We are using x-ray crystallography to determine the structure of therapeutically relevant proteins in complex with natural substrates and products, substrate and cofactor analogs and inhibitors of enzyme action. Our lab seeks to understand the binding of these small molecules to their protein targets at a level sufficient to permit the design of potent new inhibitors which may serve as lead compounds in the development of useful drugs and the improvement of currently existing drugs. Our research at UCSF is focused on two targets of structure based drug design, HIV-1 protease and E. coli thymidylate synthase. We have determined many structures of these enzymes complexed with a variety of ligands including substrate, reaction products, cofactors and cofactor analogs, lead compounds and clinically tested drugs. A stable, non-peptide inhibitor (UCSF8) of the HIV-1 protease has been developed, and the stereochemistry of binding defined through crystallographic three-dimensional structure determination. The initial compound, haloperidol, was discovered through computational screening of the Cambridge Structural Database using a shape complementarity algorithm. The subsequent modification is a non-peptidic lateral lead which belongs to a family of compounds with well characterized pharmacological properties. This thioketal derivative of haloperidol (UCSF8) and a halide counterion are bound within the enzyme active site in a mode distinct from that observed for peptide-based inhibitors. A variant of the protease cocrystallized with UCSF8 shows binding in the manner predicted during the initial computer based search. The structures provide the context for subsequent synthetic modifications of the inhibitor. Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) by 5,10-methylenetetrahydrofolate (CH2THF) to form deoxythymidine monophosphate (dTMP) and dihydrofolate (H2-folate). TS is essential for the biosynthesis of DNA and non-essential for the biosynthesis of RNA. This unique role makes TS an ideal candidate inhibitors which will serve as lead compounds in the development of anti-cancer drugs. Furthermore, structural differences between human TS and TS from certain infectious agents, such as Pneumocystis carinii which infects the lungs of AIDS patients, may be exploited to design species specific inhibitors. To facilitate the design of such compounds, we seek to develop an understanding of the forces at play at the enzyme.substrate interface and establish a precise relationship between macromolecular structure and the thermodynamics of substrate binding. We have studied TS to develop a method for the rational design and improvement of potent inhibitors, and also to understand on an atomic level the kinetics and catalysis of this enzyme. Throughout these studies, we use the program MidasPlus and the Computer Graphics Laboratory at UCSF to vizualize small molecule ligands, electron density, and atomic resolution protein structures determined by x-ray crystallography.
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