1108
D. Vidal et al. / European Journal of Medicinal Chemistry 42 (2007) 1102e1108
[6] T.N. Doman, S.L. McGovern, B.J. Witherbee, T.P. Kasten, R. Kurumbail,
W.C. Stallings, D.T. Connolly, B.K. Shoichet, J. Med. Chem. 45 (2002)
2213e2221.
concentration determined by the relationship 1AU (A280) ¼
0.84(ꢃ0.05) g/l [40].
[7] C.N. Cavasotto, M.A. Ortiz, R.A. Abagyan, F.J. Piedrafita, Bioorg. Med.
Chem. Lett. 16 (2006) 1969e1974.
5.2. Enzymatic activity
[8] D.B. Kitchen, H. Decornez, J.R. Furr, J. Bajorath, Nat. Rev. Drug Discov.
3 (2004) 935e949.
Kinetic parameters were measured at 37 ꢄC in 40 mM so-
dium acetate buffer at pH 5.0 containing 150 mM NaCl and
1 mM TCEP (tris[2-carboxyethyl]phosphine) assuring com-
plete reduction of the active site Cys12. p-Nitrophenyl phos-
phate ( pNPP) was used as the substrate and the reaction rate
was determined by monitoring the release of p-nitrophenolate
by measuring the absorbance at a wavelength of 405 nm as de-
scribed before [39]. A constant concentration of 10 nM bovine
lmwPTP was used at the five final pNPP concentrations of 0.4,
0.8, 1.2, 1.6 and 2.0 mM. Each velocity was measured three
times and the Vmax and Km values were obtained giving
Km ¼ 0.57 ꢃ 0.03 mM and Vmax ¼ 21.3 ꢃ 0.6 mmol/min, which
is in good agreement with previous results [39,41].
[9] J.S. Mason, A.C. Good, E.J. Martin, Curr. Pharm. Des. 7 (2001)
567e597.
[10] R.D. Cramer, D.E. Patterson, J.D. Bunce, J. Am. Chem. Soc. 110 (1988)
5959e5967.
[11] P. Willett, J.M. Barnard, G.M. Downs, J. Chem. Inf. Comput. Sci. 38
(1998) 983e996.
[12] R. Macarron, Drug Discov. Today 11 (2006) 277e279.
[13] W.P. Walters, M.T. Stahl, M.A. Murcko, Drug Discov. Today 3 (1998)
160e178.
[14] T. Lengauer, C. Lemmen, M. Rarey, M. Zimmermann, Drug Discov. To-
day 1 (2004) 27e34.
[15] D. Vidal, M. Thormann, M. Pons, J. Chem. Inf. Model. 45 (2005)
386e393.
[16] J.A. Grant, J.A. Haigh, B.T. Pickup, A. Nicholls, R.A. Sayle, J. Chem.
Inf. Model. 46 (2006) 1912e1918.
[17] D. Vidal, M. Thormann, M. Pons, J. Chem. Inf. Model. 46 (2006)
836e843.
5.3. Measurement of inhibition constants Ki
[18] M. Zhang, R.L. Van Etten, C.V. Stauffacher, Biochemistry 33 (1994)
11097e11105.
[19] T. Hunter, Cell 100 (2000) 113e127.
[20] L. Bialy, H. Waldmann, Angew. Chem. Int. Ed. 44 (2005) 3814e3839.
[21] P. Tailor, J. Gilman, S. Williams, T. Mustelin, Eur. J. Biochem. 262
(1999) 277e282.
Measurements of the dissociation constants (Ki) of the en-
zymeeinhibitor complexes were carried out at pH 5.0 and
37 ꢄC using the same reaction buffer as mentioned before.
All inhibitors were stored at 100 mM in DMSO. Inhibition
constants were determined at constant concentration of
10 nM bovine lmwPTP, five different pNPP concentrations
(0.4, 0.8, 1.2, 1.6 and 2.0 mM) and four to five suitable inhib-
itor concentrations. DMSO concentration was kept constant at
1% throughout all measurements. Details, such as Linewea-
vereBurk plots and experimental data, for the measurement
of Ki can be found in the Supplementary material.
[22] K.D. Kikawa, D.R. Vidale, R.L. Van Etten, M.S. Kinch, J. Biol. Chem.
277 (2002) 39274e39279.
[23] Y.A. Puius, Y. Zhao, M. Sullivan, D.S. Lawrence, S.C. Almo,
Z.Y. Zhang, Proc. Natl. Acad. Sci. 94 (1997) 13420e13425.
[24] P.J. Scrivens, M.A. Alaoui-Jamali, G. Giannini, T. Wang, M. Loignon,
G. Batist, V.A. Sandor, Mol. Cancer Ther. 2 (2003) 1053e1059.
[25] K.R. Kim, J.L. Kwon, J.S. Kim, Z. No, H.R. Kim, H.G. Cheon, Eur.
J. Pharmacol. 528 (2005) 37e42.
[26] R.A. Urbanek, S.J. Suchard, G.B. Steelman, K.S. Knappenberger,
L.A. Sygowski, C.A. Veale, M.J. Chapdelaine, J. Med. Chem. 44
(2001) 1777e1793.
Acknowledgements
[28] ChemBridge Research Laboratories, Inc. San Diego (CA) USA.http://
This work was partially supported by funds from the Span-
´
ish Ministerio de Educacion y Ciencia-FEDER (BIO2004-
5436, PTR1995-0795-OP, GEN2003-20642-C09-04). D.V. and
J.B. acknowledge predoctoral fellowships from the Spanish
[29] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart,
R.K. Belew, A.J. Olson, J. Comput. Chem. 19 (1998) 1639e1662.
[30] M. Thormann, M. Pons, J. Comput. Chem. 22 (2001) 1971e1982.
[31] M. Zhang, M. Zhou, R.L. Van Etten, C.V. Stauffacher, Biochemistry 36
(1997) 15e23.
´
Ministerio de Educacion y Ciencia.
[32] B. Evans, P.A. Tishmack, C. Pokalsky, M. Zhang, R.L. Van Etten, Bio-
chemistry 35 (1996) 13609e13617.
Appendix A. Supplementary material
[34] D.J. Weininger, Chem. Inf. Comput. Sci. 28 (1988) 31e36.
cessed Mar 2005).
Supplementary data associated with this article can be
[36] A.C. Wallace, R.A. Laskowski, J.M. Thornton, Prot. Eng. 8 (1995)
127e134.
[38] A.P.R. Zabell, S. Corden, P. Helquist, C.V. Stauffacher, O. Wiest, Bioorg.
Med. Chem. 12 (2004) 1867e1880.
References
[1] K.H. Bleicher, H.J. Bohm, K. Muller, A.I. Alanine, Nat. Rev. Drug Dis-
¨
¨
cov. 2 (2003) 369e378.
[39] Y.Y.P. Wo, M.M. Zhou, P. Stevis, J.P. Davis, Z.Y. Zhang, R.L. Van Etten,
Biochemistry 31 (1992) 1712e1721.
[2] J.C. Alvarez, Curr. Opin. Chem. Biol. 8 (2004) 365e370.
[3] J. Mestres, Biochem. Soc. Trans. 30 (2002) 797e799.
[4] M.H.J. Seifert, K. Wolf, D. Vitt, BIOSILICO 1 (2003) 143e149.
[5] P. Burkhard, U. Hommel, M. Sanner, M.D. Walkinshaw, J. Mol. Biol. 287
(1999) 853e858.
[40] T. Akerud, E. Thulin, R.L. Van Etten, M. Akke, J. Mol. Biol. 322 (2002)
137e152.
[41] J.P. Davis, M.-M. Zhou, R.L. Van Etten, Biochemistry 33 (1994) 1278e
1286.