US2007100560A1PendingUtilityA1

Crystal structures of MK2 and uses thereof

Assignee: WYETH A DELAWARE CORPPriority: Nov 13, 2002Filed: Dec 6, 2006Published: May 3, 2007

Est. expiryNov 13, 2022(expired)· nominal 20-yr term from priority

Inventors:Kevin D. Parris Kathryn Underwood Mark Stahl Lidia Mosyak Kristine Svenson Tania Shane Meggin Taylor

Y02A90/10C07H 21/04C07K 2299/00C12N 9/1205

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References

Claims

Abstract

This invention is directed to the crystal structures of MK2, and to the use of these structures in rational drug design methods to identify agents that may interact with active sites of MK2 proteins. Such agents may be useful as therapeutic agents.

Claims

exact text as granted — not AI-modified

1 - 10 . (canceled)  
     
     
         11 . A method for obtaining crystallized native MK2, crystallized MK2 polypeptide or crystallized MK2 analogue comprising contacting native MK2, MK2 polypeptide or MK2 analogue with a buffer solution comprising at least one of cacodylate, Tris, Tris-HCL, acetate, malonate, sodium phosphate, potassium phosphate, citrate, HEPES and MES, at a salt concentration of 0.1 M to 2.4 M, and at a pH of 4.5 to 8.5, under conditions permitting the formation of crystallized MK2, crystallized MK2 polypeptide or crystallized MK2 analogue.  
     
     
         12 . The method of  claim 11 , wherein the salt has an anion selected from the group consisting of sulfate, citrate, chloride, acetate, phosphate, malonate and tartrate.  
     
     
         13 . The method of  claim 11 , wherein the salt concentration is 0.8 M or higher.  
     
     
         14 . The method of  claim 11 , wherein native MK2, MK2 polypeptide or MK2 analogue is contacted with the buffer solution in the presence of PEG or a PEG substitute having a molecular weight up to 3350.  
     
     
         15 . The method of  claim 14 , wherein PEG or PEG substitute is selected from the group consisting of PEG-200, PEG-400, PEG-500-MME, PEG-1000, PEG-1500, PEG-2000-MME, PEG-3350, Jeffamine M-600, ethylene glycol, glycerol and 1-6 hexanediol, 2-methyl-2,4-pentanediol (MPD).  
     
     
         16 . The method of  claim 14 , wherein PEG is PEG-400.  
     
     
         17 . A crystallized complex comprising an MK2 polypeptide and staurosporine.  
     
     
         18 . The crystallized complex of  claim 17 , characterized as having space group P6 3 , unit cell parameters of a=b=160.20 Å, c=133.48 Å.  
     
     
         19 . A crystallized complex comprising an MK2 polypeptide and ADP.  
     
     
         20 . The crystallized complex of  claim 19 , characterized as having space group F4 1 32, unit cell parameters of a=b=c=253.05 Å.  
     
     
         21 - 57 . (canceled)  
     
     
         58 . The crystallized complex of  claim 17 , wherein the complex comprises four molecules of MK2 polypeptide per asymmetric unit.  
     
     
         59 . The crystallized complex of  claim 17 , wherein the complex further comprises selenomethionine.  
     
     
         60 . The crystallized complex of  claim 17 , wherein the complex diffracts to at least 3.1 Å resolution.  
     
     
         61 . The crystallized complex of  claim 17 , wherein the complex diffracts to at least 1.1 Å resolution.  
     
     
         62 . The crystallized complex of  claim 19 , wherein the complex comprises one molecule of MK2 polypeptide per asymmetric unit.  
     
     
         63 . The crystallized complex of  claim 19 , wherein the complex diffracts to at least 1.0 Å resolution.  
     
     
         64 . The crystallized complex of  claim 17 , wherein the MK2 polypeptide comprises amino acid residues Leu70, Gly71, Leu72, Gly73, Val78, Ala91, Val118, Mse138, Glu139, Cys140, Leu141, Glu145, Glu190, Asn191, Leu192, Thr206, and Asp207 as set forth in SEQ ID NO:1.  
     
     
         65 . The crystallized complex of  claim 64 , wherein the MK2 polypeptide further comprises amino acid residues Val69, Ile74, Gly76, Ala77, Leu79, Gln80, Lys89, Phe90, Leu92, Lys93, Leu95, Glu104, His108, Arg 119, Ile136, Val137, Asp142, Gly143, Gly144, His184, Asp186, Lys188, Pro189, Leu193, Tyr194, Thr195, Lys204, Leu205, Phe208, and Gly209 as set forth in SEQ ID NO: 1.  
     
     
         66 . The crystallized complex of  claim 17 , wherein the crystallized complex has the structural coordinates set forth in  FIGS. 2 through 2 A- 204  ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å.  
     
     
         67 . The crystallized complex of  claim 17 , wherein the MK2 polypeptide comprises an active site comprising the relative structural coordinates of amino acid residues Leu70, Gly71, Leu72, Gly73, Val78, Ala91, Val118, Mse138, Glu139, Cys140, Leu141, Glu145, Glu190, Asn191, Leu192, Thr206, and Asp207 of molecules A, B, C or D according to the sequence set forth in SEQ ID NO:1, and the structural coordinates as set forth in  FIGS. 2 through 2 A- 204  ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å.  
     
     
         68 . The crystallized complex of  claim 67 , wherein the active site of the MK2 polypeptide further comprises the relative structural coordinates of amino acid residues Val69, Ile74, Gly76, Ala77, Leu79, Gln80, Lys89, Phe90, Leu92, Lys93, Leu95, Glu104, His108, Arg119, Ile136, Val137, Asp142, Gly143, Gly144, His184, Asp186, Lys188, Pro189, Leu193, Tyr194, Thr195, Lys204, Leu205, Phe208, and Gly209 of molecules A, B, C or D according to the sequence set forth in SEQ ID NO:1, and the structural coordinates as set forth in  FIGS. 2 through 2 A- 204  ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å.  
     
     
         69 . The crystallized complex of  claim 19 , wherein the MK2 polypeptide comprises amino acid residues Leu70, Gly71, Leu72, Gly73, Val78, Ala91, Val118, Mse138, Glu139, Cys140, Leu141, Glu145, Glu190, Asn191, Leu192, Thr206, and Asp207 as set forth in SEQ ID NO:1.  
     
     
         70 . The crystallized complex of  claim 69 , wherein the MK2 polypeptide further comprises amino acid residues Val69, Ile74, Gly76, Ala77, Leu79, Gln80, Lys89, Phe90, Leu92, Lys93, Leu95, Glu104, His108, Arg119, Ile136, Val237, Asp142, Gly143, Gly144, His184, Asp186, Lys188, Pro189, Leu193, Tyr194, Thr195, Lys204, Leu205, Phe208, and Gly209 as set forth in SEQ ID NO:1.  
     
     
         71 . The crystallized complex of  claim 19 , wherein the crystallized complex has the structural coordinates set forth in  FIGS. 3 through 3 A- 40  ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å.  
     
     
         72 . The crystallized complex of  claim 19 , wherein the MK2 polypeptide comprises an active site comprising the relative structural coordinates of amino acid residues Leu70, Gly71, Leu72, Gly73, Val78, Ala91, Val118, Mse138, Glu139, Cys140, Leu141, Glu145, Glu190, Asn191, Leu192, Thr206, and Asp207 of molecule A according to the sequence set forth in SEQ ID NO:1, and the structural coordinates as set forth in  FIGS. 3 through 3 A- 40  ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å.  
     
     
         73 . The crystallized complex of  claim 72 , wherein the active site of the MK2 polypeptide further comprises the relative structural coordinates of amino acid residues Val69, Ile74, Gly76, Ala77, Leu79, Gln80, Lys89, Phe90, Leu92, Lys93, Leu95, Glu104, His108, Arg119, Ile136, Val137, Asp142, Gly143, Gly144, His184, Asp186, Lys188, Pro189, Leu193, Tyr194, Thr195, Lys204, Leu205, Phe208, and Gly209 of molecule A according to the s set forth in SEQ ID NO:1, and the structural coordinates as set forth in  FIGS. 3 through 3 A- 40  ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å.

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