Crystal Structure of PTP1B Complexed with a Potent and Selective Bidentate Inhibitor

Abstract

Protein-tyrosine phosphatase 1B (PTP1B) has been implicated as an important regulator in several signaling pathways including those initiated by insulin and leptin. Potent and specific PTP1B inhibitors could serve as useful tools in elucidating the physiological functions of PTP1B and may constitute valuable therapeutics in the treatment of several human diseases. We have determined the crystal structure of PTP1B in complex with compound 2, the most potent and selective PTP1B inhibitor reported to date. The structure at 2.15-Å resolution reveals that compound 2 simultaneously binds to the active site and a unique proximal noncatalytic site formed by Lys-41, Arg-47, and Asp-48. The structural data are further corroborated by results from kinetic analyses of the interactions of PTP1B and its site-directed mutants with compound 2 and several of its variants. Although many of the residues important for interactions between PTP1B and compound 2 are not unique to PTP1B, the combinations of all contact residues differ between PTP isozymes, which provide a structural basis for potent and selective PTP1B inhibition. Our data further suggest that potent, yet highly selective, PTP1B inhibitory agents can be acquired by targeting the area defined by residues Lys-41, Arg-47, and Asp-48, in addition to the previously identified second aryl phosphate-binding pocket.1N6W Protein-tyrosine phosphatase 1B (PTP1B) has been implicated as an important regulator in several signaling pathways including those initiated by insulin and leptin. Potent and specific PTP1B inhibitors could serve as useful tools in elucidating the physiological functions of PTP1B and may constitute valuable therapeutics in the treatment of several human diseases. We have determined the crystal structure of PTP1B in complex with compound 2, the most potent and selective PTP1B inhibitor reported to date. The structure at 2.15-Å resolution reveals that compound 2 simultaneously binds to the active site and a unique proximal noncatalytic site formed by Lys-41, Arg-47, and Asp-48. The structural data are further corroborated by results from kinetic analyses of the interactions of PTP1B and its site-directed mutants with compound 2 and several of its variants. Although many of the residues important for interactions between PTP1B and compound 2 are not unique to PTP1B, the combinations of all contact residues differ between PTP isozymes, which provide a structural basis for potent and selective PTP1B inhibition. Our data further suggest that potent, yet highly selective, PTP1B inhibitory agents can be acquired by targeting the area defined by residues Lys-41, Arg-47, and Asp-48, in addition to the previously identified second aryl phosphate-binding pocket.1N6W protein-tyrosine phosphatase epidermal growth factor receptor nuclear magnetic resonance phosphotyrosine protein-tyrosine phosphatase 1B p-nitrophenyl phosphate N-(9-fluorenyl)methoxycarbonyl phosphonodifluoromethylphenylalanine 4-morpholineethanesulfonic acid T cell Protein-tyrosine phosphatases (PTPs)1 are signaling enzymes that regulate a wide variety of cellular processes including cell growth, differentiation, metabolism, progression through the cell cycle, cell-cell communication, cell migration, gene transcription, ion channel activity, the immune response, and apoptosis/survival decisions. Evidence suggests that PTPs can exert both positive and negative effects on signaling pathways and play crucial physiological roles in a variety of mammalian tissues and cells. Defective or inappropriate regulation of PTP activity can lead to aberrant tyrosine phosphorylation, which contributes to the development of many human diseases including cancers and diabetes (1Neel B.G. Tonics N.K. Curr. Opin. Cell Biol. 1997; 9: 193-204Google Scholar, 2Zhang Z.-Y. Curr. Opin. Chem. Biol. 2001; 5: 416-423Google Scholar). Given the complexity of cellular signaling and the large number of phosphoproteins in the cell, it is likely that a single PTP may be involved in the regulation of multiple signaling pathways, and that multiple PTPs may act cooperatively to regulate a particular pathway. For example, PTP1B is regarded as a major negative regulator of both insulin- and leptin-stimulated signal transduction pathways (3Elchebly M. Payette P. Michaliszyn E. Cromlish W. Collins S. Loy A.L. Normandin D. Cheng A. Himms-Hagen J. Chan C.C. Ramachandran C. Gresser M.J. Tremblay M.L. Kennedy B.P. Science. 1999; 283: 1544-1548Google Scholar, 4Klaman L.D. Boss O. Peroni O.D. Kim J.K. Martino J.L. Zabolotny J.M. Moghal N. Lubkin M. Kim Y.B. Sharpe A.H. Stricker-Krongrad A. Shulman G.I. Neel B.G. Kahn B.B. Mol. Cell. Biol. 2000; 20: 5479-5489Google Scholar, 5Zabolotny J.M. Bence-Hanulec K.K. Stricker-Krongrad A. Haj F. Wang Y. Minokoshi Y. Kim Y.B. Elmquist J.K. Tartaglia L.A. Kahn B.B. Neel B.G. Dev. Cell. 2002; 2: 489-495Google Scholar, 6Cheng A. Uetani N. Simoncic P.D. Chaubey V.P. Lee-Loy A. McGlade C.J. Kennedy B.P. Tremblay M.L. Dev. Cell. 2002; 2: 497-503Google Scholar), which suggests that specific PTP1B inhibitors may enhance insulin and leptin sensitivity and act as effective therapeutics for the treatment of type II diabetes, insulin resistance, and obesity. In addition to a role in insulin and leptin signaling, PTP1B is also implicated in several other physiological and pathological processes including transformation by the Neu oncogene (7Wiener J.R. Kerns B.J.M. Harvey E.L. Conaway M.R. Iglehart J.D. Berchuck A. Bast Jr., R.C. J. Natl. Cancer Inst. 1994; 86: 372-378Google Scholar), Src kinase activation (8Bjorge J.D. Pang A. Fujita D.J. J. Biol. Chem. 2000; 275: 41439-41446Google Scholar), antagonizing signaling by the EGFR (9Liu F Chernoff J. Biochem. J. 1997; 327: 139-145Google Scholar, 10Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685Google Scholar), and the oncoprotein p210bcr-abl (11LaMontagne Jr., K.R. Flint A.J. Franza Jr., B.R. Pandergast A.M. Tonks N.K. Mol. Cell. Biol. 1998; 18: 2965-2975Google Scholar), and the negative regulation of integrin-mediated adhesion and signaling by binding to and dephosphorylating ॆ-catenin (12Balsamo J. Arregui C. Leung T. Lilien J. J. Cell Biol. 1998; 143: 523-532Google Scholar) and p130cas (Crk-associated substrate) (13Liu F. Hill D.E. Chernoff J. J. Biol. Chem. 1996; 271: 31290-31295Google Scholar). These results, taken together, suggest that PTP1B may be a participant in several signaling pathways. Conversely, in addition to PTP1B, both leukocyte common antigen-related phosphate and PTPα have been implicated as negative regulators of insulin signaling (2Zhang Z.-Y. Curr. Opin. Chem. Biol. 2001; 5: 416-423Google Scholar). A major goal in the PTP field is to establish the precise functional roles for individual PTPs, both in normal cellular physiology and in pathogenic conditions. This is an important prerequisite for PTP-based drug discovery in order to minimize unwanted side effects. Potent and selective PTP inhibitors would be extremely useful in helping to delineate the physiological roles of PTPs and to validate them as therapeutic targets. Furthermore, these inhibitors could provide the foundation upon which therapeutically useful agents can be devised. Unfortunately, as a consequence of the conserved nature of the PTP-active sites (i.e. Tyr(P)-binding sites), there are currently few PTP inhibitors that exhibit the potency and specificity required for biological and pharmacological investigation. Fortunately, PTP substrate specificity studies have shown that Tyr(P) alone is not sufficient for high affinity binding and that residues flanking Tyr(P) furnish additional interactions for specific and high affinity substrate recognition (14Zhang Z.-Y. Maclean D. Thieme-Sefler A.M. McNamara D. Dobrusin E.M. Sawyer T.K. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4446-4450Google Scholar, 15Zhang Z.-Y. Maclean D. McNamara D.J. Sawyer T.K. Dixon J.E. Biochemistry. 1994; 33: 2285-2290Google Scholar, 16Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Google Scholar, 17Wu L. Buist A. den Hertog J. Zhang Z.-Y. J. Biol. Chem. 1997; 272: 6994-7002Google Scholar, 18Sarmiento M. Zhao Y. Gordon S.J. Zhang Z.-Y. J. Biol. Chem. 1998; 273: 26368-26374Google Scholar, 19Vetter S.W. Keng Y.F. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2000; 275: 2265-2268Google Scholar, 20Sarmiento M. Puius Y.A. Vetter S.W. Keng Y.-F. Wu L. Zhao Y. Lawrence D.S. Almo S.C. Zhang Z.-Y. Biochemistry. 2000; 39: 8171-8179Google Scholar). These studies, together with the discovery of a second aryl phosphate-binding site adjacent to the active site in PTP1B (21Puius Y.A. Zhao Y. Sullivan M. Lawrence D.S. Almo S.C. Zhang Z.-Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13420-13425Google Scholar), demonstrated the potential for multiple site recognition of substrates by PTPs. They also suggested a novel paradigm for the design of potent and specific PTP inhibitors, that to both the active site and a unique adjacent have acquired a highly potent and selective PTP1B inhibitor which a of for PTP1B and several of in of PTP1B a of PTPs Keng Y.-F. Wu L. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2001; Scholar). is the most potent and selective PTP1B inhibitor reported to date. This as a in PTP inhibitor as it the of potent, yet highly selective, PTP inhibitory In order to the structural basis of the potency and of for PTP1B, and to the for the design of potent and specific inhibitors that can with the processes by PTP1B and other PTPs, have determined the crystal structure of PTP1B in complex with compound 2 and 2 exhibit potency and In a of kinetic studies and site-directed to the binding interactions between compound several and phosphate from of PTP1B the from Wang F. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2002; Scholar). The for from of the by The of PTP1B and its mutants and to as previously (21Puius Y.A. Zhao Y. Sullivan M. Lawrence D.S. Almo S.C. Zhang Z.-Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13420-13425Google Scholar, Wang F. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2002; Scholar). The of compound reported in Keng Y.-F. Wu L. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2001; The of in M. Keng Y.F. Wu L. Lawrence D.S. Zhang Z.-Y. Biochemistry. 1999; and Wang F. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2002; Scholar, 2 and on the a for activation of The in for a of acid to The to the to furnish compound and by and side by treatment with acid with and at for of the in a that with The the by and the by high in 2 as a by a of and at The to the to furnish compound with an side with side and acid Wang F. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2002; Scholar). by and side by treatment with a of and acid at for The by and the The with in and by high acid in and to the The and data for compound at and at for of and for PTP1B inhibitors determined as previously Wang F. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2002; Scholar). at in with an of with at the or the PTP1B to D. Flint A.J. Tonks N.K. Science. 1994; Scholar). In PTP1B in PTP1B inhibitors a of to For of PTP1B in and with PTP1B In 2 of PTP1B with an of the and and the of the A single crystal several in type and in a of at a of and of on a at of the The data with the and Z. W. 1997; Scholar). are in data and cell unique for for inhibitor and are the and structure factor for all in is for of the data that and not in and are for all data and for all data in the resolution and are the and structure factor for all in is for of the data that and not in in a and are for all data and for all data in the resolution from the is with the and Although to of PTP1B to the is M. Puius Y.A. Vetter S.W. Keng Y.-F. Wu L. Zhao Y. Lawrence D.S. Almo S.C. Zhang Z.-Y. Biochemistry. 2000; 39: 8171-8179Google Scholar), a in the structure by the structure by the structure of with the as the M. Puius Y.A. Vetter S.W. Keng Y.-F. Wu L. Zhao Y. Lawrence D.S. Almo S.C. Zhang Z.-Y. Biochemistry. 2000; 39: 8171-8179Google Scholar), and the and with P.D. P. J. M. T. Biol. 1998; Scholar) data in the resolution an with a of and a of of and both for compound 2 in and adjacent to the active site of of and individual factor all data from to S.W. M. A. Scholar). and to and The residues inhibitor 2 and 2 with compound the of with compound 2 a to or of compound 2 the potency and PTP1B as compound This is with that of an to the of Tyr(P) not the affinity of substrates for PTP1B Keng Y.-F. Wu L. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2001; Scholar). PTP1B with compound the that both inhibitors could PTP1B in the these data to to high of the PTP1B could be as in the of the inhibitor at a in the of the D. Flint A.J. Tonks N.K. Science. 1994; Scholar). or the crystal of to an of the and in high for the complex of PTP1B with compound 2 that to 2.15-Å The for the complex PTP1B residues and all in compound 2 the in the The structure to an of for data from to 2 to defined The structure of PTP1B in the complex is to those in the between PTP1B and Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Google Scholar, 20Sarmiento M. Puius Y.A. Vetter S.W. Keng Y.-F. Wu L. Zhao Y. Lawrence D.S. Almo S.C. Zhang Z.-Y. Biochemistry. 2000; 39: 8171-8179Google Scholar, A. Tonks N.K. Barford D. Mol. Cell. 2000; Scholar). in other the a in the with PTP1B in a defined that phosphonodifluoromethylphenylalanine the active the interactions with residues and Asp-48, and the both and with and These are in with on and analyses Wang F. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2002; Scholar). Although the from the of are in the interactions with PTP1B, with the that exhibit the potency and binding of compound 2 of the interactions between and The contact residues from PTP1B are shown 2 is to type and The is as a A. J. Chem. Scholar) of PTP1B in complex with The and and The in compound 2 the in the active site of PTP1B as that for Tyr(P) in The an of with the of the phosphate-binding and the side of The of is between the side of and and with the side of and These interactions are in all PTP1B Tyr(P) in the active to which is a of is the that the in The at the are to be important for high affinity binding to For example, Tyr(P) in the is with the for the for is potent the Jr., Biochem. 1994; Scholar, L. Wu L. A. den Hertog J. Zhang Z.-Y. Biochem. 1995; Scholar, M. Wu L. Jr., Zhang Z.-Y. J. Biol. Chem. 2000; 275: Scholar). The structure of for the specific interactions between the and PTP1B active site residues L. Wu L. A. den Hertog J. Zhang Z.-Y. Biochem. 1995; Scholar). shown in the and of the of the are to an that is by the of and the side of A is also in several and M. Puius Y.A. Vetter S.W. Keng Y.-F. Wu L. Zhao Y. Lawrence D.S. Almo S.C. Zhang Z.-Y. Biochemistry. 2000; 39: 8171-8179Google M.R. Jr., Barford D. Biochemistry. 1998; Scholar, Z. Wang D. Payette P. Ramachandran C. Kennedy J. Chem. 2001; Scholar, E. S. C. P. Wang Ramachandran C. Y. Kennedy B.P. Biochemistry. 2002; Scholar). In addition to the the are also involved in interactions of which are in the of the are with the side of and In addition to the interactions between and the active there are and specific interactions between several PTP1B residues the active site with the of compound specific interactions between compound 2 and to the and the of In a role for high affinity binding of The side of is highly and can on the nature of the residues to Tyr(P) M. Puius Y.A. Vetter S.W. Keng Y.-F. Wu L. Zhao Y. Lawrence D.S. Almo S.C. Zhang Z.-Y. Biochemistry. 2000; 39: 8171-8179Google Scholar). In the structure of is in a that is from those in studies, to with both the and the aryl the of a with the of the the of with the side of compound A between the of and a of the is also from the active the of a with and in a with a of the In addition to there are also between PTP1B and the and in compound 2 and of are to be of of and and of the in compound The at the of the are in interactions with the of both and the of the side of is involved in interactions with the of the The of several residues to binding by site-directed with several PTP1B inhibitors including The and in E. and to as Wang F. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2002; Scholar). kinetic at at and an of shown in with the of and in the kinetic and and the for for the and structural in the active site by the and are involved in with of the side may the precise of active site residues important for M. Zhao Y. Gordon S.J. Zhang Z.-Y. J. Biol. Chem. 1998; 273: 26368-26374Google Wang F. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2002; of and of and for the and for as a substrate at and in a as a substrate at and This of mutants also for the binding of and is a previously inhibitor of PTP1B with of M. Keng Y.F. Wu L. Lawrence D.S. Zhang Z.-Y. Biochemistry. 1999; Scholar). from compound in that the is of the by and and with to the For the and for PTP1B and the mutants are in The of for PTP1B a of phosphatases is shown in of compound a of at at at at at at at in a identified a second aryl phosphate-binding site in PTP1B and that of the could be for the design of potent and selective PTP1B inhibitors (21Puius Y.A. Zhao Y. Sullivan M. Lawrence D.S. Almo S.C. Zhang Z.-Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13420-13425Google Scholar). This has in and to PTP1B inhibitors by together that could both the active site and the second aryl phosphate-binding results from S. T.K. J. Y. M.J. Biochemistry. 2001; Scholar) and J.M. Curr. Opin. 2002; Scholar) demonstrated that is a to potent and selective PTP1B at targeting both the active site and the second aryl phosphate-binding site have inhibitors compound that affinity and for PTP1B M. Keng Y.F. Wu L. Lawrence D.S. Zhang Z.-Y. Biochemistry. 1999; Scholar, C.C. Wang Ramachandran C. Z. Chem. 1998; Scholar, S. Ramachandran C. Biochem. J. 1999; Scholar). The structure of PTP1B in complex with a that the not to the second aryl phosphate-binding site the and interactions with the of Z. Wang D. Payette P. Ramachandran C. Kennedy J. Chem. 2001; Scholar). The in the inhibitor also binds in a area to E. S. C. P. Wang Ramachandran C. Y. Kennedy B.P. Biochemistry. 2002; Scholar). Unfortunately, specific interactions between inhibitor and PTP1B could be defined to a high of in that reported to exhibit an of for both PTP1B and E. C. Payette P. C. Ramachandran C. Kennedy B.P. J. Biol. Chem. 2001; affinity may have been as the or conditions. We that the for PTPs are highly to the of Keng Y.-F. Wu L. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2001; Scholar). the of for PTP1B physiological of is to be This affinity is those of 2, which may the of interactions between the and PTP1B in the structure of with the affinity of for PTP1B, the to and its contact residues in PTP1B are defined in the structure of and interactions both the and the aryl in addition to those between the active site and the The potency and of and 2 for PTP1B are the results of specific interactions with is from the structure of that the active site in a to that of interactions for both and Tyr(P) with the active site interactions between the and the side of and and interactions between the and the of the phosphate-binding and the side of to are additional interactions an that the of and the side of with the and a In the are also involved in interactions with the side of and of these interactions to the high affinity of compound for PTP1B, and the interactions with the in particular may is to Tyr(P) or for binding to the PTP active these residues that with are major to the by compound are highly conserved PTPs. may be an as both and residues can be at residues adjacent to the active including Lys-41, Arg-47, and Asp-48, are important for both potency and of and The of to the on both of in compound In the of is also of the of and and of the in compound of by an the affinity of PTP1B for compound by which is with the of these interactions not between and PTP1B Z. Wang D. Payette P. Ramachandran C. Kennedy J. Chem. 2001; Scholar), of the side of not the binding affinity for compound the affinity of compound for is by which may from a of between PTP1B and compound This the that could be as a of both affinity and S. W. J. Biol. Chem. 2000; 275: Scholar). is not the for the affinity and of compound for PTP1B, other PTPs leukocyte common antigen-related and of compound PTP1B Keng Y.-F. Wu L. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2001; Scholar). and are in a unique of and interactions with the and the These residues are highly the PTPs and are likely for the potency and by interactions are between and the including the of and a and a of the and the of the and the of of by an of compound for PTP1B by the in the in compound to an in results in an of inhibitory activity Wang F. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2002; Scholar). addition or of a single from of the also in a in affinity Keng Y.-F. Wu L. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2001; Scholar). these results suggest that the interactions of the with are of side from adjacent and in the for compound or compound Wang F. Lawrence D.S. Zhang Z.-Y. J. Biol. Chem. 2002; and data not PTP1B and an of a in to an a affinity for compound for that the structural at may be for the of in of an important role in binding both the and the aryl in compound shown in the of a with the of the of in with the of the in Furthermore, there are also interactions between the of the side of and the and the of the in compound of by an or residues at in other PTPs, results in and in binding for These results suggest that is the most important for PTP1B potency and binds to the the This may be to effects on both of the between the side of and the of and of interactions of and with the of the between the side of and the of the aryl contributes to the binding affinity between PTP1B and compound effects on the binding of compound for the mutants This is with the interactions between the and the Z. Wang D. Payette P. Ramachandran C. Kennedy J. Chem. 2001; Scholar). a in the second aryl phosphate-binding of PTP1B, is to an a in the affinity of compound for PTP1B is it not have on the and binding In in binding affinity of is for in with the that the acid in not with with the of the active site of the S. C. and Z.-Y. the results the that the second in may be of binding in and may be with residues and on the of the PTP1B is in its substrate as it a wide variety of substrates with Z.-Y. 2002; Scholar). The structural basis for the in PTP1B substrate recognition to be by a single Arg-47, which can both and residues M. Puius Y.A. Vetter S.W. Keng Y.-F. Wu L. Zhao Y. Lawrence D.S. Almo S.C. Zhang Z.-Y. Biochemistry. 2000; 39: 8171-8179Google Scholar). in and in on the nature of the residues to the side of can binding For example, a is at the of the EGFR a for a second is at the Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Google Scholar). a the in the PTP1B can to in interactions with both the and M. Puius Y.A. Vetter S.W. Keng Y.-F. Wu L. Zhao Y. Lawrence D.S. Almo S.C. Zhang Z.-Y. Biochemistry. 2000; 39: 8171-8179Google of in the of PTP1B with the EGFR the and compound in a The of an at the and a at the a of in that to that in the complex with a major in the and This interactions between with both the and the aryl the in compound is by a the affinity of the compound for PTP1B is potent that of compound is also highly selective for PTP1B, and a of for PTP1B all phosphatases In the of a crystal structure for to the structure of PTP1B in the The suggests that the interactions between and compound are in nature the of both the and The also a between a of the and the of Lys-41, which is by the and in the binding affinity of compound for and The effects on binding of compound with compound for the mutants suggest that most at can of the The results the of in PTP1B We have determined the crystal structure of PTP1B in complex with the most potent and selective PTP1B inhibitor identified to date. This together with results from kinetic analyses of the interactions of PTP1B and its site-directed mutants with the inhibitor and several of its a for the potency and of compound The results that the Tyr(P) the active the of the with a area residues Lys-41, Arg-47, and Asp-48. in addition to the second aryl phosphate-binding have identified the of the active which can also be to enhance potency and for Furthermore, that inhibitors with potency and can be by simultaneously both the area by Lys-41, Arg-47, and and the second aryl phosphate-binding the of be to in the design in order to the of PTP1B inhibitors cell In an for PTP1B (i.e. to inhibitors that both the active site as as unique adjacent could also be to specific inhibitors for all of the PTP that would the pharmacological of signaling pathways for treatment of diseases.