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(Minneapolis, MN) (MMP-2), Chemicon International
(Temecula, CA) (MMP-14), or Biomol (Plymouth Meet-
ing, PA) (MMP-8). MMP-2 and MMP-14 were activated
with AMPA. Enzymatic assay conditions for MMP-1, -2,
-7, -9, -13, and -14 have been previously described in Ref.
20c and identical assay conditions were used for MMP-3
and -12, except MMP-3 used the substrate Mca-PKPLAL-
Dpa-AR-NH2 (Bachem, King of Prussia, PA). Measure-
ments were performed in fluorimeter plate readers at the
excitation and emission wavelengths of the fluorophore.
Note that while IC50 values were reported, the assays were
established in such a way as to keep the IC50 and Ki within
2-fold of each other by manipulating the relationship
between a given MMPs substrate concentration and its Km
as dictated by the Cheng–Prusoff equation (see Ref. 20b).
Note also that the mode of inhibition is competitive as
illustrated by the crystal structure of compound 2 and
MMP-13.(b) Cheng, Y.-C.; Prusoff, W. Biochem. Phar-
macol. 1973, 22, 3099; (c) Zhang, Y.; Xu, J.; Levin, J.;
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22. (a) Bovine articular cartilage explants were harvested
from carpal joints up to 4 h after death. The explants
were cultured in DulbeccoÕs modified EagleÕs medium
(DMEM) in the presence or absence of 5 ng IL-1/mL to
induce synthesis and activation of proteolytic enzymes
including MMP-13. Various concentrations of small
molecule inhibitors were added to the culture wells.
Culture medium was collected after 3, 7, 10, 14, and 17
days in culture and replaced with identical cytokine and
inhibitor concentrations. After day 17, cartilage explants
were collected and total collagen content in media and
remaining in the explant were quantitated by biochem-
ical analysis of hydroxyproline content using a modifi-
cation of previously described in Refs. (b) and (c).
Percent inhibition of collagen degradation by a small
molecule inhibitor was calculated as percent reduction in
collagen release over the 17-day experiment compared to
collagen release with IL-1 alone and no inhibitor; (b)
Bergman, I.; Loxley, R. Anal. Chem. 1963, 35, 1961; (c)
Brown, S. W.; Worsfold, M.; Sharp, C. BioTechniques
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Soc. 2000, 122, 9648.
16. Small molecule building and minimizations were per-
formed in Sybyl (Tripos Inc) with the MMFF94s force-
field. Differences in the size, shape, and chemical
environment of the MMP active sites were identified with
the assistance of GRID (Molecular Discovery, Ltd.).
Simulations of solvent-based electrostatics were accom-
plished with Zap_Bind (OpenEye Scientific Software),
which employs a Poisson–Boltzmann continuum electro-
statics method.
17. Skiles, J. W.; Gonnella, N. C.; Jeng, A. Y. Curr. Med.
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18. Results not published.
19. All new compounds gave satisfactory 1H NMR, MS in
accord with their assigned structure.
20. (a) The inhibitory ability of small molecules was tested in a
continuous fluorescent assay designed for each enzyme in
which the substrate is a synthetic peptide containing a
fluorescent group (7-methoxycoumarin or 2-amino-
benzoyl) that is quenched by energy transfer to 2,4-
dinitrophenyl. Enzymes were either prepared at Wyeth
Research (MMP-1, -12, -13) or purchased from Calbio-
chem (San Diego, CA) (MMP-3, -7, -9), R&D Systems
23. Computational studies to be reported separately.
24. Berman, H. M.; Feng, J. W. Z.; Gilliland, G.; Bhat, T. N.;
Weissig, H.; Shindyalov, I. N.; Bourne, P. E. Nucleic Acids
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Johnson, L. L.; Ortwine, D. F.; Pavlovsky, A.; Rubin, J.
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