Mechanisms of catalysis and inhibition of carboxyketose synthases.

目的*我们的目标是为理解和控制微生物致病过程做出贡献。联合国大会将抗菌素耐药性称为“一场全球危机--一场慢动作海啸”。我们将(A)探索α-羧基酮糖合成酶(αCKs)的催化和抑制，以及蛋白质结构/动力学对功能的影响，以及(B)开发新的αCKs抑制剂。在另一个单独的项目中，我们将(C)探索微生物的铁吸收。αCKS是一种酶，它能合成对细菌生存至关重要的不寻常的糖分子，并已成为制药业的抑制目标。阻断其活性的酶抑制剂可以被开发成抗生素。*我们已经开发了以肟为基础的αCKS抑制剂。它们是有效的、慢结合的抑制剂和过渡态的模拟物。这一点很重要，因为过渡态是酶发生反应所必须攀登的能量屏障的顶端，而且酶与过渡态的结合比其他任何物种都要紧密。因此，许多过渡态的模拟物都是有效的抑制剂。令人意外的是，肟基是模拟的磷酸基团，并且潜在地非常有价值。磷酸基团无处不在，但在药物中极难模仿，因为它们在体内不稳定，而且不意味着细胞。作为小而中性的磷酸盐模拟物，肟类药物可以避免这些问题。我们已经生产了一种可以杀死培养中细菌的衍生物，证明了肟基是细胞内的有效抑制剂。*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。*成像细菌感染*细菌感染的成像部位是一个主要的临床挑战，大约3%的住院是因为发烧，在一周内无法诊断出发烧。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让细菌饥饿，而铁是一种重要的营养物质。因此，细菌使用被称为铁载体的分子来寻找铁。*我们已经创建了放射性标记的铁载体，它将定位于感染部位，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，以提高它们的灵敏度，并将与其他组织的非特异性结合降至最低。

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 α-carboxyketose synthases (αCKS), and the effects of protein structure / dynamics on function, and (b) develop new αCKS inhibitors. In a separate project, we will (c) probe microbial iron uptake.******αCKSs ***The αCKSs 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 αCKS 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.