Curriculum Vitae

Toshihiko Takeuchi

Address:

University of California, San Francisco

Department of Pharmaceutical Chemistry, Box 0446

San Francisco, CA 94143-0446

(415) 476-8147

Citizenship:

United States

Education:

1986-90 The Ohio State University

B.S. in Chemistry

1990-1996 California Institute of Technology

Ph. D. Chemistry

Research:

1987-1990: The Ohio State University

Laboratory of Professor Bruce Bursten

Project: Electrochemical and spectroscopic studies of dirhodium tetracarboxylate systems.

1990-2/96 California Institute of Technology

9/90 to 5/93: Laboratory of Professors Harry B. Gray and William A. Goddard

Project: Electronic structure calculations of distorted porphyrins.

5/93 to 2/96: Laboratory of Professors Thomas J. Meade and Harry B. Gray

Project: The development of cobalt schiff base enzyme inhibitors.

2/96 to present University of California, San Francisco

Laboratory of Professor Charles Craik

Project: Engineering Inhibitors for a Cancer-Associated Protease, Cloning

and Characterization of a Membrane-Type Serine Protease

Honors and Awards:

National Merit Scholar (1986-1990)

Battelle Scholar (full tuition Ohio State scholarship) (1986-1990)

Outstanding Student in Chemistry (1987,1988)

Elected to Phi Beta Kappa (1989)

Outstanding Graduating Senior in the Physical Sciences (1990)

Graduated Summa Cum Laude with Distinction in Chemistry and Honors in the Liberal Arts (1990)

National Defense Science and Engineering Fellowship (1990-1993)

National Institutes of Health Postdoctoral Fellowship (1996-present)

Member of American Chemical Society (1988)

Member of the American Association fo the Advancement of Science (1994)

Research Papers

T. Takeuchi, H. B. Gray, and W. A. Goddard III "Electronic Structures of Halogenated Porphyrins: Spectroscopic Properties of ZnTFPPX8 (TFPPX8 = Octa-b-halotetrakis)pentafluorophenyl)porphyrin; X = Cl, Br)" J. Am. Chem. Soc. 116 (1994) 9730-9732.

W. Jentzen, J. D. Hobbs, X. Song, M. C. Simpson, T. Ema, N. Y. Nelson, C. J. Medforth, K. M. Smith, M. Veyrat, M. Mazzanti, R. Ramasseul, J.-C. Marchon, T. Takeuchi, W. A. Goddard III, and J. A. Shelnutt. "Ruffling in a Series of Nickel(II) Meso-Tetrasubstituted Porphyrins as a Model for the Conserved Ruffling of the Heme of Cytochromes c" J. Am. Chem. Soc. 117 (1995) 11085-11097.

A. Böttcher, T. Takeuchi, K. I. Hardcastle, Z. Dori, H. B. Gray, and T. J. Meade. "Spectroscopic and Electrochemical Study on Axial and In-Plane Ligand Effects in Cobalt(III)-Schiff-Base Complexes", Inorg. Chem. 36 (1997) 2498-2504.

T. Takeuchi, A. Böttcher, C. M. Quezada, M. I. Simon, T. J. Meade, and H. B. Gray. "Selective Inhibition of Human a-Thrombin by Cobalt (III) Schiff Base Complexes", J. Am. Chem. Soc. 120 (1998) 8555-8556.

T. Takeuchi, A. Böttcher, C. M. Quezada, H. B. Gray, and T. J. Meade. "Cobalt Complexes as Irreversible Enzyme Inhibitors: Inhibition of Thrombin and Thermolysin with Co(acacen) Derivatives", Bioorg. Med. Chem., 6 (1999) 1-5.

T. Takeuchi, M. A. Shuman, C. S. Craik. "Identification of a Membrane-Type Serine Protease in Epithelial Cancer and Normal Tissue", Proc. Natl. Acad. Sci., in preparation.

Patents

Meade, T. J., Gray, H. B., Takeuchi, T., "Bioinorganic Redox Coupled Drugs.", U.S.S.N. 08/473,072, filed June 7, 1995, status: pending; U.S.S.N. 08/477,859, filed June7, 1995, status: pending

Research Summary

Undergraduate Research at The Ohio State University, 1986-1990

Electrochemical and spectroscopic studies of dirhodium tetracarboxylate systems.

Laboratory of Professor Bruce Bursten

    My research focused upon the electrochemical and spectrophotometric characterization of dirhodium tetracarboxylate complexes that showed limited effectiveness in antitumor therapy. It was proposed that the antitumor activity was a result of axial coordination of the dirhodium complexes to the tumor cells, resulting in toxicity. In order to characterize the nature of axial coordination to the rhodium complexes cyclic voltammetric and UV-vis spectrophotometric studies were performed with axial ligands of varying coordination strength. It was found that ligands of strong donor strength coordinated most effectively to the dirhodium complexes, suggesting that strong nitrogenous donors such as histidines are a likely target for metal binding.

Graduate Research at California Institute of Technology, 1990-1996

A. Electronic structure calculations of distorted porphyrins, 9/90-5/93

Laboratory of Professors Harry Gray and Bill Goddard

    Halogenated porphyrins show utility as catalysts for the oxidation of organic substrates. One limitation of these systems is the inactivation of the catalysts due to oxidation of the porphyrin macrocycle. In order to further understand factors involved in the degradation of these catalysts, UV-vis spectrophotometric studies and semiempirical quantum mechanical calculations were performed on a series of zinc(II) halogenated porphyrins. It was found that although halogenation increases the resistance of the porphyrin macrocycle to oxidation, the strong distortion of the porphyrin ring, due to bulky chlorine and bromine atoms, results in a destabilization of the porphyrins to ring oxidation. The net result is only a slight increase in stability to oxidation. These studies suggest that more robust catalysts may be derived from porphyrins substituted with less bulky substituents such as fluorines. These predictions recently have been verified by experimental results.

B. The development of cobalt schiff base enzyme inhibitors, 5/93 to 2/96

Laboratory of Professors Thomas J. Meade and Harry B. Gray

    Cobalt (III) schiff base complexes were previously found to be efficient inhibitors of the herpes simplex virus. It was speculated that inhibition of viral enzyme activity resulted in a decrease in virus proliferation. One possible mechanism of inhibition is binding of the cobalt complex to an active site residue, resulting in the loss of enzyme activity. Two enzymes were used as model targets for inhibition with cobalt (III) complexes: thermolysin and thrombin. These enzymes contain active site histidines that are crucial to enzyme catalysis, they are of therapeutic interest, and they are extensively characterized.

    These studies demonstrated that cobalt schiff base complexes can irreversibly inhibit thermolysin and thrombin by binding to the enzyme active site. The data suggest the mechanism of inhibition is due to the loss of an axial ligand from the coordinatively saturated cobalt species, followed by binding to an active site histidine, resulting in an inactive enzyme-inhibitor complex. The reactivity of the cobalt complexes can be tuned by changing the nature of the axial ligand or by changing the periphery of the schiff base equatorial ligand. The activity of the inhibitors against thermolysin appears to be hindered by unfavorable steric interactions with the active site zinc, however, prolonged incubation with the inhibitor results in loss of zinc from the enzyme and complete abrogation of enzyme activity. Inhibition of thrombin requires much less cobalt complex; this increased potency of inhibition is most likely due to greater accessibility of the histidine in the thrombin active site. In the subsequent study, the potency and selectivity of thrombin inhibition was enhanced by covalently attaching a short peptide (-dFPR-) that is known to have a high affinity for the human a-thrombin active site. Our work demonstrates that an active-site-directed peptide linked to a cobalt chelate selectively inhibits thrombin. This mode of antienzymatic activity may be generalized to a large variety of therapeutically interesting enzymes containing active site histidines.

Postdoctoral Research at the University of California, San Francisco

Project: Engineering Inhibitors for a Cancer-Associated Protease, Cloning

and Characterization of a Membrane-Type Serine Protease (2/96-present)

Laboratory of Professor Charles Craik

    The primary focus of my work has been to use macromolecular inhibitors of serine proteases to probe the role of these proteases in primary tumor growth and metastasis. Our initial protease target was urokinase-type plasminogen activator (uPA), which is thought to play a central role in the invasion and metastasis of cancer cells. The macromolecular protease inhibitor ecotin was engineered for specificity and activity against uPA. In particular, I worked on optimizing the extended primary binding site of ecotin targeted against uPA using the combinatorial technique of phage display. Combination of this optimized primary interaction sequence and the optimized secondary interaction sequence, done by Qing Yang, resulted in a 50 pM inhibitor of uPA.

    In order to dissect the role of serine proteases in tumor growth and metastasis this inhibitor and other ecotin variants were tested in animal model studies in collaboration with the Shuman laboratory. Preliminary studies with SCID mice have shown inhibition of primary tumor growth and metastasis of PC-3 prostate cancer cells when the mice were treated with ecotin and ecotin variants. These studies suggested that proteases distinct from uPA contribute to primary tumor growth and possibly metastasis of the PC-3 cells. To identify possible protease targets of the ecotins, RT-PCR using degenerate primers was performed. Five serine proteases were identified: urokinase-type plasminogen activator, factor XII, protein C, trypsinogen IV, and a protease that we refer to as membrane-type serine protease 1 (MT-SP1). The cloning and characterization of this cDNA shows that it encodes a mosaic protein of 855 amino acids that contains a transmembrane signal anchor, two CUB domains, four LDLR repeats, and a serine protease domain. Northern blotting shows broad expression of MT-SP1 in a variety of epithelial tissues with high levels of expression in the human gastrointestinal tract and the prostate. Immunoblotting shows the presence of the protein in PC-3 prostate cancer cells with a molecular mass of 90 kilodaltons, while immunofluorescence localizes the protein to the extracellular surface of the PC-3 cells. A His-tagged fusion of the MT-SP1 protease domain was expressed in E. coli, purified from inclusion bodies, and refolded from denaturant to a yield of 3 mg/L of culture. The inhibition profile of MT-SP1 by ecotin is consistent with the possibility that MT-SP1 is the protease that is involved in primary tumor growth and metastasis of PC-3 cells.