Covalent and Transition State Inhibitor Design

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This award funds Professor Andrew Murkin of the University at Buffalo, The State University of New York, to develop a series of compounds that may serve as specific inhibitors of an enzyme critical to metabolism in plants and bacteria, known as isocitrate lyase (ICL). One of the obstacles to inhibitor design is ensuring selective binding to ICL over other potential targets. Meeting this goal usually requires a special interaction or group of interactions between the inhibitor and the enzyme. One strategy for accomplishing this is to introduce a part of an inhibitor that only sticks to the target if it is in just the right place for an interaction. A second strategy is to have the inhibitor resemble what is happening during the catalyzed conversion. This tricks the enzyme into binding to the inhibitor tightly and with great specificity. The current research aims to develop compounds that inhibit ICL by one of these two strategies, which may prove to be a general means for inhibiting numerous enzymes. Educational development is provided to undergraduate and graduate students, and local area teachers will be trained to perform hands-on lessons that introduce enzymes to K-12 students.This project exploits two properties of aliphatic nitro compounds that may lead to potent enzyme inhibition. The first of these is previously unknown and therefore investigated in detail. Specifically, the carbon adjacent to the nitro group can act as an electrophile for covalent reaction with a neighboring cysteine residue under certain conditions when bound to an enzyme. Kinetic, mass spectral, and X-ray crystallographic experiments are proposed to elucidate the mechanism by which this reaction occurs between a simple nitro compound and ICL. In an attempt to improve selectivity, more elaborate nitro compounds will be prepared and tested. The second property of nitro compounds that will be utilized here is the resemblance of their conjugate bases, called nitronates, to the aci enolate form of their carboxylic acid equivalents. Through the use of kinetic isotope effects and computational modeling, the transition state of ICL will be determined. Nitronate analogues of the transition state will be prepared, with the goal of obtaining strong binding with ICL.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.