Carbohydrate Mimicry and Enzyme Inhibition

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I will finish my term as Vice President, Research in July, 2014 and will return full time to research since I have accumulated two years of administrative leave. Our laboratory is active in understanding the molecular mechanisms of carbohydrate mimicry by other ligands, such as synthetic carbohydrate mimics (glycomimetics) or functional (glyco)peptide mimics, and in their exploitation for the design of improved diagnostic agents, vaccines, or therapeutics. Over the last grant period, significant progress has been made in all of these areas, as evident from the published work; this has been summarized in the common CV (CCV). The present proposal focuses on carbohydrate-processing enzymes of mammalian and microbial origin, and presents a primarily synthetic and mechanistic inquiry, with the ultimate readout being in vitro enzyme inhibition and/or in vitro cell-based growth inhibition. The mechanistic insights derive from our knowledge of putative transition states in enzymatic reactions, informed by our previous successes. Our design strategy is based on the well accepted premise that a transition-state (TS) analogue would be the optimal candidate as an enzyme inhibitor, but in the absence of detailed knowledge of the TS, an approximate and viable strategy is to estimate as best one can an intermediate resembling the TS. However, we propose further to harness the energy gained from additional interactions between inhibitor and enzyme, not necessarily in the catalytic site. Furthermore, through interaction with both the primary catalytic site and other subsites, we contend that the compounds will act with high affinity, and potentially evade the development of new resistance as multiple mutations (in several subsites) may be required to prevent the compound’s interaction with the target enzymes. We present here two enzyme systems for which we seek effective inhibitors by use of this approach. In the case of Golgi Mannosidase II (GMII), in addition to the catalytic site, an anchoring site , a holding site, and Zn coordination have been identified as critical components. In the case of influenza A neuraminidases, in addition to the catalytic site, the 150 and 430 subsites could be utilized to provide additional contacts. In the case of GMII, we have capitalized on our earlier discovery that a new class of sulfonium ions are nanomolar inhibitors of intestinal glucosidase enzymes, and propose to use this design concept for binding in the catalytic subsite. Remarkably, these molecules do not act as alkylating agents or irreversible inhibitors. In a separate strategy with a third enzyme, UDP-Galp mutase (UGM), we propose to dupe the enzyme by supplying a surrogate substrate that leads to a non-productive event. The mechanistic insights will be coupled with molecular dynamics calculations, complemented by STD NMR or X-ray structural data when possible, to design the next-generation candidates. The results of this research have implications for the fundamental understanding of carbohydrate processing enzymes and the nature and origin of carbohydrate mimicry.