Crystal structure of human Janus Kinase 2 (JAK2) and uses thereof
The present invention relates to X-ray crystallography studies of a human Janus Kinase 2 (JAK2) domain. More particularly, it relates to the crystal structure of a JAK2 kinase domain bound to a inhibitor. The invention further relates to the use of the crystal and related structural information to select and screen for compounds that interact with JAK2 and related proteins and to compounds that could be used for the treatment of diseases mediated by inappropriate JAK2 activity.
1. A method of selecting or designing a candidate compound or compounds that interact with human Janus Kinase 2 (JAK2) and modulate human JAK2 kinase activity, the method comprising the steps of:
(a) generating a three-dimensional model of the kinase domain of said human JAK2 defined by the three-dimensional structural coordinates according to Appendix I, wherein said kinase domain consists of SEQ ID NO: 7 and/or SEQ ID NO: 8;
(b) selecting the region that form the ATP binding pocket and sugar binding pocket from the three-dimensional model in (a) which comprise amino acid acids Glu930, Leu932, Asp939, Ser936, Leu855, Arg980, Gly993, Asp994, Ala880, Val911, Leu983, Gly935, Met929 and Tyr931, Gln853, Gly856, Lys857, Gly858, Asn859, Phe860, Gly861, Ser862, Va1863, Met865, Va1878, Lys882, Glu898, Leu902, Tyr913, Leu927, Pro933, Tyr934, Asn981, 11e982, Phe995, Gly996, including the sugar binding pocket amino acids Arg938, Ala978, Thr979 to generate a three-dimensional target; wherein the numbering of human JAK 2 is shown in either sequence A (SEQ ID NO: 7) or sequence B (SEQ ID NO: 8) of Appendix 1,
(c) assessing the stereochemical complementarity between the candidate compound(s) and said region; and
(d) testing the compound in vitro or in vivo for its capacity to modulate the kinase activity of human JAK2;
wherein the structural coordinates of the amino acids and water molecules in (a) and/or (b) have a root mean square deviation of no more than 1.0 Å from the backbone atoms in the amino acids shown in Appendix 1.
2. The method according to claim 1 wherein the root mean square deviation is not more than 0.7 Å.
3. The method according to claim 1 comprising selecting a compound which forms hydrogen bonds or water-mediated hydrogen bonds with at least one amino acid selected from the group consisting of Glu930, Leu932, Asp939, Ser936, Leu855, Arg980, Gly993 and Asp994 wherein the positions set forth are defined by either sequence A or sequence B of Appendix 1.
4. The method according to claim 1 or claim 3 comprising selecting a compound which forms hydrophobic contacts with the side chains of at least one amino acid residue selected from the group consisting of Leu855, Ala880, Va1911, Leu983, Gly935, Met929, Tyr 931, Pro933, Asn981, Ala993, Asp994, Gly856, Lys857 and Va1863 wherein the positions set forth are defined by either sequence A or sequence B of Appendix 1.
5. The method according to claim 1 wherein the compound is of formula I:
where:
R is one to three groups selected from H, halogen, OH, OR 2 , NR 2 R 3 , CN, NO 2 , CO 2 R 2 , CONR 2 R 3 , NR 4 CONR 2 R 3 , OCONR 2 R 3 , NR 2 COOR 3 , NR 2 COR 3 , NR 2 SO 2 R 3 , SO 2 R 2 , OC 2-6 alkylOH, OC 2-6 alkylNR 2 R 3 , OC 1-6 alkylCN, C 1-6 alkylOH, C 1-6 alkylNR 2 R 3 , C 1-6 alkylCN;
where R 2 and R 3 are independently H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, aryl, hetaryl, C 1-6 alkylCN, C 2-6 alkylNR 5 R 6 , or may be joined to form a 4-7-membered ring which may contain a heteroatom selected from O, S, SO 2 or NR 7 ;
where R 4 is H, C 1-6 alkyl;
where R 5 and R 6 are independently H, C 1-6 alkyl, or may be joined to form a 4-7-membered ring which may contain a heteroatom selected from O, S, SO 2 or NR 7 ;
where R 7 is H, C 1-6 alkyl, C 1-6 alkylOH;
W, X, Y form a 5- or 6-membered aromatic ring, selected from furan, pyrrole, imidazole, oxazole, thiazole, pyrazine, pyridazine, pyridine; and
R 1 is selected from H, halogen, OH, OC 1-6 alkyl, C 1-6 alkyl, C 1-6 alkylCN, NR 2 R 3 , C 2-6 alkylNR 2 COR 3 , aryl, and hetaryl.