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: 10501171
• 负责人: Seok-Yong Lee
• 金额: $ 63.91万
• 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/10501171
• 关键词: Active Sites; Adverse effects; Agriculture; Anabolism; Antifungal Agents; Antifungal Antibiotics; 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; Industrial fungicide; Kinetics; Knock-out; Knowledge; Length; Methods; Molecular; Molecular Conformation; Mycoses; Nucleosides; Oligosaccharides; Organism; Physiology; Polysaccharides; Predisposition; Public Health; Reaction; Regulation; Research; Resistance; Resolution; Structure; Structure-Activity Relationship; Testing; Therapeutic; Toxic effect; Travel; Variant; analog; base; clinical development; clinical investigation; divalent metal; drug sensitivity; 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.

项目概要/摘要 由于选择性治疗的挑战，针对侵袭性真菌感染的可用抗真菌药物有限。 杀死真核病原体而不伤害人类。几丁质合酶 (CHS) 是少数经过验证的酶之一 其抑制作用可提供高度选择性的抗真菌作用，而对人类没有任何可检测到的毒性。 CHS 是跨膜进行性糖基转移酶 (GT)，负责几丁质的生物合成，几丁质是一种 真菌细胞壁的必需多糖成分。由于它们在真菌生理学中的重要功能， CHS 是天然存在的肽基核苷 (PN) 抗真菌剂的靶标。 PNs表现出体内活性 对抗多种地方性真菌病原体，对人类没有不良影响，并与 目前 FDA 批准的抗真菌药物。但由于其发展水平不高，发展缓慢。 针对临床上更常见的真菌病原体（例如白色念珠菌和 缺乏对 CHS 的原子级理解。我们的长期目标是提供全面的了解 CHS 在原子水平上的催化、调节和抑制。目前的应用主要集中在结构和 白色念珠菌 CHS 催化和抑制的机制研究。白色念珠菌有四个 CHS，其中 CaCHS1 和 CaCH2 需要同时抑制才能发挥杀菌作用。虽然现有的 PN 可以有效抑制 CaCHS2，它们对 CaCHS1 的作用要弱得多，因此对念珠菌仅表现出中等的抗真菌活性。 白色念珠菌。 CaCHS1 和 CaCH2 PN 效力差异背后的分子机制是 目前未知。对于未来开发针对 CHS 的抗念珠菌药物，至关重要的是 了解它们在催化和抑制方面的结构和机制差异。作为初步研究， 我们异源表达并纯化了具有催化活性的CaCHS1和CaCHS2，并解决了cryo-EM CaCHS2 的载脂蛋白结合、底物结合和 PN（尼可霉素 Z 和多氧霉素 D）结合形式的结构。我们 还开发了新的活性测定法，用于测定几丁质链长度和长链的定量 不溶性甲壳素和短可溶性壳寡糖，并建立了化学酶法 尼可霉素类似物的合成。基于这些进展，我们建议研究甲壳素的作用机制 CaCHS2 的形成和挤出（目标 1），是较低敏感性的功能和结构基础 CaCHS1 与 PN 的结合以及使用 CaCHS1 和 CaCHS2 抑制剂的协同作用（目标 2），以及详细的 PNs 的系统结构-活性关系（目标 3）。对于目标 2 和 3，白色念珠菌的临床分离株 和非白色念珠菌菌株也将被纳入抑制剂测试，增加转化潜力 我们的研究计划。拟议的研究意义重大，因为它将提供分子基础 针对临床未开发靶点的新型抗真菌药物的未来开发。