Mechanisms of catalysis and inhibition of carboxyketose synthases.

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Objectives ***Our objective is to make contributions to understanding and controlling processes involved in microbial pathogenesis. The UN General Assembly labelled antimicrobial resistance as “a global crisis - a slow motion tsunami”. We will (a) explore catalysis and inhibition of -carboxyketose synthases (CKS), and the effects of protein structure / dynamics on function, and (b) develop new CKS inhibitors. In a separate project, we will (c) probe microbial iron uptake.******CKSs ***The CKSs are enzymes that synthesize unusual sugar molecules essential to bacterial survival, and have been targeted by the pharmaceutical industry for inhibition. Enzyme inhibitors that block their activity could be developed into antibiotics.******We have developed oxime-based CKS inhibitors. They are potent, slow-binding inhibitors and transition state mimics. This is important because the transition state is the top of the energetic barrier that enzymes must climb for a reaction to occur, and enzymes bind transition states more tightly than any other species. Thus, many transition state mimics are potent inhibitors.******The oxime group is, unexpectedly, a phosphate group mimic, and potentially very valuable. Phosphate groups are ubiquitous, but exceedingly difficult to mimic in drugs because of they are unstable in vivo, and cell impermeant. Oximes, as small, neutral phosphate mimics, avoid these problems. We have produced a derivative that kills bacteria in culture, demonstrating that oxime groups are cell permeant and effective inhibitors in the cell.******We will continue to develop inhibitors and study their mechanisms, probe how the proteins' structures and dynamics are correlated with inhibition, and determine the enzymes' transition state structures to allow more targeted development of new inhibitors that mimic the transition state.******Imaging bacterial infections***Imaging sites of bacterial infection is a major clinical challenge, with ~3% of all hospitalizations being for fever that cannot be diagnosed within a week. Of the cases that are eventually diagnosed, bacterial infection is the most common cause. One of the body's main antimicrobial strategies is to starve bacteria of iron, an essential nutrient. As a result, bacteria scavenge for iron using molecules called siderophores. ******We have created radioactively-labelled siderophores that will be localized to the sites of infection and reveal hidden bacterial infections by PET scanning. We will continue to develop these imaging agents to improve their sensitivity and minimize non-specific binding to other tissues.