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 由于有选择的挑战，可用的抗真菌真菌感染的抗真菌药物受到限制 杀死无危害人类的真核病原体。几丁质合成酶（CHSS）代表了少数经过验证的 抑制作用的靶标提供高度选择性的抗真菌作用，而没有对人类的任何可检测毒性。 CHS是跨膜加工糖基转移酶（GTS） 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是由天然存在的肽核苷（PN）抗真菌剂靶向的。 PNS展示体内活动 针对多种流行真菌病原体对人类没有不利影响，并与 当前FDA批准的抗真菌药物。但是，由于中等程度，它们的发展很慢 抗真菌活性抗真菌性真菌病原体，例如白色念珠菌和 对CHS的缺乏原子级别的理解。我们的长期目标是提供全面的理解 CHS催化，调节和原子水平的抑制作用。当前的应用集中于结构和 白色念珠菌CHS的催化和抑制的机理研究。白色念珠菌有四个CHS CACHS1和CACH2需要同时抑制杀菌作用。而现有的PNS有效抑制 cachs2，它们针对cachs1弱得多，因此仅表现出对C的中度抗真菌活性。 白色唱片。 CACHS1和CACH2之间PN效力差的分子机制为 目前未知。为了将CHS靶向反加拿大代理商的未来发展，必须 了解它们在催化和抑制方面的结构和机械差异。作为初步研究， 我们异源表达和纯化的催化活性CACHS1和CACHS2，并解决了冷冻EM apo-，底物结合和pn（尼克霉素Z和多氧蛋白D）结合形式的CACHS2结构。我们 还开发了新的活性测定法，以确定几丁质链长度和两者的定量 不溶性几丁质和短的可溶性三含糖，并建立了一种化学酶的方法 Nikkomycin类似物的合成。基于这些发展，我们建议研究几丁质的机制 CACHS2的形成和挤出（AIM 1），较低易感性的功能和结构基础 CACHS1至PNS以及同时使用CACHS1和CACHS2抑制剂的协同作用（AIM 2）和详细的 PNS的系统结构活性关系（AIM 3）。对于目标2和3，白色念珠菌的临床分离株 也将包括用于抑制剂测试的非阿尔比克念珠菌菌株，增加了翻译潜力 我们的研究计划。拟议的研究很重要，因为它将为分子基础提供 针对临床上未开发的靶标的新型抗真菌剂的未来开发。