Mechanisms of catalysis and inhibition of carboxyketose synthases.

目标 我们的目标是为理解和控制微生物致病过程做出贡献。联合国大会将抗生素耐药性称为“全球危机-慢动作海啸”。我们将（a）探索α-羧基酮糖脱氢酶（CKS）的催化和抑制，以及蛋白质结构/动力学对功能的影响，（B）开发新的CKS抑制剂。在一个单独的项目中，我们将（c）探测微生物的铁吸收。 CKS CKS是合成对细菌生存至关重要的不寻常糖分子的酶，并且已被制药工业作为抑制的目标。阻断其活性的酶抑制剂可以被开发成抗生素。 我们已经开发了基于肟的CKS抑制剂。它们是有效的、缓慢结合的抑制剂和过渡态模拟物。这一点很重要，因为过渡态是酶必须攀登的能量屏障的顶部，酶比任何其他物种都更紧密地结合过渡态。 因此，许多过渡态模拟物是有效的抑制剂。 肟基出乎意料地是磷酸基模拟物，并且可能非常有价值。磷酸基团是普遍存在的，但由于它们在体内不稳定并且不渗透细胞，因此在药物中非常难以模拟。肟，作为小的中性磷酸盐模拟物，避免了这些问题。我们已经产生了一种在培养物中杀死细菌的衍生物，证明肟基是细胞渗透性的，并且是细胞中的有效抑制剂。 我们将继续开发抑制剂并研究其机制，探索蛋白质的结构和动力学如何与抑制相关，并确定酶的过渡态结构，以便更有针对性地开发模拟过渡态的新抑制剂。 细菌感染成像 细菌感染部位的成像是一个主要的临床挑战，约3%的住院治疗是因为发烧，无法在一周内诊断。在最终确诊的病例中，细菌感染是最常见的原因。人体的主要抗菌策略之一是使细菌缺乏铁，铁是一种必需的营养素。因此，细菌利用一种叫做铁载体的分子吸收铁。 我们已经创建了放射性标记的铁载体，将被定位到感染部位，并通过PET扫描揭示隐藏的细菌感染。我们将继续开发这些显像剂，以提高其灵敏度，并尽量减少与其他组织的非特异性结合。

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.