Conformation-assisted Inhibition of Protein-tyrosine Phosphatase-1B Elicits Inhibitor Selectivity over T-cell Protein-tyrosine Phosphatase

PTP-1B represents an attractive target for the treatment of type 2 diabetes and obesity. Given the role that protein phosphatases play in the regulation of many biologically relevant processes, inhibitors against PTP-1B must be not only potent, but also selective. It has been extremely difficult to synthesize inhibitors that are selective over the highly homologous TCPTP. We have successfully exploited the conservative Leu119 to Val substitution between the two enzymes to synthesize a PTP-1B inhibitor that is an order of magnitude more selective over TCPTP. Structural analyses of PTP-1B/inhibitor complexes show a conformation-assisted inhibition mechanism as the basis for selectivity. Such an inhibitory mechanism may be applicable to other homologous enzymes. PTP-1B represents an attractive target for the treatment of type 2 diabetes and obesity. Given the role that protein phosphatases play in the regulation of many biologically relevant processes, inhibitors against PTP-1B must be not only potent, but also selective. It has been extremely difficult to synthesize inhibitors that are selective over the highly homologous TCPTP. We have successfully exploited the conservative Leu119 to Val substitution between the two enzymes to synthesize a PTP-1B inhibitor that is an order of magnitude more selective over TCPTP. Structural analyses of PTP-1B/inhibitor complexes show a conformation-assisted inhibition mechanism as the basis for selectivity. Such an inhibitory mechanism may be applicable to other homologous enzymes. The quest for agents that can intervene in type 2 diabetes continues to be a major research focus and challenge in many laboratories. As impaired insulin action is an underlying mechanism in type 2 diabetes, the insulin signaling pathway has naturally been the focus of research in attempts to identify suitable therapeutic target(s) for drug intervention against the disease. Insulin signaling begins with the activation of the insulin receptor (IR) via tyrosine phosphorylation and culminates in the uptake of glucose into cells by the glucose transporter, Glut4 (1Saltiel A.R. Kahn C.R. Nature. 2001; 414: 799-806Crossref PubMed Scopus (3973) Google Scholar). The activated IR must then be deactivated and returned to a basal state, a process that is believed to involve protein-tyrosine phosphatase-1B (PTP-1B). 4The abbreviations used are: PTP-1B, protein-tyrosine phosphatase 1B; TCPTP, T-cell protein-tyrosine phosphatase; FDP, fluorescein diphosphate; Rsmd, root-mean-square deviation; WT, wild type. PTP-1B has been shown to directly interact with the activated insulin receptor (2Romsicki Y. Reece M. Gauthier J-Y. Asante-Appiah E. Kennedy B.P. J. Biol. Chem. 2004; 279: 12868-12875Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 3Salmeen A. Jannik N. Andersen M. Meyers P. Tonks N.K. Barford D. Mol. Cell. 2000; 6: 1401-1412Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 4Ahmad F. Meyerovitch J. Goldstein B.J. J. Biol. Chem. 1995; 270: 20503-20508Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 5Seely B.L. Staubs P.A. Reichart D.R. Berhanu P. Milarsky K.L. Saltiel A.R. Kusari J. Olefsky J.M. Diabetes. 1996; 45: 1379-1385Crossref PubMed Google Scholar, 6Dadke J. Kusari J. Chernoff J. J. Biol. Chem. 2000; 275: 23642-23647Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Disruption of the gene that codes for PTP-1B in mice results in sensitivity to insulin and also increased resistance to diet-induced obesity (7Elchebly 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: 1544-1548Crossref PubMed Scopus (1926) Google Scholar, 8Klaman 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-krongard A. Shulman G.I. Neel B.G. Kahn B.B. Mol. Cell. Biol. 2000; 20: 5479-5489Crossref PubMed Scopus (1130) Google Scholar). This phosphatase is therefore a very attractive therapeutic target for the treatment not only of type 2 diabetes but also of obesity. Predictably, there has been an intense research effort by many groups to identify potent and selective inhibitors of PTP-1B (for recent reviews, see Refs. 9Taylor S.D. Hill B. Exp. Opin. Investig. Drugs. 2004; 13: 199-214Crossref PubMed Scopus (88) Google Scholar and 10Asante-Appiah E. Kennedy B.P. Am. J. Physiol. Endocrinol. Metab. 2003; 284: E663-E670Crossref PubMed Scopus (168) Google Scholar). PTP-1B belongs to the protein-tyrosine phosphatase (PTP) superfamily of enzymes, which includes ∼100 members involved in signal transduction and regulation of cellular processes such as growth, differentiation, and proliferation. Inhibitors against the enzyme must thus be not only potent but also selective. Sequence alignment of amino acids residues within the PTPs catalytic domain shows that, in general, all phosphatases have less than 40% identity to PTP-1B: a remarkable exception is the T-cell protein-tyrosine phosphatase (TCPTP), which has a 72% identity (11Iversen L.F. Moller K.B. Pedersen A.K. Peters G.H. Petersen A.S. Andersen H.S. Branner S. Mortensen S.B. Moller N.P. J. Biol. Chem. 2002; 277: 19982-19990Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 12Cool D.E. Tonks N.K. Carbonneau H. Walsh K.A. Fisher E.H. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5257-5261Crossref PubMed Scopus (230) Google Scholar, 13Guan K.L. Haun R.S. Watson S.J. Geahlen R.L. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1501-1505Crossref PubMed Scopus (154) Google Scholar, 14Chernoff J. Schievalla A.R. Jost C.A. Erikson R.L. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2735-2739Crossref PubMed Scopus (168) Google Scholar). Inhibitors that target PTP-1B and incorporate previously identified selectivity determinants (15Asante-Appiah E. Ball K. Bateman K. Skorey K. Friesen R. Desponts C. Payette P. Bayly C. Zamboni R. Scapin G. Ramachandran C. Kennedy B.P. J. Biol. Chem. 2001; 276: 26036-26043Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 16Puius Y.A. Zhao Y. Sullivan M. Lawrence D.S. Almo S.C. Zhang Z.Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13420-13425Crossref PubMed Scopus (404) Google Scholar, 17Sun J.-P. Fedorov A.A. Lee S.-Y. Guo X.-L. Shen Lawrence D.S. Almo S.C. Zhang Z.-Y. J. Biol. Chem. 2003; 278: 12406-12414Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) are generally selective (>30-fold) over all PTPs tested but are equipotent on TCPTP. Although it is not evident that co-inhibition of TCPTP (with PTP-1B) will result in serious adverse effects, mice lacking the TCPTP gene die within 3-5 weeks after birth from defects in hematopoiesis and immune function (18You-Ten K.E. Muise E.S. Itie A. Michaliszyn E. Wagner J. Jothy S. Lapp W.S. Tremblay M.L. J. Exp. Med. 1997; 186: 683-693Crossref PubMed Scopus (318) Google Scholar). Hence, it is highly desirable to design PTP-1B inhibitors that are selective over TCPTP as potential therapeutic agents particularly as type 2 diabetes is a chronic disease. The only demonstrable structural determinant(s) of inhibitor selectivity between PTP-1B and TCPTP reported to date is located in the enzyme secondary binding site (16Puius Y.A. Zhao Y. Sullivan M. Lawrence D.S. Almo S.C. Zhang Z.Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13420-13425Crossref PubMed Scopus (404) Google Scholar), and inhibitors targeting that area have been shown to have some discriminatory capability between the two enzymes (19Erlanson D.A. McDowell R.S. He M.M. Randal M. Simmons R.L. Kung J. Waight A. Hansen S.K. J. Am. Chem. Soc. 2003; 125: 5602-5603Crossref PubMed Scopus (52) Google Scholar, 20Liu G. Szczepankiewicz B.G. Pei Z. Janowick D.A. Xin Z. Liang H. Hajduk P.J. Abad-Zapatero C. Hutchins C.W. Fesik S.W. Ballaron S.J. Stashko M.A. Lubben T. Mika A.K. Zinker B.A. Trevillyan J.M. Jirousek M.R. J. Med. Chem. 2003; 46: 2093-2103Crossref PubMed Scopus (70) Google Scholar, 21Xin Z. Oost T.K. Abad-Zapatero C. Hajduk P.J. Pei Z. Szczepankiewicz B.G. Hutchins C.W. Ballaron S.J. Stashko M.A. Lubben T. Trevillyan J.M. Jirousek M.R. Liu G. Bioorg. Med. Chem. Lett. 2003; 13: 1887-1890Crossref PubMed Scopus (66) Google Scholar, 22Lau C.K. Bayly C.I. Gauthier J.Y. Li C.S. Therien M. Asante-Appiah E. Cromlish W. Boie Y. Forghani F. Desmarais S. Wang Q. Skorey K. Waddleton D. Payette P. Ramachandran C. Kennedy B.P. Scapin G. Bioorg. Med. Chem. Lett. 2004; 14: 1043-1048Crossref PubMed Scopus (74) Google Scholar, 23Scapin G. Patel S.B. Becker J.W. Wang Q. Desponts C. Waddleton D. Skorey K. Cromlish W. Bayly C. Therien M. Gauthier J.Y. Li C.S. Lau C.K. Ramachandran C. Kennedy B.P. Asante-Appiah E. Biochemistry. 2003; 42: 11451-11459Crossref PubMed Scopus (101) Google Scholar). Very recently, Wiesmann et al. (24Wiesmann C. Barr K.J. Kung J. Zhu J. Erlanson D.A. Shen W. Fahr B.J. Zhong M. Taylor L. Randal M. McDowell R.S. Hansen S.K. Nat. Struct. Mol. Biol. 2004; 11: 730-737Crossref PubMed Scopus (408) Google Scholar) reported the discovery of a novel allosteric site that could be used for PTP-1B inhibition. Although the discovery of this site appears to be promising for the design of inhibitors with enhanced pharmacological properties, the issue of selectivity versus TCPTP remains partially unaddressed. The two enzymes share over 95% of sequence similarity in this area, and are structurally identical, although TCPTP lacks the phenylalanine at position 280 that appears to play a role in inhibitor binding. In order to further address the selectivity problem, we report here the identification of a previously unknown structural determinant between PTP-1B and TCPTP, and the biochemical and mutagenesis data that confirmed it. This new site may be exploited, alone or in conjunction with other selectivity determinants, to increase potency and elicit selectivity. Mutagenesis, Protein Expression, and Purification—A plasmid that expresses the isolated catalytic domain of human PTP-1B, pFlag2-hPTP-1B-(1-298), was used as template DNA for the mutagenesis. The Chameleon™ double-stranded mutagenesis kit (Stratagene) was employed for all site-directed mutagenesis by following the manufacturer's instructions. Oligonucleotides used for the site-directed mutagenesis were custom-synthesized by Invitrogen. Codons specifying the targeted amino acid changes were incorporated into the oligonucleotides as shown below (the codon changes that were introduced have been underlined): V113I, 5′-GTCATGCTCAACAGAATAATGGAGAAAGGTTCG-3′; M114V, 5′-ATGCTCAACAGAGTGGTTGAGAAAGGTTCGTTA-3′; V113I,M114V, 5′-GTCATGCTCAACAGAATAGTTGAGAAAGGTTCGTTA-3′; G117E, 5′-AGAGTGATGGAGAAAGAATCGTTAAAATGCGCA-3′; L119V, 5′-ATGGAGAAAGGTTCGGTTAAATGCGCACAATAC 3′. For TCPTP, the mutations were introduced into pFlag2-hTCPTP-(1-296) that was used as template DNA. The oligonucleotides used for introduction of the mutation that codes for the V121L substitution were as follows: 5′-GTGGAGAAAGAATCGCTGAAATGTGCACAGTAC-3′. The correct construction of the mutant derivatives was verified by DNA sequencing. DNA was subsequently isolated from the positive clones and introduced into Escherichia coli BL21 cells for protein expression. Protein expression was assessed by growing E. coli BL21 cells to an optical density of 0.7 at 600 nm in Luria Bertani broth supplemented with 100 μg/ml ampicillin (LB-Amp)2. The culture was then induced by addition of isopropyl-1-thio-β-d-galactopyranoside to a final concentration of 1 mm. Cells were harvested 3-h postinduction and the lysate analyzed by electrophoresis on a 10-20% (w/v) polyacrylamide gradient gel containing sodium dodecyl sulfate (SDS-PAGE). The separated bacterially expressed proteins were transferred onto nitrocellulose and probed with an anti-FLAG monoclonal antibody by chemiluminescence. The Super Signal™ West Pico kit was used for the detection of proteins by following the suggested protocol. Activity Assays—The FLAG-tagged catalytic domains of WT and mutant PTP-1B and TCPTP proteins were expressed in E. coli and purified as reported previously (15Asante-Appiah E. Ball K. Bateman K. Skorey K. Friesen R. Desponts C. Payette P. Bayly C. Zamboni R. Scapin G. Ramachandran C. Kennedy B.P. J. Biol. Chem. 2001; 276: 26036-26043Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 25Romsicki Y. Kennedy B.P. Asante-Appiah E. Arch. Biochem. Biophys. 2003; 414: 40-50Crossref PubMed Scopus (23) Google Scholar). The activity of all enzymes was assayed with fluorescein diphosphate (FDP) as substrate as previously reported (23Scapin G. Patel S.B. Becker J.W. Wang Q. Desponts C. Waddleton D. Skorey K. Cromlish W. Bayly C. Therien M. Gauthier J.Y. Li C.S. Lau C.K. Ramachandran C. Kennedy B.P. Asante-Appiah E. Biochemistry. 2003; 42: 11451-11459Crossref PubMed Scopus (101) Google Scholar, 26Huang Z. Wang Q. Ly H.D. Gorvindarajan A. Scheigetz J. Zamboni R. Desmarais S. Ramachandran C. J. Biomol. Screen. 1999; 4: PubMed Scopus (52) Google Scholar). were by the of to the with the of the inhibitor that inhibition of enzyme activity were by in the and of inhibitor to a the of the all of of was as previously C.K. Bayly C.I. Gauthier J.Y. Li C.S. Therien M. Asante-Appiah E. Cromlish W. Boie Y. Forghani F. Desmarais S. Wang Q. Skorey K. Waddleton D. Payette P. Ramachandran C. Kennedy B.P. Scapin G. Bioorg. Med. Chem. Lett. 2004; 14: 1043-1048Crossref PubMed Scopus (74) Google Scholar). of the isolated catalytic domains of WT PTP-1B and mutant in with 1 were as previously reported (23Scapin G. Patel S.B. Becker J.W. Wang Q. Desponts C. Waddleton D. Skorey K. Cromlish W. Bayly C. Therien M. Gauthier J.Y. Li C.S. Lau C.K. Ramachandran C. Kennedy B.P. Asante-Appiah E. Biochemistry. 2003; 42: 11451-11459Crossref PubMed Scopus (101) Google Scholar). The data were from a at the in the of the at the were for in in in the The data were and from the Scholar). The WT 1 was by the J. A. Scopus Google Scholar) and the from (16Puius Y.A. Zhao Y. Sullivan M. Lawrence D.S. Almo S.C. Zhang Z.Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13420-13425Crossref PubMed Scopus (404) Google Scholar), and as of the was by of of the in M. 1997; 277: PubMed Scopus Google Scholar) and P. J. M. T. D. PubMed Scopus Google Scholar). For all data of the data were for Nature. PubMed Scopus Google was the and the was the A. 1996; Scopus Google Scholar). was at the of the and the two of Struct. PubMed Scopus Google Scholar), and were in were used to of the protein that were in the 1 the For the of the mutant A. 42: Scopus Google Scholar) 2 and density were in from the WT 1 with and to for the of the was then as for the WT but for the of D. PubMed Scopus Google Scholar, A.A. D. 1997; PubMed Scopus Google Scholar, D. PubMed Scopus Google Scholar, A.A. A. D. 1999; PubMed Scopus Google Scholar) as the 1 the for data and and for the data used to the of PTP-1B WT and in with of protein in a new The and for the here have been with the Protein codes and of of not inhibitors that were selective for PTP-1B over TCPTP. to identify between TCPTP and PTP-1B by enzyme not between the two enzymes that could be exploited for inhibitor design to selectivity (23Scapin G. Patel S.B. Becker J.W. Wang Q. Desponts C. Waddleton D. Skorey K. Cromlish W. Bayly C. Therien M. Gauthier J.Y. Li C.S. Lau C.K. Ramachandran C. Kennedy B.P. Asante-Appiah E. Biochemistry. 2003; 42: 11451-11459Crossref PubMed Scopus (101) Google Scholar). a structural of the of enzymes not determinants of selectivity within the site The structural a of identity between the enzymes the residues of the inhibitor binding we to PTP-1B inhibitor data to identify that a of selectivity between the two enzymes. a and an of the data it that the of an in a inhibitors as by 1 in 1 in a very selectivity over TCPTP. of 1 a selectivity between the enzymes. that the of the was not selective between the enzymes of the of PTP-1B in with other inhibitors as (23Scapin G. Patel S.B. Becker J.W. Wang Q. Desponts C. Waddleton D. Skorey K. Cromlish W. Bayly C. Therien M. Gauthier J.Y. Li C.S. Lau C.K. Ramachandran C. Kennedy B.P. Asante-Appiah E. Biochemistry. 2003; 42: 11451-11459Crossref PubMed Scopus (101) Google Scholar), we that the be the residues to as We that this may as a potential of at from the identify the for the selectivity we the amino acid of PTP-1B and TCPTP to in this were at and in PTP-1B is with in is with a is to and is a The amino acid changes in TCPTP were then introduced into PTP-1B by site-directed mutagenesis. The proteins were purified to and residues and are and could a the was also The introduction of the amino acid not the of PTP-1B The for for all the mutant the was within of the for the the structural of the we also the of the amino acid on the inhibitory potency of a binding on PTP-1B has been reported and not involve E. Patel S. C. P. Wang Q. Patel Friesen Ramachandran C. Becker J.W. Y. Kennedy B.P. Scapin G. Biochemistry. 2002; PubMed Scopus Google Scholar). were between the and PTP-1B with to inhibitory potency of we that the amino acid changes in not the of the we the of the amino acid on the potency of the selective As shown in the mutant proteins for 1 with the exception of the The to amino acid substitution at position in a in potency of to the selectivity that was over of PTP-1B with as in a new for on PTP-1B wild type and in a new for 1 on in a new of PTP-1B WT and in with further the of Leu119 in the selectivity and a structural basis for effects, we the of the isolated catalytic domains of and of the mutant in with 1 the for the In the protein in with two In the WT the inhibitor with the in the binding or in in the catalytic is The is located the in it with the of The the secondary binding site by et 16Puius Y.A. Zhao Y. Sullivan M. Lawrence D.S. Almo S.C. Zhang Z.Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13420-13425Crossref PubMed Scopus (404) Google in and the is located in the of the residues to type in This in of the reported to was by with in an and In the of the is not involved in with other and appears to be very E. Patel S. C. P. Wang Q. Patel Friesen Ramachandran C. Becker J.W. Y. Kennedy B.P. Scapin G. Biochemistry. 2002; PubMed Scopus Google Scholar). In the of other inhibitors to 1 but with a in of the (23Scapin G. Patel S.B. Becker J.W. Wang Q. Desponts C. Waddleton D. Skorey K. Cromlish W. Bayly C. Therien M. Gauthier J.Y. Li C.S. Lau C.K. Ramachandran C. Kennedy B.P. Asante-Appiah E. Biochemistry. 2003; 42: 11451-11459Crossref PubMed Scopus (101) Google Scholar), it appears to be to a from that in the of inhibitors show selectivity between PTP-1B and TCPTP as by 2 In the 1 the two in the two but it appears in to be in a with the of Leu119 within from the The mutant is extremely to the WT and the inhibitor in the The only is that in the mutant the is highly and density for the residues was not This further that the of Leu119 is for of the of the position of the in the WT PTP-1B: 1 and PTP-1B 1 of the in the WT the density at is and the of the of the in the the density is as The as in the WT enzyme is shown in for In this the is highly and density for residues is not The V121L in suggested a structural mechanism for the role of Leu119 as a selectivity determinant between PTP-1B and TCPTP. or not that alone was to elicit the selectivity between the two enzymes, we the Val to substitution into TCPTP. As shown in substitution of Val at position to position in PTP-1B) selectivity between the and the The selectivity was between the two derivatives as was for PTP-1B that the at position is and to selectivity. shown in is a PTP-1B inhibitor that of the and is an order of magnitude more selective over TCPTP. of the from this inhibitor selectivity between the two phosphatases not In this the also as a more for the of As substitution of Leu119 to Val the selectivity with PTP-1B over TCPTP the V121L a in TCPTP. the amino acid from to Val PTP-1B and TCPTP with to the and to Val between PTP-1B and inhibitor selectivity in a new We have previously reported on structural determinants on PTP-1B that selectivity over other PTPs with the exception of TCPTP (15Asante-Appiah E. Ball K. Bateman K. Skorey K. Friesen R. Desponts C. Payette P. Bayly C. Zamboni R. Scapin G. Ramachandran C. Kennedy B.P. J. Biol. Chem. 2001; 276: 26036-26043Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). determinants it was to synthesize inhibitors that were selective over other PTPs and of magnitude more selective over a we have reported the identification of a selectivity determinant between PTP-1B and the structurally highly homologous TCPTP by a previously unknown of PTP-1B the to an of the data that the of the inhibitor the the position of the The of Leu119 with the some of binding the 1 the has an of versus the nm of The may be by the that, although the is there is a in by the from the and it into a more of with Val in the TCPTP has the of the of between the and the with of binding and increase in the of the two and that a in position is not to the function as the thus that Leu119 is the selectivity determinant in this of between PTP-1B and TCPTP. we the Val to substitution into TCPTP. The was that the substitution a selectivity between TCPTP and that alone to elicit selectivity and it In this we also the to the structural determinant to design a more selective PTP-1B inhibitor over TCPTP. of the potential of this we have also at this in all PTPs in the data not all the PTP-1B is the only enzyme that at position Hence, it that other PTPs to this may be exploited for selectivity. We will the for the design and of more potent and selective PTP-1B As the is to the site of PTP-1B, targeting Leu119 the design and of selective inhibitors that are less with inhibitors to target the secondary binding site by Such inhibitors will have a of issue with the design and of PTP-1B inhibitors as therapeutic The identification of Leu119 as a determinant of inhibitor selectivity was not from a structural of the of and TCPTP alone (11Iversen L.F. Moller K.B. Pedersen A.K. Peters G.H. Petersen A.S. Andersen H.S. Branner S. Mortensen S.B. Moller N.P. J. Biol. Chem. 2002; 277: 19982-19990Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). We to such the targeted structural is not from the as it may not be induced substrate binding to inhibitor is a to the remarkable of protein and to We that conformation-assisted inhibition may be applicable to other structurally highly homologous proteins We the at the at the for data The at are by the of the a with of for and of the was by the U. S. of of

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