Mechanism and Inhibition for LuxS: A Biodefense Target

描述(申请人提供)：细菌感染，特别是那些出现抗生素耐药性的细菌感染，对公众健康构成令人担忧的威胁。我们的长期目标是识别、表征和验证新的抗菌靶标。传统抗生素的作用方式是杀死或抑制细菌，从而产生抗药性。因此，必须探索替代办法或补充办法。在过去的几年里，普遍存在的细菌酶LuxS被发现在细菌群体感应、毒力调节、毒素分泌和生物被膜形成中发挥着各种关键作用。此外，还提出了LuxS的中枢代谢作用。该酶存在于A类病原体中，包括炭疽杆菌和鼠疫耶尔森氏菌；存在于B类病原体中，包括霍乱弧菌、沙门氏菌和致泻性肠出血性肠杆菌。在人类中不存在的LuxS是抗感染药物开发的一个有吸引力的靶点。 LUXS的酶机制仍然不清楚。根据我们的初步研究，我们认为LuxS既具有醛糖-酮糖异构酶的功能，又具有裂解酶的功能。LUXS的双重功能在机械上是耐人寻味的。我们提出的机制包括最初的醛糖-酮糖异构化在碳水化合物部分的C3位生成酮，以及最终的β-消除以裂解S-核糖同型半胱氨酸中的C-S键。我们的具体目标1是用化学方法合成所建议的中间体及其类似物，并测试它们作为LuxS底物或抑制剂的作用。我们还将尝试诱捕或直接观察拟议的中间体。我们的目标2和3是通过诱变和化学拯救来研究Glu57和Cys84在枯草杆菌LuxS中的催化作用，以及Cys84氧化的生物学意义。最后，我们计划设计、合成和测试基于机制的LUXS抑制剂，特别是那些与活性中心锌离子相互作用的抑制剂。我们还将研究一组天然抗菌剂-卤代呋喃酮是如何灭活LuxS的，特别是对霍乱弧菌的酶。此外，我们还将测试LuxS抑制剂对群体感应、生物膜形成和相关细菌生理的影响。

DESCRIPTION (provided by applicant): Bacterial infections, particularly those emerging with antibiotic resistance, pose an alarming threat to public health. Our long-term objective is to identify, characterize and validate new antibacterial targets. Traditional antibiotics act by killing or inhibiting bacteria, hence inducing antibiotic resistance. It is therefore imperative to explore alternative or complementary approaches. In the past few years, the ubiquitous bacterial enzyme LuxS has been found to play diverse and pivotal roles in bacterial quorum sensing, virulence regulation, toxin secretion and biofilm formation. In addition, central metabolic roles for LuxS are also proposed. This enzyme is found in Category A pathogens, including B. anthracis and Yersinia pestis; and in Category B pathogens, including Vibrio cholerae, Salmonella and diarrheagenic E. coil. Absent in humans, LuxS is an attractive target for anti-infective agent development. The enzymatic mechanism of LuxS remains elusive. Based on our preliminary studies, we propose that LuxS possesses functions of both an aldose-ketose isomerase and a lyase. The dual function of LuxS is mechanistically intriguing. Our proposed mechanism involves an initial aldose-ketose isomerization to generate a ketone at the C3 position on the carbohydrate moiety, and a final beta-elimination to cleave the C-S bond in S-ribosylhomocysteine. Our Specific Aim 1 is to chemically synthesize the proposed intermediates and their analogs, and test them as LuxS substrates or inhibitors. We will also attempt to trap or directly observe the proposed intermediates. Our Specific Aims 2 and 3 are to investigate the catalytic roles of Glu57 and Cys84 in B. subtilis LuxS by mutagenesis and chemical rescue, and the biological relevance of Cys84 oxidation. Lastly, we plan to design, synthesize and test mechanism-based inhibitors for LuxS, particularly those interacting with the active site zinc ion. We will also investigate how halogenated furanones, a group of natural antibacterial agents, inactivates LuxS, particularly for the V. cholerae enzyme. Additionally, we will test the effects of LuxS inhibitors on quorum sensing, biofilm formation and related bacterial physiology.