Studies on Enzyme Mechanism and Inhibitor Design

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This proposal is divided into three sections. The first section describes our studies on enzymes that produce alkaloids. Alkaloids are nitrogen-containing natural products primarily produced by plants and fungi. They often possess potent biological activities and many have been used as valuable pharmaceuticals (morphine/pain, vinblastine/cancer, ergotamine/migraines). We are studying the enzymes involved in the biosynthesis of the indole alkaloids such as ergotamine. We have discovered that these enzymes can produce novel alkaloids that may have unique biological properties. By understanding the details of how these enzymes function, we may be able to utilize them in the production of new bioactive compounds. The second section involves studies on the biosynthesis of menaquinone (vitamin K2) in the pathogenic bacteria that are responsible for ulcers and food poisoning. These bacteria require menaquinone for respiration and without it they cannot survive. We are investigating the enzymes involved in menaquinone biosynthesis in these organisms since these are attractive targets for inhibitor design. Such inhibitors would prevent the enzymes from operating and would therefore serve as antibiotics against these organisms. In our initial studies we outlined the correct biosynthetic pathway for the formation of this vitamin, and in ongoing work we are studying individual enzymes in the pathway so that inhibitors can be designed. The third section involves studies on tubulin-modifying enzymes. Microtubules form the cytoskeleton of human cells and are absolutely required for proper cell division. For this reason, compounds that interfere with proper microtubule formation act as potent anticancer agents (vinblastine, taxol). Microtubules are generated by the spontaneous self-assembly of tubulin. We are studying a group of enzymes that chemically modify tubulin and therefore control its physical properties. We are preparing inhibitor molecules that will interfere with the action of these enzymes and we anticipate that such compounds will be cytotoxic. This could potentially serve as a new approach towards treating cancer.