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Increased expression and activity of nuclear cathepsin in cancer cells
crystal structure 1ICF [24], the system was modeled using Insight II
2005 software from Accelrys [9]. Two hydrogen bonds, one
between the terminal NH of KGP94 and the Gln19 side-chain car-
boxamide, and the other between the phenolic OH of KGP94 and
Asp162, are important in orienting the inhibitor at the active site.
This conformation, places the active site Cys25 thiolate within 5 A
of the thiosemicarbazone thiocarbonyl carbon of KGP94, thus
available to form a transient covalent bond.
L
suggests a novel mechanism of cell transformation, Mol. Cancer Res. 5 (2007)
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ꢀ
3. Conclusions
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KGP94,
a benzophenone thiosemicarbazone inhibitor of
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Lett. 20 (2010) 1415e1419;
cathepsin L, was evaluated to characterize its mode of action and
assess its performance and efficacy in a series of in vitro and in vivo
studies. Kinetic analysis was used to investigate the mechanism of
action of this non-peptidic inhibitor. KGP94 was determined to be
a slow, time-dependent, reversible, competitive inhibitor of the
fluorogenic substrate, Z-FR-AMC. It was also able to significantly
inhibit the activity of cathepsin L toward type I collagen. Molecular
modeling indicates KGP94 binds to cathepsin L in a manner that
positions the inhibitor thiocarbonyl moiety for attack by the
enzyme thiolate to form, at least, a transient tetrahedral interme-
diate. The kinetic studies support this proposed mechanism. The
possibility that a thiocarbamoylated enzyme is formed followed by
slow hydrolysis cannot be ruled out, and future studies will address
this issue. KGP94 was a potent inhibitor of the invasion and
migration of the breast cancer MDA-MB-231 and the prostate DU-
145 cell lines (see Supplementary Material). The effects of five daily
doses of 20 mg/kg given to mice with treatment initiated either on
the day of tumour inoculation or once the tumours reached
200 mm3 in size were assessed. Results indicated significant
growth retardation is achieved against both recently implanted and
established tumours. These preliminary studies have contributed to
understanding the characteristics of this thiosemicarbazone as
a potential anti-cancer agent.
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Acknowledgments
For the in vivo experiments, the authors thank Ms. Dorthe Grand,
Ms. Inger Marie Horsman, and Ms. Pia Schjerbeck for technical
assistance and both the Danish Agency for Science Technology &
Innovation and the Danish Cancer Society for financial support
(MRH). We would also like to thank Dr. Michelle Nemec, Director, for
use of the Molecular Biosciences Center, and Dr. Craig Moehnke for
his assistance with instrumentation, and Ms. Alison Garzone for
assistance in preparation of this manuscript (Baylor University). This
study was supported by OXiGENE, Inc. (grants to MLT and KGP).
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
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