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Inhibition of Yersinia Tyrosine Phosphatase by Furanyl Salicylate Compounds

2004; Elsevier BV; Volume: 280; Issue: 10 Linguagem: Inglês

10.1074/jbc.m413122200

ISSN

1083-351X

Autores

Lutz Tautz, Shane Bruckner, Sina Sareth, Andrés Alonso, Jori Bogetz, Nunzio Bottini, Maurizio Pellecchia, Tomas Mustelin,

Tópico(s)

Chromatography in Natural Products

Resumo

To avoid detection and targeting by the immune system, the plague-causing bacterium Yersinia pestis uses a type III secretion system to deliver a set of inhibitory proteins into the cytoplasm of immune cells. One of these proteins is an exceptionally active tyrosine phosphatase termed YopH, which paralyzes lymphocytes and macrophages by dephosphorylating critical tyrosine kinases and signal transduction molecules. Because Y. pestis strains lacking YopH are avirulent, we set out to develop small molecule inhibitors for YopH. We used a novel and cost-effective approach, in which leads from a chemical library screening were analyzed and computationally docked into the crystal structure of YopH. This resulted in the identification of a series of novel YopH inhibitors with nanomolar Ki values, as well as the structural basis for inhibition. Our inhibitors lack the polar phosphate-mimicking moiety of rationally designed tyrosine phosphatase inhibitors, and they readily entered live cells and rescued them from YopH-induced tyrosine dephosphorylation, signaling paralysis, and cell death. These inhibitors may become useful for treating the lethal infection by Y. pestis. To avoid detection and targeting by the immune system, the plague-causing bacterium Yersinia pestis uses a type III secretion system to deliver a set of inhibitory proteins into the cytoplasm of immune cells. One of these proteins is an exceptionally active tyrosine phosphatase termed YopH, which paralyzes lymphocytes and macrophages by dephosphorylating critical tyrosine kinases and signal transduction molecules. Because Y. pestis strains lacking YopH are avirulent, we set out to develop small molecule inhibitors for YopH. We used a novel and cost-effective approach, in which leads from a chemical library screening were analyzed and computationally docked into the crystal structure of YopH. This resulted in the identification of a series of novel YopH inhibitors with nanomolar Ki values, as well as the structural basis for inhibition. Our inhibitors lack the polar phosphate-mimicking moiety of rationally designed tyrosine phosphatase inhibitors, and they readily entered live cells and rescued them from YopH-induced tyrosine dephosphorylation, signaling paralysis, and cell death. These inhibitors may become useful for treating the lethal infection by Y. pestis. To survive in humans, pathogenic bacteria have evolved numerous mechanisms to evade the immune response in the host (1Ernst J.D. Cell. Microbiol. 2000; 2: 379-386Crossref PubMed Scopus (91) Google Scholar, 2DeVinney I. Steele-Mortimer I. Finlay B.B. Trends Microbiol. 2000; 8: 29-33Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). One of the most successful strategies was adopted by Yersinia pestis, namely a type III secretion system that injects a set of paralyzing proteins directly into the cytoplasm of macrophages and lymphocytes that the bacterium encounters in the lymph nodes of infected individuals (3Persson C. Nordfelth R. Holmstrom A. Hakansson S. Rosqvist R. Wolf-Watz H. Mol. Microbiol. 1995; 18: 135-150Crossref PubMed Scopus (207) Google Scholar, 4Cheng L.W. Schneewind O. J. Bacteriol. 2000; 182: 3183-3190Crossref PubMed Scopus (70) Google Scholar). As a result, the targeted cells become unable to respond, and the bacteria can multiply unopposed by the normal mechanisms of host defense.The natural route of Y. pestis infection is by transmission from infected rats or other animals by blood-sucking fleas, which are weakened by the bacteria in their gut and therefore expel bacterial mass into the epidermis of their next victim when trying to feed (5Titball R.W. Leary S.E. Br. Med. Bull. 1998; 54: 625-633Crossref PubMed Scopus (46) Google Scholar, 6Hinnebusch B.J. J. Mol. Med. 1997; 75: 645-652Crossref PubMed Scopus (46) Google Scholar). From these flea bites, the bacteria travel to local lymph nodes (7Autenrieth I.B. Vogel U. Preger S. Heymer B. Heesemann J. Infect. Immun. 1993; 61: 2585-2595Crossref PubMed Google Scholar, 8Autenrieth I.B. Hantschmann P. Heymer B. Heesemann J. Immunobiology. 1993; 187: 1-16Crossref PubMed Scopus (56) Google Scholar, 9Autenrieth I.B. Firsching R. J. Med. 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Infect. 1997; 12: 12-23PubMed Google Scholar). By this route of infection, the number of bacteria entering the body can be much larger than from microscopic flea bites, and the bacteria are efficiently disseminated to the peritracheal, mediastinal, and other central lymph nodes, from which they gain access to the bloodstream much earlier. Although several vaccines exist (11Titball R.W. Williamson E.D. Vaccine. 2001; 19: 4175-4184Crossref PubMed Scopus (156) Google Scholar, 12Friedlander A.M. Welkos S.L. Worsham P.L. Andrews G.P. Heath D.G. Anderson Jr., G.W. Pitt M.L. Estep J. Davis K. Clin. Infect. Dis. 1995; 21: 178-181Crossref PubMed Scopus (116) Google Scholar), and Yersinia usually is sensitive to antibiotics, the pneumonic form of the disease is difficult to diagnose and still often results in death (10Cleri D.J. Vernaleo J.R. Lombardi L.J. Rabbat M.S. Mathew A. Marton R. Reyelt M.C. Semin. Respir. Infect. 1997; 12: 12-23PubMed Google Scholar).Despite efforts to eradicate the disease, natural reservoirs of Y. pestis still exist in wild rats and other rodent populations in parts of Africa, southeast Asia, and southwestern United States (13Christie A.B. Ecol. Dis. 1982; 1: 111-115PubMed Google Scholar), and sporadic human cases of plague still occur every year. Although these cases pale by comparison to the devastating pandemics that killed an estimated 200 million people, mostly in Europe, during historical times (5Titball R.W. Leary S.E. Br. Med. Bull. 1998; 54: 625-633Crossref PubMed Scopus (46) Google Scholar, 6Hinnebusch B.J. J. Mol. Med. 1997; 75: 645-652Crossref PubMed Scopus (46) Google Scholar), the World Health Organization now recognizes plague as a reemerging public health concern. There are also increasing fears that Y. pestis may be used for biological warfare or bioterrorism (14McGovern T.W. Christopher G.W. Eitzen E.M. Arch. Dermatol. 1999; 135: 311-322Crossref PubMed Scopus (62) Google Scholar, 15Inglesby T.V. Dennis D.T. Henderson D.A. Bartlett J.G. Ascher M.S. Eitzen E. Fine A.D. Friedlander A.M. Hauer J. Koerner J.F. Layton M. McDade J. Osterholm M.T. O'Toole T. Parker G. Perl T.M. Russell P.K. Schoch-Spana M. Tonat K. J. Am. Med. Assoc. 2000; 283: 2281-2290Crossref PubMed Scopus (839) Google Scholar, 16Hawley R.J. Eitzen Jr., E.M. Annu. Rev. Microbiol. 2001; 55: 235-253Crossref PubMed Scopus (120) Google Scholar). The potential threat is heightened by the existence of multidrug-resistant strains of Y. pestis (17Galimand M. Guiyoule A. Gerbaud G. Rasoamanana B. Chanteau S. Carniel E. Courvalin P. N. Engl. J. Med. 1997; 337: 677-680Crossref PubMed Scopus (358) Google Scholar, 18Guiyoule A. Gerbaud G. Buchrieser C. Galimand M. Eahalison L. Chanteau S. Courvalin P. Carniel E. Emerg. Infect. Dis. 2001; 7: 43-48Crossref PubMed Scopus (182) Google Scholar) and the rapidly lethal course of the pneumonic form of the disease caused by aerosolized Yersinia. Clearly, new approaches to combat plague are urgently needed.The molecular mechanisms employed by all virulent strains of Y. pestis and the two related species, Yersinia pseudotuberculosis and Yersinia enterocolitica, are based on an extrachromosomal virulence plasmid (19Cornelis G.R. Wolf-Watz H. Mol. Microbiol. 1997; 23: 861-867Crossref PubMed Scopus (474) Google Scholar), which encodes a type III secretion system and several effector proteins called Yops (Yersinia outer membrane proteins) (20Cornelis G.R. Boland A. Boyd A.P. Geuijen C. Iriarte M. Neyt C. Sory M.P. Stainier I. Microbiol. Mol. Biol. Rev. 1998; 62: 1315-1352Crossref PubMed Google Scholar). The type III secretion system is a highly conserved macromolecular machinery found in many pathogenic Gram-negative bacteria and is induced by contact with a eukaryotic cell to inject effector Yops into the cytoplasm of the target cells (21Juris S.J. Shao F. Dixon J.E. Cell. Microbiol. 2002; 4: 201-211Crossref PubMed Scopus (68) Google Scholar). In the host cell, the Yops disrupt signaling cascades responsible for initiating key immune functions, such as phagocytosis (22Black D.S. Bliska J.B. EMBO J. 1997; 16: 2730-2744Crossref PubMed Scopus (287) Google Scholar, 23Persson C. Carballeira N. Wolf-Watz H. Fallman M. EMBO J. 1997; 16: 2307-2318Crossref PubMed Scopus (298) Google Scholar, 24Andersson K. Carballeira N. Magnusson K.E. Persson C. Stendahl O. Wolf-Watz H. Fallman M. Mol. Microbiol. 1996; 20: 1057-1069Crossref PubMed Scopus (133) Google Scholar), respiratory burst (25Aepfelbacher M. Zumbihl R. Ruckdeschel K. Jacobi C.A. Barz C. Heesemann J. Biol. 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Dis. 1995; 21: 178-181Crossref PubMed Scopus (116) Google Scholar).A key Yop protein is YopH, a 468-amino acid, exceptionally active protein-tyrosine phosphatase (PTP) 1The abbreviations used are: PTP, protein-tyrosine phosphatase; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; pNPP, p-nitrophenyl phosphate; mAb, monoclonal antibody.1The abbreviations used are: PTP, protein-tyrosine phosphatase; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; pNPP, p-nitrophenyl phosphate; mAb, monoclonal antibody. (29Guan K. Dixon J.E. Science. 1990; 249: 553-556Crossref PubMed Scopus (412) Google Scholar, 30Bliska J.B. Guan K. Dixon J.E. Falkow S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1187-1191Crossref PubMed Scopus (313) Google Scholar) with a C-terminal catalytic domain and a multifunctional N-terminal domain, which binds tyrosine-phosphorylated target proteins (31Montagna L.G. Ivanov M.I. Bliska J.B. J. Biol. Chem. 2001; 276: 5005-5011Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 32Evdokimov A.G. Tropea J.E. Routzahn K.M. Copeland T.D. Waugh D.S. Acta Crystallogr. 2001; 57: 793-799Crossref PubMed Scopus (42) Google Scholar). The catalytic domain of YopH is structurally similar to that of eukaryotic PTPs (33Stuckey J.A. Schubert H.L. Fauman E.B. Zhang Z.Y. Dixon J.E. Nature. 1994; 370: 571-575Crossref PubMed Scopus (376) Google Scholar). A marked dephosphorylation of proteins in human epithelial cells and murine macrophages has been observed during infection with live bacteria (24Andersson K. Carballeira N. Magnusson K.E. Persson C. Stendahl O. Wolf-Watz H. Fallman M. Mol. Microbiol. 1996; 20: 1057-1069Crossref PubMed Scopus (133) Google Scholar, 30Bliska J.B. Guan K. Dixon J.E. Falkow S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1187-1191Crossref PubMed Scopus (313) Google Scholar, 34Bliska J.B. Clemens J.C. Dixon J.E. Falkow S. J. Exp. Med. 1992; 176: 1625-1630Crossref PubMed Scopus (119) Google Scholar, 35Hartland E.L. Green S.P. Phillips W.A. Robins-Browne R.M. Infect. Immun. 1994; 62: 4445-4453Crossref PubMed Google Scholar). In macrophages and neutrophils, the targets include the focal adhesion proteins Cas, focal adhesion kinase, and paxillin (22Black D.S. Bliska J.B. EMBO J. 1997; 16: 2730-2744Crossref PubMed Scopus (287) Google Scholar, 23Persson C. Carballeira N. Wolf-Watz H. Fallman M. EMBO J. 1997; 16: 2307-2318Crossref PubMed Scopus (298) Google Scholar), providing a molecular mechanism for inhibition of migration and phagocytosis by these cells (22Black D.S. Bliska J.B. EMBO J. 1997; 16: 2730-2744Crossref PubMed Scopus (287) Google Scholar, 23Persson C. Carballeira N. Wolf-Watz H. Fallman M. EMBO J. 1997; 16: 2307-2318Crossref PubMed Scopus (298) Google Scholar, 37Black D.S. Montagna L.G. Zitsmann S. Bliska J.B. Mol. Microbiol. 1998; 29: 1263-1274Crossref PubMed Scopus (82) Google Scholar, 38Ruckdeschel K. Roggenkamp A. Schubert S. Heesemann J. Infect. Immun. 1996; 64: 724-733Crossref PubMed Google Scholar).YopH also inhibits the activation of T and B lymphocytes (27Yao T. Mecsas J. Healy J.I. Falkow S. Chien Y. J. Exp. Med. 1999; 190: 1343-1350Crossref PubMed Scopus (119) Google Scholar, 39Alonso A. Bottini N. Bruckner S. Rahmouni S. Williams S. Schoenberger S.P. Mustelin T. J. Biol. Chem. 2004; 279: 4922-4928Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). We recently reported (39Alonso A. Bottini N. Bruckner S. Rahmouni S. Williams S. Schoenberger S.P. Mustelin T. J. Biol. Chem. 2004; 279: 4922-4928Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) that YopH in T cells directly dephosphorylated the Src family tyrosine kinase Lck at its positive regulatory site, Tyr-394, resulting in a complete loss of Lck activity. Because this kinase is the first upstream signal-generating molecule for the T cell antigen receptor, signaling from this receptor was completely abrogated. As a consequence, all tyrosine phosphorylation of downstream signaling proteins was inhibited; the T cells failed to form immune synapses with antigen-presenting cells, and they were unable to secrete any interleukin-2 into the medium (39Alonso A. Bottini N. Bruckner S. Rahmouni S. Williams S. Schoenberger S.P. Mustelin T. J. Biol. Chem. 2004; 279: 4922-4928Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Similarly, T cells exposed to live Y. enterocolitica became unable to flux calcium and produce cytokines (40Sauvonnet N. Lambermont I. van der Bruggen P. Cornelis G.R. Mol. Microbiol. 2002; 45: 805-815Crossref PubMed Scopus (73) Google Scholar).Because Yersinia strains that carry a pYV plasmid with a nonfunctional yopH gene are avirulent (41Bolin I. Wolf-Watz H. Mol. Microbiol. 1988; 2: 237-245Crossref PubMed Scopus (108) Google Scholar, 42Michielis T. Cornelis G. Microb. Pathog. 1988; 5: 449-459Crossref PubMed Scopus (52) Google Scholar, 43Rosqvist R. Bolin I. Wolf-Watz H. Infect. Immun. 1988; 56: 2139-2143Crossref PubMed Google Scholar, 44Straley S.C. Bowmer W.S. Infect. Immun. 1986; 51: 445-454Crossref PubMed Google Scholar) and even a point mutation that changes the catalytic Cys-403 to an alanine eliminates the virulence of Y. pseudotuberculosis in a murine infection model (30Bliska J.B. Guan K. Dixon J.E. Falkow S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1187-1191Crossref PubMed Scopus (313) Google Scholar, 34Bliska J.B. Clemens J.C. Dixon J.E. Falkow S. J. Exp. Med. 1992; 176: 1625-1630Crossref PubMed Scopus (119) Google Scholar), it is clear that the catalytic activity of YopH is critical for the lethality of Yersinia infection. We therefore set out to develop small molecule inhibitors of YopH by a combination of chemical library screening, structure-activity analysis, and in silico docking of lead compounds.MATERIALS AND METHODSReagents—p-Nitrophenyl phosphate (pNPP) was purchased from Sigma. BIOMOL GREEN™ reagent was from BIOMOL Research Laboratories (Plymouth Meeting, PA). All other chemicals and reagents were of the highest grade available commercially. Anti-phosphotyrosine mAb 4G10 was from Upstate Biotechnology, Inc. (Lake Saranac, NY), and mAb PY20 was from BD Biosciences.Plasmids and Protein Purification—The eukaryotic and prokaryotic expression plasmids for YopH were as described previously (39Alonso A. Bottini N. Bruckner S. Rahmouni S. Williams S. Schoenberger S.P. Mustelin T. J. Biol. Chem. 2004; 279: 4922-4928Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). YopH was expressed and purified as before (39Alonso A. Bottini N. Bruckner S. Rahmouni S. Williams S. Schoenberger S.P. Mustelin T. J. Biol. Chem. 2004; 279: 4922-4928Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The PTPs VHX (51Liang F. Huang Z. Lee S.-Y. Liang J. Alonso A. Bliska J.B. Lawrence D.S. Mustelin T. Zhang Z.-Y. J. Biol. Chem. 2003; 278: 41734-41741Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), VHR, VH1, LMPTP, and HePTP were expressed in Escherichia coli and purified as described previously (45Alonso A. Merlo J.J. Na S. Kholod N. Jaroszewski L. Kharitonenkov A. Williams S. Godzik A. Posada J.D. Mustelin T. J. Biol. Chem. 2002; 277: 5524-5528Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 46Alonso A. Rahmouni S. Williams S. van Stipdonk M. Jaroszewski L. Godzik A. Abraham R.T. Schoenberger S.P. Mustelin T. Nat. Immun. 2003; 4: 44-48Crossref Scopus (89) Google Scholar, 47Kholod N. Mustelin T. BioTechniques. 2001; 31: 322-328Crossref PubMed Scopus (56) Google Scholar, 48Saxena M. Williams S. Taskén K. Mustelin T. Nat. Cell Biol. 1999; 1: 305-311Crossref PubMed Scopus (184) Google Scholar, 49Saxena M. Williams S. Brockdorff J. Gilman J. Mustelin T. J. Biol. Chem. 1999; 274: 11693-11700Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Recombinant CD45, PTP1B, and LAR were purchased from BIOMOL Research Laboratories.Chemical Library Screening for YopH Inhibitors—A subset of 10,000 compounds from the DIVERSet™ library of 50,000 drug-like molecules (ChemBridge, Inc.) was screened in a 96-well format in vitro assay. Each reaction contained 50 nm YopH, 1 mmpNPP, and 0.03 mg/ml compound in 0.1 m BisTris, pH 6.0, reaction buffer. The final volume amounted to 50 μl and contained 2% Me2SO. The reaction was initiated by addition of pNPP after a preincubation of the enzyme with the compounds for 10 min at room temperature. After 7 min, the reaction was quenched by addition of 100 μl of BIOMOL GREEN™ reagent, and the pNPP hydrolysis was determined by measuring the absorbance of the complexed free phosphate at 620 nm. The nonenzymatic hydrolysis of the substrate was corrected by measuring the control without addition of enzyme. To quantitate the inhibitory efficacy of the library compounds, we determined the ratio of inhibition in comparison to 200 μm orthovanadate, a PTP inhibitor. Every compound with a ratio of >1 was considered as a hit. ClogP for each compound was calculated with ChemDraw8.Ki Determination—The YopH PTP-catalyzed hydrolysis of pNPP in the presence of inhibitors was assayed at 30 °C in 0.1 m BisTris, pH 6.0, assay buffer containing 1 mm dithiothreitol and 5% Me2SO. The ionic strength was adjusted to 150 mm with NaCl. The enzyme was preincubated with various fixed concentrations of inhibitors for 10 min. The reaction was initiated by the addition of various concentrations of pNPP (ranging from 0.2 to 10 Km) to the reaction mixtures to a final volume of 100 μl. The reaction was quenched by addition of 100 μl of 1 m NaOH. The nonenzymatic hydrolysis of the substrate was corrected by measuring the control without addition of enzyme. The amount of product p-nitrophenol was determined from the absorbance at 405 nm detected by a PowerWaveX340 microplate spectrophotometer (Bio-Tek Instruments, Inc.) using a molar extinction coefficient of 18,000 m-1 cm-1. The inhibition constant and inhibition pattern were evaluated by fitting the data to the Michaelis-Menten equations for either competitive (Equation 1), uncompetitive (Equation 2), or mixed (Equation 3) inhibition, using nonlinear regression and the program GraphPad Prism® (version 4.0).v0=Vmax[S]/(Km(app)+[S]) with Km(app)=Km(1+[I]/Ki) (Eq. 1) v0=Vmax[S]/(Km+(1+[I]/Ki)[S]) with Km(app)=Km/(1+[I]/Ki)(Eq. 2) v0=Vmax[S]/((1+[I]/Kic)Km+(1+[I]/Kiu)[S]) with Km(app)=Km(1+[I]/Kic)/(1+[I]/Kiu)(Eq. 3) In the case of the mixed inhibition model, Kic is the inhibition constant for the competitive participation, and Kiu is the inhibition constant for the uncompetitive participation. For a comparison of the fitting results, the second-order Akaike's Information Criterion (AICc) was calculated with Equation 4, where N is the number of data points, SS the absolute sum of squares, and K the number of parameters fit by nonlinear regression plus 1.AICc=Nln(SS/N)+2K+(2K(K+1))/(N−K−1)(Eq. 4) The probability to have chosen the right model can be computed by Equation 5, where Δ is the difference between Akaike's Information Criterion (AICc) scores.probability=exp(−0.5Δ)/(1+exp(−0.5Δ))(Eq. 5) IC50 Measurements—The PTP-catalyzed hydrolysis of pNPP in the presence of inhibitor was assayed at 30 °C in a 100-μl reaction system in the same assay buffer described above. At various concentrations of the compound, the initial rate at fixed pNPP concentrations (equal to the corresponding Km values for each PTP) was measured by determining the free phosphate with the BIOMOL GREEN™ reagent, as described above. The IC50 value was determined by plotting the relative pNPP activity versus inhibitor concentration and fitting to Equation 6 using GraphPad Prism®.Vi/V0=IC50/(IC50+[I])(Eq. 6) In this case, Vi is the reaction velocity when the inhibitor concentration is [I]; V0 is the reaction velocity with no inhibitor, and IC50 = Ki + Ki[S]/Km.Molecular Modeling—Molecular modeling studies were conducted on several R12000 SGI Octane workstations with the software package Sybyl version 6.9 (TRIPOS). Energy-minimized molecular models of the compounds were generated by the Sybyl/MAXIMIN2 routine. Flexible ligand docking calculations were performed with FlexX as implemented in Sybyl. For each compound, 20 solutions were generated and rank-ordered via FlexX score and CSCORE. In all cases, there was a high degree of convergence for the salicylic acid-furanyl moiety and more variability in the position of the remaining molecules. The coordinates of three-dimensional structure of catalytic domain of YopH (Protein Data Bank codes 1YTS and 1QZ0) were used in the docking studies, and the binding pocket was defined as composed of the following amino acid residues: Arg-205, Arg-228, Phe-229, Ile-232, Asn-245, Ala-258, Cys-259, Gln-260, Tyr-261, Val-284, Leu-285, Ala-286, Ser-287, Glu-290, Ile-291, Phe-296, Met-298, Val-351, Trp-354, Pro-355, Asp-356, Gln-357, Thr-358, Ala-359, Val-360, Ile-401, His-402, Ser-403 (Cys-403 in wild-type YopH), Arg-404, Ala-405, Gly-406, Val-407, Gly-408, Arg-409, Thr-410, Ala-411, Gln-412, Leu-413, Ile-414, Arg-440, Asn-441, Ile-443, Met-444, Val-445, Gln-446, Lys-447, and Gln-450. Molecular surfaces were generated with MOLCAD as implemented in Sybyl. Comparisons with other PTPs were made by using the x-ray coordinates for PTP1B (Protein Data Bank code 1PA1), VHR (Protein Data Bank code 1VHR), and bovine LMPTPB (1DG9) and the computer models of the membrane-proximal domains of CD45, VHX, and HePTP. These were generated as described (50Alonso A. Sasin J. Bottini N. Friedberg I. Friedberg I. Osterman A. Godzik A. Hunter T. Dixon J.E. Mustelin T. Cell. 2004; 117: 699-711Abstract Full Text Full Text PDF PubMed Scopus (1503) Google Scholar).Cells and Cell Treatments—Normal T lymphocytes were isolated from venous blood of healthy volunteers by Ficoll gradient centrifugation. Monocytes/macrophages were eliminated by adherence to plastic for 1 h at 37 °C. Jurkat T leukemia cells were kept at logarithmic growth in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mm l-glutamine, 1 mm sodium pyruvate, nonessential amino acids, and 100 units/ml each of penicillin G and streptomycin. For T cell receptor- and CD28-induced tyrosine phosphorylation responses, normal T lymphocytes were incubated in ice for 15 min with 10 μg/ml OKT3 and anti-CD28 mAbs, washed, and incubated with a cross-linking rabbit anti-mouse Ig for 15 min, washed, and transferred to 37 °C for 5 min. Cells were pelleted and lysed in 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA containing 1% Nonidet P-40, 1 mm Na3VO4, 10 μg/ml aprotinin and leupeptin, 100 μg/ml soybean trypsin inhibitor, and 1 mm phenylmethylsulfonyl fluoride and clarified by centrifugation at 15,000 rpm for 20 min. Lysate was mixed with an equal volume of twice concentrated SDS sample buffer, boiled for 1 min, and resolved by SDS-PAGE.SDS-PAGE and Immunoblotting—These procedures were done as before (39Alonso A. Bottini N. Bruckner S. Rahmouni S. Williams S. Schoenberger S.P. Mustelin T. J. Biol. Chem. 2004; 279: 4922-4928Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar).Interleukin-2 Secretion Assay—5 × 106 human T lymphocytes were treated with 6 μm ANT-YopH for5hat37 °Cin RPMI medium, washed, and stimulated with C305, anti-CD28 mAb, plus a cross-linking anti-mouse Ig for 15 h in 250 μl of medium with 10% fetal calf serum. 20 μl of the supernatant was used for measurement of the amount of interleukin-2 using an enzyme-linked immunosorbent assay kit from Roche Applied Science, as before (39Alonso A. Bottini N. Bruckner S. Rahmouni S. Williams S. Schoenberger S.P. Mustelin T. J. Biol. Chem. 2004; 279: 4922-4928Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Results are given as pg/ml of secreted interleukin-2/2 × 105 cells.RESULTSIdentification of Lead Compounds by Chemical Library Screening—A 96-well format in vitro assay was used to screen the first 10,000 compounds of the DIVERSet™ library (ChemBridge, Inc.) of drug-like compounds. A total of 10 compounds inhibited YopH to a higher extent than 200 μm orthovanadate, a general PTP inhibitor. After determination of the kinetic parameters of these 10 first hits, we selected four compounds, which were showing a competitive or mixed inhibition pattern with a Ki value 100>100>256>26LMPTP B>100>100>256>26VH1>100>100>256>26LAR>100>100>256>26 Open table in a new tab Table IIKinetic data of 2 initial hits and 21 representative analogs Open table in a new tab Structure-Activity Relationship Analysis of Compound 1 and 2—Encouraged by these findings, we investigated the structure-activity relationships for a total of 61 analogs that all contained a substituted phenyl ring linked via a furanyl moiety to a more diverse entity at the other end of the molecule, preferentially a 5-methylenethiazolidine ring. A total of 44 compounds inhibited YopH in a competitive manner with Ki values of <100 μm. The structures and kinetic data for a representative set of 21 analogs are given in Table II. Significantly, 13 of the 16 salicylic acid analogs were among the 26 best compounds (competitive inhibition with Ki values <10 μm). Elimination of the salicylic acid moiety (compound 23) led to a complete loss of YopH inhibition, whereas salicyl furyl aldehyde (compound 13) shows good competitive inhibition (Ki = 2.08 μm). However, salicylic acid alone was a poor inhibitor (Ki = 882 μm) compared with the most potent inhibitor, compound 3, which has a 6,211-fold lower inhibitory constant (Ki = 0.143 μm). The very similar compound 7, which only differs in the location of a single methyl group, shows almost 10 times less activity, suggesting some steric constraints for the putative binding site or some very specific interactions of that methyl group. A comparison of the Ki values for compounds 12 and 20, which differ by a methyl group in a very similar position, supports the latter.Another example of a unique binding mode is presented by the second best inhibitor, compound 4 (Ki = 0.208 μm). In this case, the structurally very similar compound 18 (Ki = 6.60 μm), in which the positions of the carboxylic and the hydroxyl groups have been switched, has a 32 times higher Ki value, supporting the notion of specific binding.Substitution of the hydroxyl group in the original hit compound 1 by a chlorine in compound 16 led to a 14.5 times less inhibitory activity. Elimina

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