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.

项目摘要/摘要 可用于治疗侵袭性真菌感染的抗真菌药物是有限的，因为选择性地 在不伤害人类的情况下杀死真核病原体。几丁质合成酶(CHSS)是为数不多的已证实的 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到的毒性的靶标。 CHs是负责甲壳素生物合成的跨膜过程性糖基转移酶(GTS)。 真菌细胞壁的基本多糖组分。由于它们在真菌生理学中的基本功能， CHSS是天然的多肽核苷(PN)抗真菌药物的靶标。三七总皂甙在体内表现出活性 对抗多种地方性真菌病原体，对人类没有不良影响，并显示出很强的协同作用 目前FDA批准的抗真菌药物。然而，它们的发展一直很缓慢，因为只有适度的 对临床常见真菌病原体的抗真菌活性，如白色念珠菌和 缺乏对CHS的原子水平的理解。我们的长期目标是提供一个全面的理解 在原子水平上对CHS的催化、调节和抑制。当前的应用主要集中在结构和 白色念珠菌CHSS催化和抑制的机理研究。白色念珠菌有四个CHS，其中 CaCHS1和CaCH2需要同时抑制杀菌作用。而现有的PNS有效地抑制了 CaCHS2对CaCHS1的抗菌活性要弱得多，因此对C. 白念珠菌。CaCHS_1和CaCH_2 Pn活性差异的分子机制是 目前尚不清楚。对于CHS靶向抗念珠菌药物的未来发展，至关重要的是 了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， 我们异源表达和纯化了具有催化活性的CaCHS1和CaCHS2，并解决了低温EM CaCHS2在脱氧核糖核酸、底物结合和PN(尼克霉素Z和多氧菌素D)结合形式中的结构。我们 还开发了新的活性分析方法来测定甲壳素的链长和两者的定量 不溶于水的甲壳素和短溶壳寡糖，建立了一种化学-酶促反应的方法 尼可霉素类似物的合成。基于这些发展，我们建议研究甲壳素的作用机理。 CaCHS_2的形成和挤出(目标1)，低磁化率的功能和结构基础 CaCHS1到PNS以及同时使用CaCHS1和CaCHS2抑制剂的协同作用(目标2)，以及详细的 和三七总皂苷的系统构效关系(目标3)。对于AIMS 2和AIMS 3，临床分离的白色念珠菌 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。这项拟议的研究具有重要意义，因为它将为 针对临床未开发靶点的新型抗真菌药物的未来发展。