Catalysis and inhibition of chitin synthesis from pathogenic fungi

病原真菌几丁质合成的催化和抑制

• 批准号: 10640198
• 负责人: Seok-Yong Lee
• 金额: $ 61.14万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-07 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10640198
• 关键词: Active Sites; Adverse effects; Agriculture; Anabolism; Antifungal Agents; Antifungal Antibiotics; Binding; Biological Assay; Candida; Candida albicans; Catalysis; Cell Wall; Cell division; Cellular Structures; Chitin; Chitin Synthase; Clinical; Coccidioidomycosis; Code; Complex; Cryoelectron Microscopy; Dependence; Development; Drug resistance; Enzyme Inhibitor Drugs; Enzyme Interaction; Enzymes; Evaluation; Exhibits; FDA approved; Foundations; Fungal Components; Future; Genes; Genome; Goals; Human; Immunocompromised Host; Kinetics; Knock-out; Knowledge; Length; Membrane; Methods; Molecular; Molecular Conformation; Mycoses; Nucleosides; Organism; Physiology; Polysaccharides; Predisposition; Public Health; Reaction; Regulation; Research; Resistance; Resolution; Structure; Structure-Activity Relationship; Testing; Therapeutic; Toxic effect; Travel; Variant; analog; clinical development; clinical investigation; divalent metal; drug sensitivity; fungicide; fungus; glycosyltransferase; in vivo; inhibitor; insight; nikkomycin; novel; pathogen; pathogenic fungus; periplasm; programs; resistance mechanism; synergism; translational potential

Project Summary/Abstract The available antifungal drugs against invasive fungal infections are limited due to the challenge in selectively killing eukaryotic pathogens without harming humans. Chitin synthases (CHSs) represent one of few proven targets whose inhibition provides highly selective antifungal effects without any detectable toxicity to humans. CHSs are transmembrane processive glycosyltransferases (GTs) responsible for the biosynthesis of chitin, an essential polysaccharide component of the fungal cell wall. Due to their essential function in fungal physiology, CHSs are targeted by naturally occurring peptidyl nucleoside (PN) antifungal agents. PNs exhibit in vivo activities against multiple endemic fungal pathogens without adverse effects on humans, and exhibit a strong synergy with current FDA-approved antifungal drugs. However, their development has been slow because of only moderate antifungal activities against more clinically prevalent fungal pathogens such as Candida albicans and the absence of atomic-level understanding of CHS. Our long-term goal is to provide a comprehensive understanding of CHS catalysis, regulation, and inhibition at the atomic level. The current application focuses on structural and mechanistic studies of the catalysis and inhibition of C. albicans CHSs. C. albicans has four CHSs of which CaCHS1 and CaCH2 require simultaneous inhibition for fungicidal effects. While existing PNs potently inhibit CaCHS2, they are much weaker against CaCHS1 and thus exhibit only moderate antifungal activity against C. albicans. The molecular mechanism behind the difference in PN potency between CaCHS1 and CaCH2 is currently unknown. For the future development of CHS-targeting anti-candida agents, it is essential to understand their structural and mechanistic differences for both catalysis and inhibition. As a preliminary study, we heterologously expressed and purified catalytically active CaCHS1 and CaCHS2, and solved the cryo-EM structures of CaCHS2 in the apo-, substrate-bound, and PN (nikkomycin Z and polyoxin D)-bound forms. We also developed novel activity assays for the determination of chitin chain length and quantitation of both long insoluble chitin and short soluble chito-oligosaccharides, and established a method for chemo-enzymatic synthesis of nikkomycin analogs. Based on these developments, we propose to study the mechanism of chitin formation and extrusion by CaCHS2 (Aim 1), the functional and structural basis of the lower susceptibility of CaCHS1 to PNs as well as the synergy of using both CaCHS1 and CaCHS2 inhibitors (Aim 2), and the detailed and systematic structure-activity relationships of PNs (Aim 3). For Aims 2 and 3, clinical isolates of C. albicans and non-albicans candida strains will also be included for inhibitor testing, increasing the translational potential of our research program. The proposed research is significant because it will provide the molecular basis for future development of novel antifungals against a clinically unexploited target.

项目总结/摘要 针对侵袭性真菌感染的可用抗真菌药物是有限的，这是由于选择性抗真菌药物的挑战。 杀死真核病原体而不伤害人类。几丁质酶（CHSs）是少数几种已证实的 其抑制作用提供高度选择性的抗真菌作用，而对人类没有任何可检测的毒性。 CHS是跨膜进行性糖基转移酶（GT），负责几丁质的生物合成， 真菌细胞壁的基本多糖成分。由于它们在真菌生理学中的基本功能， CHS被天然存在的肽基核苷（PN）抗真菌剂靶向。PN表现出体内活性 对抗多种地方性真菌病原体，而对人类没有不利影响，并表现出与 目前FDA批准的抗真菌药物。然而，由于只有适度的发展， 抗真菌活性针对临床上更普遍的真菌病原体如白色念珠菌， 缺乏对CHS的原子级理解。我们的长期目标是提供一个全面的了解 CHS催化，调节和抑制在原子水平上。目前的应用集中在结构和 对C.白色念珠菌C.白色念珠菌有四种CHS， CaCHS 1和CaCH 2需要同时抑制杀真菌作用。虽然现有的PN有效抑制 CaCHS 2，它们对CaCHS 1弱得多，因此对C. 白色念珠菌CaCHS 1和CaCH 2之间PN效力差异背后的分子机制是 目前未知。对于CHS靶向抗念珠菌药物的未来发展， 理解它们在催化和抑制方面的结构和机理差异。作为初步研究， 我们异源表达并纯化了具有催化活性的CaCHS 1和CaCHS 2， CaCHS 2在载脂蛋白、底物结合和PN（尼可霉素Z和多氧霉素D）结合形式中的结构。我们 还开发了新的活性测定法，用于测定几丁质链长和定量两种长 不溶性甲壳素和短溶性甲壳低聚糖，并建立了化学-酶法 尼可霉素类似物的合成。在此基础上，我们提出了研究甲壳素的作用机理 CaCHS 2的形成和挤出（目的1），低敏感性的功能和结构基础， CaCHS 1对PN的协同作用以及使用CaCHS 1和CaCHS 2抑制剂的协同作用（目的2），以及详细的 以及PNs的系统构效关系（Aim 3）。对于目标2和3，C.白色 和非白色念珠菌菌株也将被纳入抑制剂测试，增加翻译潜力 我们的研究项目。这项研究具有重要意义，因为它将为以下方面提供分子基础： 针对临床未开发目标的新型抗真菌药物的未来发展。