Targeting evolutionarily acquired insertion sequences in Candida species, for development of antifungal drugs

针对念珠菌物种中进化获得的插入序列，用于开发抗真菌药物

• 批准号: 10592776
• 负责人: PRIYA UPPULURI
• 金额: $ 20.19万
• 依托单位: LUNDQUIST INSTITUTE FOR BIOMEDICAL INNOVATION AT HARBOR-UCLA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-11-18 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10592776
• 关键词: Affinity; Affinity Chromatography; Amino Acid Sequence; Amino Acids; Antifungal Agents; Antifungal Therapy; Biodistribution; Biological; Biological Assay; Biological Availability; Candida; Candida albicans; Candida auris; Candidiasis; Carbon; Cell Wall; Cells; Clinical; Co-Immunoprecipitations; Codon Nucleotides; DNA Insertion Elements; Databases; Development; Disseminated candidiasis; Docking; Dose; Drug Controls; Drug Kinetics; Drug Targeting; Encapsulated; Epitopes; Eudragit; Eukaryota; FDA approved; Filament; Formulation; Frequencies; Gel; Gene Family; Gene Proteins; Generic Drugs; Genes; Genetic Screening; Growth; Human; Hydrophobicity; Immune Evasion; In Vitro; Individual; Invaded; Leucine; Ligands; Mass Spectrum Analysis; Medical Device; Membrane; Microbial Biofilms; Mitochondria; Modeling; Mucous Membrane; Multi-Drug Resistance; Mus; Mutagenesis; Mycoses; NMR Spectroscopy; Open Reading Frames; Oral; Oral Administration; Oral candidiasis; Oral mucous membrane structure; Organism; Pathogenicity; Penetration; Phagocytosis; Pharmaceutical Preparations; Phosphorylation Site; Phylogenetic Analysis; Polymers; Property; Protein Biochemistry; Proteins; Proteome; Reporting; Respiration; Role; Sepsis; Serine; Solubility; Source; Spectroscopy, Fourier Transform Infrared; Stress; Stretching; Structure; Surface; Testing; Time; Toxic effect; Translating; Treatment Protocols; Virulence; Water; Yeasts; aqueous; biological adaptation to stress; drug development; fascinate; forward genetics; fungus; gain of function; genome-wide; improved; in silico; in vivo; in vivo evaluation; inhibitor; link protein; member; mortality; mouse model; mutant; nanoparticle; novel; oropharyngeal thrush; pathogen; pharmacologic; prevent; protein protein interaction; public health relevance; screening; success; surfactant; trait; vaginal candidiasis

Abstract Candida albicans causes half of all invasive fungal infections in humans. Its success as a pathogen is due to its ability to adapt and thrive in various microenvironments in the host. Using a forward genetics screen, we identified an uncharacterized gene NDU1 whose loss caused C. albicans to lose viability on alternative carbon sources and made the organism completely avirulent in vivo. We found that Candida-Ndu1 protein possesses three evolutionarily acquired stretches of amino acid inserts (~64 amino acids), absent from all other eukaryotes including humans. We reported for the first time that ~17% of the Candida proteome possess “additional” sets of evolutionarily acquired amino-acid inserts, that are absent from all non-CTG clade eukaryotes, including humans. The role of insert regions in C. albicans proteome remains to be unraveled. We have shown that the Ndu1 insertion sequences are vital and required for Ndu1’s activity. In aim 1, the mechanistic contribution of Ndu1 inserts will be investigated by protein biochemistry studies. The role of insert regions in other Candida proteins will also be evaluated , to understand if inserts are universally important for function. Considering Ndu1 is important for growth and virulence in vivo, we targeted Ndu1 and its insert regions for development of antifungal molecules. We identified a repurposed FDA approved drug niclosamide (NCL), and developed Eudragit EPO nanoparticle formulations of this molecule to enhance its solubility and bioavailability. NCL nanoparticles (NCL-EPO-NP) could prevent growth of Candida in alternative carbon sources, penetrate and dismantle mature biofilms in vitro, and eradicate it in vivo in biofilm models of mucosal candidiasis. The activity of NCL-NP was 6-10 fold higher than the generic drug alone. In Aim 2, we further characterize and evaluate NCL-EPO-NP for their pharmacokinetics and oral biodistribution, toxicity profiles and in vivo efficacy in an oral regimen for treatment of oral candidiasis, as standalone or adjunct therapy with other antifungal drugs.

抽象的 白色念珠菌引起人类所有侵入性真菌感染的一半。它作为病原体的成功是由于其 能够在宿主中的各种微环境中适应和壮成长的能力。使用正向遗传学屏幕，我们确定了 一个未表征的基因NDU1，其损失导致白色念珠菌在替代碳源上失去生存能力 并使生物体在体内完全无动于衷。我们发现念珠菌-NDU1蛋白具有三个 进化获得的氨基酸插入物（〜64个氨基酸），所有其他真核生物都不存在 包括人类。我们首次报道了约17％的念珠菌蛋白质组具有“其他”集合 所有非CTG进化核真菌都不存在的进化获得的氨基酸插入物，包括 人类。插入区域在白色念珠菌蛋白质组中的作用尚待揭示。我们已经证明了 NDU1插入序列对于NDU1的活动至关重要，并且需要。在AIM 1中，机械贡献 NDU1插入物将通过蛋白质生物化学研究研究。插入区域在其他念珠菌中的作用 还将评估蛋白质，以了解插入物对于功能是否普遍重要。 考虑到NDU1对于体内的生长和病毒很重要，我们将NDU1及其插入区域作为目标 抗真菌分子的发展。我们确定了已重新利用的FDA批准的药物烟酰胺（NCL），并且 开发了该分子的Eudragit Epo纳米颗粒公式，以增强其溶解度和生物利用度。 NCL纳米颗粒（NCL-EPO-NP）可以防止在替代碳源中念珠菌的生长，渗透 并在体外拆除成熟的生物膜，并在粘膜念珠菌病的生物膜模型中对其进行放射性化。 NCL-NP的活性比单独使用仿制药高6-10倍。在AIM 2中，我们进一步描述和 评估NCL-EPO-NP的药代动力学和口服生物分布，毒性特征和体内效率 口服念珠菌病治疗的口服方案，作为其他抗真菌药物的独立治疗或辅助疗法。