequence at its C-terminal end, N-terminal end or both, up to a total of 45 amino acid residues, including said 9-mer sequence.
70 . The composition of claim 69 in which at least one additional amino acid flanks said 9-mer sequence.
71 . The composition of claim 69 in which at least two additional amino acids flank said 9-mer sequence.
72 . The composition of claim 69 in which at least three additional amino acids flank said 9-mer sequence.
73 . A method of disrupting protein tyrosine kinase-mediated signal transduction pathways comprising administering an effective amount of a peptide of claim 1 .
74 . A method of regulating the processing, trafficking or translation of RNA by administering an effective amount of a peptide of claim 1 .
75 . A purified peptide that binds to the SH3 domain of Cortactin, said peptide comprising the amino acid sequence ZPPφPxKPxW (SEQ ID NO:113), where Z represents K or R; φ represents a hydrophobic amino acid; and x represents any amino acid.
76 . A purified peptide that binds to the middle SH3 domain of Nck, said peptide comprising the amino acid sequence φxxxxPxPPφRZxSL (SEQ ID NO:127), where Z represents S or T; φ represents a hydrophobic amino acid; and x represents any amino acid.
77 . A purified peptide that binds to the SH3 domain of Abl, said peptide comprising the amino acid sequence PPxWxPPPφP (SEQ ID NO:141), where φ represents a hydrophobic amino acid; and x represents any amino acid.
78 . A purified peptide that binds to the SH3 domain of Src, said peptide comprising the amino acid sequence LXXRPLPXψP (SEQ ID NO:165), where ψ represents an aliphatic amino acid; and X represents any amino acid.
79 . A purified peptide that binds to the SH3 domain of Cortactin, said peptide comprising the amino acid sequence +PPψPXKPXWL (SEQ ID NO:166), where +represents a basic amino acid; ψ represents an aliphatic amino acid; and X represents any amino acid.
80 . A purified peptide that binds to the SH3 domain of Abl, said peptide comprising the amino acid sequence PPXθXPPPψP (SEQ ID NO:173) , where θ represents an aromatic amino acid; ψ represents an aliphatic amino acid; and X represents any amino acid.
81 . A purified peptide that binds to the SH3 domain of PLC said peptide comprising the amino acid sequence PPVPPRPXXTL (SEQ ID NO:175), where X represents any amino acid.
82 . A purified peptide that binds to the SH3 domain of p53bp2, said peptide comprising the amino acid sequence RPXψPψR+SXP (SEQ ID NO:196), where + represents a basic amino acid; ψ represents an aliphatic amino acid; and X represents any amino acid.
83 . A purified peptide that binds to the N terminal SH3 domain of Crk, said peptide comprising the amino acid sequence ψPψPψLPψK (SEQ ID NO:210), where ψ represents an aliphatic amino acid; and X represents any amino acid.
84 . A purified peptide that binds to the SH3 domain of Yes, said peptide comprising the amino acid sequence ψXXRPLPXLP (SEQ ID NO:222), where ψ represents an aliphatic amino acid; and X represents any amino acid.
85 . A purified peptide that binds to the N terminal SH3 domain of Grb2, said peptide comprising an amino acid sequence selected from the group consisting of: +θDXPLPXLP (SEQ ID NO:223), YθX+PLPXLP (SEQ ID NO:238), and 6DPLPXLP (SEQ ID NO:243), where θ represent an aromatic amino acid; +represents a basic amino acid; ψ represents an aliphatic amino acid; and X represents any amino acid.
86 . A purified peptide that binds to the SH3 domain of Cortactin, said peptide comprising an amino acid sequence selected from the group consisting of:
LTPQSKPPLPPKPSAV;
(a portion of SEQ ID NO:112)
SSHNSRPPLPEKPSWL;
(a portion of SEQ ID NO:111)
PVKPPLPAKPWWLPPL;
(SEQ ID NO:167)
TERPPLPQRPDWLSYS;
(a portion of SEQ ID NO:109)
LGEFSKPPIPQKPTWM;
(a portion of SEQ ID NO:108)
YPQFRPPVPPKPSLMQ;
(SEQ ID NO:168)
VTRPPLPPKPGHMADF;
(SEQ ID NO:169)
VSLGLKPPVPPKPMQL;
(SEQ ID NO:170)
LLGPPVPPKPQTLFSF;
(a portion of SEQ ID NO:107)
YKPEVPARPIWLSEL;
(SEQ ID NO:171)
GAGAARPLVPKKPLFL; and
(SEQ ID NO:172)
SREPDWLCPNCPLLLRSDSR.
(SEQ ID NO:110)
87 . A purified peptide that binds to the middle SH3 domain of Nck, said peptide comprising an amino acid sequence selected from the group consisting of:
SSLGVGWKPLPPMRTASLSR;
(SEQ ID NO:114)
SSVGFADRPRPPLRVESLSR;
(SEQ ID NO:115)
SSAGILRPPEKPXRSFSLSR;
(SEQ ID NO:116)
SSPYTGDVPIPPLRGASLSR;
(SEQ ID NO:117)
SSLMGSWPPVPPLRSDSLSR;
(SEQ ID NO:118)
SSIGEDTPPSPPTRRASLSR;
(SEQ ID NO:119)
SRSLSEVSPKPPIRSVSLSR;
(SEQ ID NO:120)
SSVSEGYSPPLPPRSTSLSR;
(SEQ ID NO:121)
SSSFTLAAPTPPTRSLSLSR;
(SEQ ID NO:122)
SSPPYELPPRPPNRTVSLSR;
(SEQ ID NO:123)
SRVVDGLAPPPPVRLSSLSR;
(SEQ ID NO:124)
SSLGYSGAPVPPHRxSSLSR; and
(SEQ ID NO:125)
SSISDYSRPPPPVRTLSLSR.
(SEQ ID NO:126)
88 . A purified peptide that binds to the SH3 domain of Abl, said peptide comprising an amino acid sequence selected from the group consisting of:
PPWWAPPPIPNSPQVL;
(SEQ ID NO:174);
PPKFSPPPPPYWQLHA;
(a portion of SEQ ID NO:132)
PPHWAPPAPPAMSPPI;
(a portion of SEQ ID NO:130)
PPTWTPPKPPGWGVVF;
(a portion of SEQ ID NO:137)
PPSFAPPAAPPRHSFG;
(a portion of SEQ ID NO:133)
PTYPPPPPPPDTAKGA;
(a portion of SEQ ID NO:135)
GPRWSPPPVPLPTSLD;
(a portion of SEQ ID NO:128)
APTWSPPALPNVAKYK;
(a portion of SEQ ID NO:138)
PPDYAAPAIPSSLWVD;
(a portion of SEQ ID NO:129)
IKGPRFPVPPVPLNGV;
(a portion of SEQ ID NO:139)
PPAWSPPHRPVAFGST;
(a portion of SEQ ID NO:140)
APKKPAPPVPMMAHVM;
(a portion of SEQ ID NO:134)
SSDRCWECPPWPAGGQRGSR;
(SEQ ID NO:131)
and
SSPPXXXPPPIPNSPQVLSR.
(SEQ ID NO:136)
89 . A purified peptide that binds to the SH3 domain of PLC said peptide comprising an amino acid sequence selected from the group consisting of:
MPPPVPPRPPGTLQVA;
(SEQ ID NO:176)
LSYSPPPVPPRPDSTL;
(SEQ ID NO:177)
VLAPPVPPRPGNTFFT;
(SEQ ID NO:178)
YRPPVAPRPPSSLSVD;
(SEQ ID NO:179)
LQCPDCPRVPPRPIPI;
(SEQ ID NO:180)
VPPLVAPRPPSTLNSL;
(a portion of SEQ ID NO:143)
LTPPPFPKRPRWTLPE;
(SEQ ID NO:181)
YWPHRPPLAPPQTTLG;
(SEQ ID NO:182)
SSMKVHNFPLPPLPSYETSR;
(SEQ ID NO:142)
SSLYWQHGPDPPVGAPQLSR;
(SEQ ID NO:144)
and
SSHPLNSWPGGPFRHNLSSR.
(SEQ ID NO:145)
90 . A purified peptide that binds to the SH3 domain of Src, said peptide comprising an amino acid sequence selected from the group consisting of:
LASRPLPLLPNSAPGQ;
(a portion of SEQ ID NO:155)
LTGRPLPALPPPFSDF;
(a portion of SEQ ID NO:152)
PAYRPLPRLPDLSVIY;
(a portion of SEQ ID NO:150)
RALRVRPLPPVPGTSL;
(a portion of SEQ ID NO:146)
DAPGSLPFRPLPPVPT;
(a portion of SEQ ID NO:148)
LKWRALPPLPETDTPY;
(a portion of SEQ ID NO:157)
ISQRALPPLPLMSDPA;
(a portion of SEQ ID NO:149)
LTSRPLPDIPVRPSKS;
(a portion of SEQ ID NO:156)
NTNRPLPPTPDGLDVR;
(a portion of SEQ ID NO:158)
MKDRVLPPIPTVESAV;
(a portion of SEQ ID NO:153)
LQSRPLPLPPQSSYPI;
(a portion of SEQ ID NO:159)
FINRRLPALPPDNSLL;
(a portion of SEQ ID NO:151)
FRALPLPPTPDNPFAG; and
(a portion of SEQ ID NO:147)
LYSAIAPDPPPRNSSS.
(a portion of SEQ ID NO:154)
91 . A purified peptide that binds to the SH3 domain of p53bp2, said peptide comprising an amino acid sequence selected from the group consisting of:
YDASSAPQRPPLPVRKSRP;
(SEQ ID NO:183)
EYVNASPERPPIPGRKSRP;
(SEQ ID NO:184)
WNGIAIPGRPEIPPRASRP;
(SEQ ID NO:185)
SMIFIYPERPSPPPRFSRP;
(SEQ ID NO:186)
GVEEWNPERPQIPLRLSRP;
(SEQ ID NO:187)
WVVDSRPDIPLRRSLP;
(SEQ ID NO:188)
VVPLGRPEIPLRKSLP;
(SEQ ID NO:189)
GGTVGRPPIPERKSVD;
(SEQ ID NO:190)
YSHAGRPEVPPRQSKP;
(SEQ ID NO:191)
FSAAARPDIPSRASTP;
(SEQ ID NO:192)
LYIPKRPEVPPRRHEA;
(SEQ ID NO:193)
NNISARPPLPSRQNPP; and
(SEQ ID NO:194)
MAGTPRPAVPQRMNPP.
(SEQ ID NO:195)
92 . A purified peptide that binds to the N terminal SH3 domain of Crk, said peptide comprising an amino acid sequence selected from the group consisting of:
GQPAGDPDPPPLPAKF;
(SEQ ID NO:197)
FEQTGVPLLPPKSFKY;
(SEQ ID NO:198)
IFGDPPPPIPMKGRSL;
(SEQ ID NO:199)
SNQGSIPVLPIKRVQY;
(SEQ ID NO:200)
NYVNALPPGPPLPAKN;
(SEQ ID NO:201)
SSDPERPVLPPKLWSV;
(SEQ ID NO:202)
HFGPSKPPLPIKTRIT;
(SEQ ID NO:203)
DWKVPEPPVPKLPLKQ;
(SEQ ID NO:204)
ATSEGLPILPSKVGSY;
(SEQ ID NO:205)
NANVSAPRAPAFPVKT;
(SEQ ID NO:206)
EMVLGPPVPPKRGTVV;
(SEQ ID NO:207)
AGSRHPPTLPPKESGG; and
(SEQ ID NO:208)
SVAADPPRLPAKSRPQ.
(SEQ ID NO:209)
93 . A purified peptide that binds to the SH3 domain of Yes, said peptide comprising an amino acid sequence selected from the group consisting of:
ITMRPLPALPGHGQIH;
(SEQ ID NO:211)
LPRRPLPDLPMAAGKG;
(SEQ ID NO:212)
LGSRPLPPTPRQWPEV;
(SEQ ID NO:213)
STIRPLPAIPRDTLLT;
(SEQ ID NO:214)
RSGRPLPPIPEVGHNV;
(SEQ ID NO:215)
IGSRPLPWTPDDLGSA;
(SEQ ID NO:216)
LAQRELPGLPAGAGVS;
(SEQ ID NO:217)
IPGRALPELPPQRALP;
(SEQ ID NO:218)
FVGRELPPTPRTVIPW;
(SEQ ID NO:219)
DPRSALPALPLTPLQT; and
(SEQ ID NO:220)
SPHDVLPALPDSHSKS.
(SEQ ID NO:221)
94 . A purified peptide that binds to the N terminal SH3 domain of Grb2, said peptide comprising an amino acid sequence selected from the group consisting of:
KWDSLLPALPPAFTVE;
(SEQ ID NO:224)
RWDQVLPELPTSKGQI;
(SEQ ID NO:225)
RFDFPLPTHPNLQKAH;
(SEQ ID NO:226)
RLDSPLPALPPTVMQN;
(SEQ ID NO:227)
RWGAPLPPLPEYSWST;
(SEQ ID NO:228)
YWDMPLPRLPGEEPSL;
(SEQ ID NO:229)
RFDYNLPDVPLSLGTA;
(SEQ ID NO:230)
TKKPNAPLPPLPAYMG;
(SEQ ID NO:231)
KWDLDLPPEPMSLGNY;
(SEQ ID NO:232)
YYQRPLPPLPLSHFES;
(SEQ ID NO:234)
YYRKPLPNLPRGQTDD;
(SEQ ID NO:235)
YFDKPLPESPGALMSL;
(SEQ ID NO:236)
YFSRALPGLPERQEAH;
(SEQ ID NO:237)
SLWDPLPPIPQSKTSV;
(SEQ ID NO:239)
SYYDPLPKLPDPGDLG;
(SEQ ID NO:240)
KLYYPLPPVPFKDTKH; and
(SEQ ID NO:241)
DPYDALPETPSMKASQ.
(SEQ ID NO:242)
95 . A purified peptide having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 250-252, 254, 256-259, 261, 262, 264-266, 269-272, 275, 280, 281, 286-289, 291, 294, and 295.
96 . A purified peptide having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 296-453.
97 . A method of identifying an inhibitor of the binding between a first molecule comprising an SH3 domain and a second molecule that binds to the SH3 domain comprising incubating one or more compounds from which it is desired to select such an inhibitor with the first molecule and the second molecule under conditions conducive to binding and detecting the one or more compounds that inhibit binding of the first molecule to the second molecule
98 . The method of claim 97 where the second molecule is obtained by:
(i) screening a peptide library with the SH3 domain to obtain peptides that bind the SH3 domain;
(ii) determining a consensus sequence for the peptides obtained in step (i);
(iii) producing a peptide comprising the consensus sequence;
wherein the second molecule comprises the peptide comprising the consensus sequence.
99 . The method of claim 97 where the second molecule is obtained by:
(i) screening a peptide library with the SH3 domain to obtain peptides that bind the SH3 domain;
(ii) determining a consensus sequence for the peptides obtained in step (i);
(iii) searching a database to identify amino acid sequences that resemble the consensus sequence of step (ii);
(iv) producing a peptide comprising an amino acid sequence identified in step (iii);
wherein the second molecule comprises the peptide comprising an amino acid sequence identified in step (iii).
100 . The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of Cortactin, said peptide comprising the amino acid sequence ZPPφPxKPxW (SEQ ID NO:113), where Z represents K or R; + represents a hydrophobic amino acid; and x represents any amino acid.
101 . The method of claim 97 where the second molecule is a peptide that binds to the middle SH3 domain of Nck, said peptide comprising the amino acid sequence φxxxxxPxPPφRZxSL (SEQ ID NO:127), where Z represents S or T; φ represents a hydrophobic amino acid; and x represents any amino acid.
102 . The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of Abl, said peptide comprising the amino acid sequence PPxWxPPPφP (SEQ ID NO:141), where φ represents a hydrophobic amino acid; and x represents any amino acid.
103 . The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of Src, said peptide comprising the amino acid sequence LXXRPLPXψP (SEQ ID NO:165), where ψ represents an aliphatic amino acid; and X represents any amino acid.
104 . The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of Cortactin, said peptide comprising the amino acid sequence +PPψPXKPXWL (SEQ ID NO:166), where + represents a basic amino acid; ψ represents an aliphatic amino acid; and X represents any amino acid.
105 . The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of Abl, said peptide comprising the amino acid sequence PPXθXPPPψP (SEQ ID NO:173), where θrepresents an aromatic amino acid; ψ represents an aliphatic amino acid; and X represents any amino acid.
106 . The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of PLCγ, said peptide comprising the amino acid sequence PPVPPRPXXTL (SEQ ID NO:175), where X represents any amino acid.
107 . The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of p53bp2, said peptide comprising the amino acid sequence RPXψPψR+SXP (SEQ ID NO:196), where + represents a basic amino acid; ψ represents an aliphatic amino acid; and X represents any amino acid.
108 . The method of claim 97 where the second molecule is a peptide that binds to the N terminal SH3 domain of Crk, said peptide comprising the amino acid sequence ψPψLPψK (SEQ ID NO:210), where ψ represents an aliphatic amino acid; and X represents any amino acid.
109 . The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of Yes, said peptide comprising the amino acid sequence ψXXRPLPXLP (SEQ ID NO:222), where ψ represents an aliphatic amino acid; and X represents any amino acid.
110 . The method of claim 97 where the second molecule is a peptide that binds to the N terminal SH3 domain of Grb2, said peptide comprising an amino acid sequence selected from the group consisting of: +θDXPLPXLP (SEQ ID NO:223), YθX+PLPXLP (SEQ ID NO:238), and θDPLPXLP (SEQ ID NO:243), where θ represent an aromatic amino acid; +represents a basic amino acid; ψ represents an aliphatic amino acid; and X represents any amino acid.
111 . The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of Cortactin, said peptide comprising an amino acid sequence selected from the group consisting of:
LTPQSKPPLPPKPSAV;
(a portion of SEQ ID NO:112)
SSHNSRPPLPEKPSWL;
(a portion of SEQ ID NO:111)
PVKPPLPAKPWWLPPL;
(SEQ ID NO:167)
TERPPLPQRPDWLSYS;
(a portion of SEQ ID NO:109)
LGEFSKPPIPQKPTWM;
(a portion of SEQ ID NO:108)
YPQFRPPVPPKPSLMQ;
(SEQ ID NO:168)
VTRPPLPPKPGHMADF;
(SEQ ID NO:169)
VSLGLKPPVPPKPMQL;
(SEQ ID NO:170)
LLGPPVPPKPQTLFSF;
(a portion of SEQ ID NO:107)
YKPEVPARPIWLSEL;
(SEQ ID NO:171)
GAGAARPLVPKKPLFL; and
(SEQ ID NO:172)
SREPDWLCPNCPLLLRSDSR.
(SEQ ID NO:110)
112 . The method of claim 97 where the second molecule is a peptide that binds to the middle SH3 domain of Nck, said peptide comprising an amino acid sequence selected from the group consisting of:
SSLGVGWKPLPPMRTASLSR;
(SEQ ID NO:114)
SSVGFADRPRPPLRVESLSR;
(SEQ ID NO:115)
SSAGILRPPEKPXRSFSLSR;
(SEQ ID NO:116)
SSPYTGDVPIPPLRGASLSR;
(SEQ ID NO:117)
SSLMGSWPPVPPLRSDSLSR;
(SEQ ID NO:118)
SSIGEDTPPSPPTRRASLSR;
(SEQ ID NO:119)
SRSLSEVSPKPPIRSVSLSR;
(SEQ ID NO:120)
SSVSEGYSPPLPPRSTSLSR;
(SEQ ID NO:121)
SSSFTLAAPTPPTRSLSLSR;
(SEQ ID NO:122)
SRVVDGLAPPPPVRLSSLSR;
(SEQ ID NO:123)
SRVVDGLAPPPPVRLSSLSR;
(SEQ ID NO:124)
SSLGYSGAPVPPHRxSSLSR; and
(SEQ ID NO:125)
SSISDYSRPPPPVRTLSLSR.
(SEQ ID NO:126)
113 . The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of Abl, said peptide comprising an amino acid sequence selected from the group consisting of:
PPWWAPPPIPNSPQVL;
(SEQ ID NO:174)
PPKFSPPPPPYWQLHA;
(a portion of SEQ ID NO:132)
PPHWAPPAPPAMSPPI;
(a portion of SEQ ID NO:130)
PPTWTPPKPPGWGVVF;
(a portion of SEQ ID NO:137)
PPSFAPPAAPPRHSFG;
(a portion of SEQ ID NO:133)
PTYPPPPPPDTAKGA;
(a portion of SEQ ID NO:135)
GPRWSPPPVPLPTSLD;
(a portion of SEQ ID NO:128)
APTWSPPALPNVAKYK;
(a portion of SEQ ID NO:138)
PPDYAAPAIPSSLWVD;
(a portion of SEQ ID NO:129)
IKGPRFPVPPVPLNGV;
(a portion of SEQ ID NO:139)
PPAWSPPHRPVAFGST;
(a portion of SEQ ID NO:140)
APKKPAPPVPMMAHVM;
(a portion of SEQ ID NO:134)
SSDRCWECPPWPAGGQRGSR;
(SEQ ID NO:131)
and
SSPPXXXPPPIPNSPQVLSR.
(SEQ ID NO:136)
114 . The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of PLCγ, said peptide comprising an amino acid sequence selected from the group consisting of:
MPPPVPPRPPGTLQVA;
(SEQ ID NO:176)
LSYSPPPVPPRPDSTL;
(SEQ ID NO:177)
VLAPPVPPRPGNTFFT;
(SEQ ID NO:178)
YRPPVAPRPPSSLSVD;
(SEQ ID NO:179)
LQCPDCPRVPPRPIPI;
(SEQ ID NO:180)
VPPLVAPRPPSTLNSL;
(a portion of SEQ ID NO:143)
LTPPPFPKRPRWTLPE;
(SEQ ID NO:181)
YWPHRPPLAPPQTTLG;
(SEQ ID NO:182)
SSMKVHNFPLPPLPSYETSR;
(SEQ ID NO:142)
SSLYWQHGPDPPVGAPQLSR;
(SEQ ID NO:144)
and
SSHPLNSWPGGPFRHNLSSR.
(SEQ ID NO:145)
115 . The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of Src, said peptide comprising an amino acid sequence selected from the group consisting of:
LASRPLPLLPNSAPGQ;
(a portion of SEQ ID NO:155)
LTGRPLPALPPPFSDF;
(a portion of SEQ ID NO:152)
PAYRPLPRLPDLSVIY;
(a portion of SEQ ID NO:150)
RALRVRPLPPVPGTSL;
(a portion of SEQ ID NO:146)
DAPGSLPFRPLPPVPT;
(a portion of SEQ ID NO:148)
LKWRALPPLPETDTPY;
(a portion of SEQ ID NO:157)
ISQRALPPLPLMSDPA;
(a portion of SEQ ID NO:149)
LTSRPLPDIPVRPSKS;
(a portion of SEQ ID NO:156)
NTNRPLPPTPDGLDVR;
(a portion of SEQ ID NO:158)
MKDRVLPPIPTVESAV;
(a portion of SEQ ID NO:153)
LQSRPLPLPPQSSYPI;
(a portion of SEQ ID NO:159)
FINRRLPALPPDNSLL;
(a portion of SEQ ID NO:151)
FRALPLPPTPDNPFAG; and
(a portion of SEQ ID NO:147)
LYSAIAPDPPPRNSSS.
(a portion of SEQ ID NO:154)
116 . The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of p53bp2, said peptide comprising an amino acid sequence selected from the group consisting of:
YDASSAPQRPPLPVRKSRP;
(SEQ ID NO:183)
EYVNASPERPPIPGRKSRP;
(SEQ ID NO:184)
WNGIAIPGRPEIPPRASRP;
(SEQ ID NO:185)
SMIFIYPERPSPPPRFSRP;
(SEQ ID NO:186)
GVEEWNPERPQIPLRLSRP;
(SEQ ID NO:187)
WVVDSRPDIPLRRSLP;
(SEQ ID NO:188)
VVPLGRPEIPLRKSLP;
(SEQ ID NO:189)
GGTVGRPPIPERKSVD;
(SEQ ID NO:190)
YSHAGRPEVPPRQSKP;
(SEQ ID NO:191)
FSAAARPDIPSRASTP;
(SEQ ID NO:192)
LYIPKRPEVPPRRHEA;
(SEQ ID NO:193)
NNISARPPLPSRQNPP; and
(SEQ ID NO:194)
MAGTPRPAVPQRMNPP.
(SEQ ID NO:195)
117 . The method of claim 97 where the second molecule is a peptide that binds to the N terminal SH3 domain of Crk, said peptide comprising an amino acid sequence selected from the group consisting of:
GQPAGDPDPPPLPAKF;
(SEQ ID NO:197)
FEQTGVPLLPPKSFKY;
(SEQ ID NO:198)
IFGDPPPPIPMKGRSL;
(SEQ ID NO:199)
SNQGSIPVLPIKRVQY;
(SEQ ID NO:200)
NYVNALPPGPPLPAKN;
(SEQ ID NO:201)
SSDPERPVLPPKLWSV;
(SEQ ID NO:202)
HFGPSKPPLPIKTRIT;
(SEQ ID NO:203)
DWKVPEPPVPKLPLKQ;
(SEQ ID NO:204)
ATSEGLPILPSKVGSY;
(SEQ ID NO:205)
NANVSAPRAPAFPVKT;
(SEQ ID NO:206)
EMVLGPPVPPKRGTVV;
(SEQ ID NO:207)
AGSRHPPTLPPKESGG; and
(SEQ ID NO:208)
SVAADPPRLPAKSRPQ.
(SEQ ID NO:209)
118 . The method of claim 97 where the second molecule is a peptide that binds to the SH3 domain of Yes, said peptide comprising an amino acid sequence selected from the group consisting of:
ITMRPLPALPGHGQIH;
(SEQ ID NO:211)
LPRRPLPDLPMAAGKG;
(SEQ ID NO:212)
LGSRPLPPTPRQWPEV;
(SEQ ID NO:213)
STIRPLPAIPRDTLLT;
(SEQ ID NO:214)
RSGRPLPPIPEVGHNV;
(SEQ ID NO:215)
IGSRPLPWTPDDLGSA;
(SEQ ID NO:216)
LAQRELPGLPAGAGVS;
(SEQ ID NO:217)
IPGRALPELPPQRALP;
(SEQ ID NO:218)
FVGRELPPTPRTVIPW;
(SEQ ID NO:219)
DPRSALPALPLTPLQT; and
(SEQ ID NO:220)
SPHDVLPALPDSHSKS.
(SEQ ID NO:221)
119 . The method of claim 97 where the second molecule is a peptide that binds to the N terminal SH3 domain of Grb2, said peptide comprising an amino acid sequence selected from the group consisting of:
KWDSLLPALPPAFTVE;
(SEQ ID NO:224)
RWDQVLPELPTSKGQI;
(SEQ ID NO:225)
RFDFPLPTHPNLQKAH;
(SEQ ID NO:226)
RLDSPLPALPPTVMQN;
(SEQ ID NO:227)
RWGAPLPPLPEYSWST;
(SEQ ID NO:228)
YWDMPLPRLPGEEPSL;
(SEQ ID NO:229)
RFDYNLPDVPLSLGTA;
(SEQ ID NO:230)
TKKPNAPLPPLPAYMG;
(SEQ ID NO:231)
KWDLDLPPEPMSLGNY;
(SEQ ID NO:232)
YYQRPLPPLPLSHFES;
(SEQ ID NO:234)
YYRKPLPNLPRGQTDD;
(SEQ ID NO:235)
YFDKPLPESPGALMSL;
(SEQ ID NO:236)
YFSRALPGLPERQEAH;
(SEQ ID NO:237)
SLWDPLPPIPQSKTSV;
(SEQ ID NO:239)
SYYDPLPKLPDPGDLG;
(SEQ ID NO:240)
KLYYPLPPVPFKDTKH; and
(SEQ ID NO:241)
DPYDALPETPSMKASQ.
(SEQ ID NO:242)
120 . The method of claim 97 where the second molecule is a peptide having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 250-252, 254, 256-259, 261, 262, 264-266, 269-272, 275, 280, 281, 286-289, 291, 294, and 295.
121 . The method of claim 97 where the second molecule is a peptide having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 296-453.
122 . A method of identifying a compound that affects the binding of a molecule comprising an SH3 domain and a ligand of the SH3 domain, the method comprising:
(a) contacting the SH3 domain and the ligand under conditions conducive to binding in the presence of a candidate compound and measuring the amount of binding between the SH3 domain and the ligand; (b) comparing the amount of binding in step (a) with the amount of binding known or determined to occur between the molecule and the ligand in the absence of the candidate compound, where a difference in the amount of binding between step (a) and the amount of binding known or determined to occur between the molecule and the ligand in the absence of the candidate compound indicates that the candidate compound is a compound that affects the binding of the molecule comprising an SH3 domain and the ligand.
123 . A kit comprising, in one or more containers:
(a) a purified first molecule comprising an SH3 domain; (b) a purified second molecule that binds to the SH3 domain.
124 . The kit of claim 123 wherein said second molecule comprises a peptide having an amino acid sequence selected from the group consisting of: SEQ ID NOs:107- 112, 114-126, 128-140, 142-159, 167, 168-172, 174, 176-195, 197-209, 211-221, 224-232, 234-237, 239-242, 250-252, 254, 256-259, 261, 262, 264-266, 269-272, 275, 280, 281 , 286-289, 291, 294-453.
125 . A purified peptide that binds to the SH3 domain of Src, said peptide comprising the amino acid sequence
LX 1 X 2 RPLPX 3 ψPX 4 X 5 (SEQ ID NO:454) where ψ represents aliphatic amino acid residues and X 1 , X 2 , X 3 , X 4 , and X 5 represent any amino acid; except that if X 3 =P, ψ=L, X 4 =P, and X 5 =P, then: where X 1 =F, then X 2 is not H or R; or where X 1 =S, then X 2 is not R, H, A, N, T, G, V, M, or W; or where X 1 =C, then X 2 is not S or G; or where X 1 =R, then X 2 is not T or F; or where X 1 =A, then X 2 is not R, Q, N, S, or L; or where X 1 =Q, then X 2 is not M; or where X 1 =L, then X 2 is not R; or where X 1 =I, then X 2 is not A; or where X 1 =P, then X 2 is not P, W, or R; or where X 1 =G, then X 2 is not S or R; or where X 1 =T, then X 2 is not T.
126 . A purified peptide that binds to the SH3 domain of Yes, said peptide comprising the amino acid sequence
ψX 1 X 2 RPLPX 3 LPX 4 X 5 (SEQ ID NO:455) where ψ represents aliphatic amino acid residues and X 1 , X 2 , X 3 , X 4 , and X 5 represent any amino acid; except that if X 3 =P, X 4 =P, and X 5 =P, then: when ψ=L, where X 1 =F, then X 2 is not H or R; or where X 1 =S, then X 2 is not R, H, A, N, T, G, V, M, or W; or where X 1 =C, then X 2 is not S or G; or where X 1 =R, then X 2 is not T or F; or where X 1 =A, then X 2 is not R, Q, N, S, or L; or where X 1 =Q, then X 2 is not M; or where X 1 =L, then X 2 is not R; or where X 1 =I, then X 2 is not A; or where X 1 =P, then X 2 is not P, W, or R; or where X 1 =G, then X 2 is not S or R; or where X 1 =T, then X 2 is not T; and when ψ=P, where X 1 =A, then X 2 is not R; or where X 1 =S, then X 2 is not R or Y; or where X 1 =M, then X 2 is not S; or where X 1 =V, then X 2 is not G; or where X 1 =R, then X 2 is not S; or where X 1 =I, then X 2 is not R; and when ψ=A, where X 1 =A, then X 2 is not K; and when ψ=V, where X 1 =A, then X 2 is not C or Q; or where X 1 =P, then X 2 is not P; and when ψ=I, where X 1 =G, then X 2 is not H; or where X 1 =T, then X 2 is not S; or where X 1 =R, then X 2 is not S.Join the waitlist — get patent alerts
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