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J. T. Palmer et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2909–2914
7. (a) Palmer, J. T.; Bryant, C.; Wang, D-X.; Davis, D. E.;
amino-4-methylcoumarin which was monitored fluoro-
metrically (excitation at 355 nm and emission at 460 nm)
using an FMAX 96-well plate reader from Molecular
Devices (Sunnyvale, CA, USA) interfaced with a Macin-
tosh computer. Under these experimental conditions 1 lM
of product produces a fluorescence signal of approximate-
ly 125 U. The velocity of the enzyme-catalyzed reaction
was obtained from the linear portion of the progress
curves (usually the first 5 min after addition of substrate).
Apparent inhibition constants, Ki(app.)’s, were calculated
from the velocity data generated at the various inhibitor
concentrations using the software package, BatchKi
(obtained from Dr. Petr Kuzmic, Biokin Ltd., Pullman,
WA, USA). BatchKi provides a parametric method for the
determination of inhibitor potency using a transformation
of the tight binding inhibition model described by Mor-
rison15 and further refines the appKi values by nonlinear
least-squares regression. The apparent inhibition constant
is related to the true thermodynamic binding constant, Ki,
by the following relationship: Ki = appKi/(1 + [substrate]/
Km) (2). Since substrates are typically supplied in the
assays at their Km, Ki = appKi/2. Specific assay conditions
for each cathepsin tested are as follows: cathepsin K: The
buffer used to assay this enzyme consisted of: 50 mM MES
(pH 5.5), 2.5 mM D,L-dithiothreitol (DTT), 2.5 mM eth-
ylenediaminetetraacetic acid (EDTA), and 10% dimethyl-
Setti, E. L.; Rydzewski, R. M.; Venkatraman, S.;
Tian, Z-Q.; Burrill, L. C.; Mendonca, R. V.; Springman,
E.; McCarter, J.; Chung, T.; Cheung, H.; McGrath, M.;
Somoza, J.; Enriquez, P.; Yu, Z. W.; Strickley, R. M.; Liu,
L.; Venuti, M. C.; Percival, M. D.; Falgueyret, J-P.; Prasit,
P.; Oballa, R.; Riendeau, D.; Young, R. N.; Wesolowski,
G.; Rodan, S. B.; Johnson, C.; Kimmel, D. B.; Rodan, G.
J. Med. Chem. 2005, 48, 7520; (b) Black, W. C.; Bayly, C.
I.; Davis, D. E.; Demarais, S.; Falgueyret, J-P.; Leger, S.;
Li, C. S.; Masse, F.; McKay, D. J.; Palmer, J. T.; Percival,
M. D.; Robichaud, J.; Tsou, N.; Zamboni, R. Bioorg.
Med. Chem. Lett. 2005, 15, 4741; (c) Robichaud, J.;
Oballa, R.; Prasit, P.; Falgueyret, J-P.; Percival, M. D.;
Wesolowski, G.; Rodan, S. B.; Kimmel, D.; Johnson, C.;
Bryant, C.; Venkatraman, S.; Setti, E.; Mendonca, R.;
Palmer, J. T. J. Med. Chem. 2003, 46, 3709.
8. Tavares, F. X.; Deaton, D. N.; Miller, A. B.; Miller, L. R.
Bioorg. Med. Chem. Lett. 2005, 15, 3891.
9. Ohmoto, K.; Yamamoto, T.; Okuma, M.; Horiuchi, T.;
Imanishi, H.; Odagaki, Y.; Kawabata, K.; Sekioka, T.;
Hirota, Y.; Matsuoka, S.; Nakai, H.; Toda, M.; Cheronis,
J. C.; Spruce, L. W.; Gyorkos, A.; Wieczorek, M. J. Med.
Chem. 2001, 44, 1268.
10. See, for example: (a) Steitz, T. A.; Shulman, R. G. Annu.
Rev. Biophys. Bioeng. 1982, 11, 419; (b) Vajda, T.; Szabo,
T. Acta Biochim. Biophys. Acad. 1976, 11, 287.
sulfoxide (DMSO). Recombinant cathepsin
K was
11. Lipinski, C. A. Adv. Drug Del. Rev. 1997, 23, 3.
12. Enzyme assays. All enzymes used in these studies, with the
exception of human cathepsin B, were produced by Celera
Genomics. Cathepsin B was from human liver and was
purchased from Athens Research and Technology (Ath-
ens, GA, USA). The substrates used in these studies were
purchased from the following vendors: Z-Phe-Arg-AMC,
Boc-Leu-Lys-Arg-AMC, and Z-Val-Val-Arg-AMC were
from Bachem (Torrance, CA, USA) and Z-Leu-Arg-AMC
was from Calbiochem-Novabiochem (San Diego, CA,
USA). Bovine serum albumin was purchased from the
Sigma Chemical Company (St. Louis, MO, USA). Rou-
tine buffer components and all other chemicals used in
these experiments were of the highest available quality.
Enzyme inhibition studies were performed under several
sets of conditions; each set was tailored to provide the
optimal activity of the given cathepsin being assayed. Each
cathepsin was incubated with the inhibitor, present at
variable concentrations, under the conditions specified
below. Enzyme and inhibitor were incubated together for
30 min at room temperature (21–24 °C) in 96-well
U-bottomed, microtiter plates (Falcon, from Becton–
Dickinson, Franklin Lakes, NJ, USA). After the pre-
incubation phase, reactions were initiated with the addi-
tion of the 7-amino-4-methylcoumarin (AMC) substrate
specified below. The hydrolysis of these substrates yields 7-
supplied at 1 nM. Substrate, Z-Phe-Arg-AMC, was sup-
plied at 40 lM. Human cathepsin B: the buffer used to
assay this enzyme consisted of: 50 mM MES (pH 6.0),
2.5 mM DTT, 2.5 mM EDTA, 0.001 Tween 20, and 10%
DMSO. Human liver cathepsin B was supplied at 1 nM.
Substrate, Boc-Leu-Lys-Arg-AMC, was supplied at
190 lM. Human cathepsin L: the buffer used to assay this
enzyme consisted of: 50 mM MES (pH 5.5), 2.5 mM DTT,
2.5 mM EDTA, and 10% DMSO. Recombinant human
cathepsin L was supplied at 500 pM. Substrate, Z-Phe-
Arg-AMC, was supplied at 10 lM. Human cathepsin S:
the buffer used to assay this enzyme consisted of: 50 mM
MES (pH 6.5), 2.5 mM b-mercaptoethanol (BME),
2.5 mM EDTA, 100 mM NaCl, 0.001% bovine serum
albumin (BSA), and 10% DMSO. Recombinant human
cathepsin S was supplied at 500 pM. Substrate, Z-Val-Val-
Arg-AMC, was supplied at 60 lM.
13. Falgueyret, J.-P.; Desmarais, S.; Oballa, R.; Black, W. C.;
Cromlish, W.; Khougaz, K.; Lamontagne, S.; Masse, F.;
Riendeau, D.; Toulmond, S.; Percival, M. D. J. Med.
Chem. 2005, 48, 7535.
14. Falgueyret, J. P.; Oballa, R. M.; Okamoto, O.; Wesolow-
ski, G.; Aubin, Y.; Rydzewski, R. M.; Prasit, P.;
Riendeau, D.; Rodan, S. B.; Percival, M. D. J. Med.
Chem. 2001, 44, 94.
15. Morrison, J. F. Biochim. Biophys. Acta 1969, 185, 269.