Molecular design of cathepsin L-specific inhibitor based on the teritiary structure.

1996 Fiscal Year Final Research Report Summary

• Project/Area Number: 06453194
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 医薬分子機能学
• Research Institution: Osaka University of Pharmaceutical Sciences
• Principal Investigator: ISHIDA Toshimasa Osaka University of Pharmaceutical Sciences, Professor, 薬学部, 教授 (00111021)
• Co-Investigator(Kenkyū-buntansha): TOMOO Koji Osaka University of Pharmaceutical Sciences, research associate, 薬学部, 助手 (70257898) ; IN Yasuko Osaka University of Pharmaceutical Sciences, research associate, 薬学部, 助手 (50257896)
• Project Period (FY): 1994 – 1996
• Keywords: cathepsin L / procathepsin L / recombinant / expression / X-ray analysis / NMR / inhibitor / molecular design

Research Abstract

In the term of 1994-1996, we went ahead with the research project according to the following research plans :
(1) Large scale expression of rat cathepsin L in Escherichia coli.
(2) Isolation and purification of recombinant rat cathepsin L from Escherichia coli.
(3) Identification of active conformation of recombinant rat cathepsin L by the comparison of native one.
(4) X-Ray crystal structure determination of recombinant rat cathepsin L.
(5) Preparation and physicochemical characterization of recombinant rat cathepsin L-inhibitor complex.
(6) X-Ray crystal structure determination of recombinant rat cathepsin L and its complex with inhibitor.
(7) NMR solution conformation of recombinant rat cathepsin L and its complex with inhibitor.
(8) Model building and characterization of cathepsin L active site and elucidation of the catalytic mechanism at atomic level.
(9) Molecular design and inhibitory measurement of cathepsin L-specific inhibitor based on the results of (4) - (8).
We succeeded already in the research plans of (1) - (3), and are now vigorously studying the projects of (4) - (7) at the same time, with hoping these accomplishments in 1997. The main reason why we could not complete the research project within 1994-1996 is due to the instability of recombinant cathepsin L.Although structure determinations by X-ray diffraction and NMR methods require the stability of sample for a long time, rat cathepsin L itself decomposes to peptide fragments within few days due to the high autocatalytic activity. In order to overcome such trouble, we prepared (i) two kinds of recombinant procathepsin L from E.coli., which are resistant against the autocatalysis, and (ii) cathepsin L-inhibitor complex, where the inhibitor was complexed with recombinant cathepsin L at its expression state. Although the tertiary structure of mature cathepsin L itself could not be analyzed, we believe that the structure analyzes of the procathepsin Ls and cathepsin L-inhibor complex would not affect the accomplishment of this research project, seriously, i.e., the molecular design of cathepsin L-specific inhibitor. At present, the tertiary structures of a procathepsin L and cathepsin L-inhibitor complex are being analyzed by X-ray diffraction and various 2D NMR spectroscopy methods, and we expect the completion in 1997.