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.

摘要 白色念珠菌导致人类所有侵袭性真菌感染的一半。它作为病原体的成功是因为它 在宿主的各种微环境中适应和茁壮成长的能力。通过正向遗传学筛选，我们发现 NDU 1基因缺失导致C.白色念珠菌在替代碳源上失去生存能力 并使该生物体在体内完全无毒。我们发现，Adipda-Ndu 1蛋白具有三个 进化获得的氨基酸插入片段（~64个氨基酸），在所有其他真核生物中缺失 包括人类我们首次报道了~17%的念珠菌蛋白质组具有“额外”集合 的进化获得的氨基酸插入，这是不存在于所有非CTG分支真核生物，包括 人类插入区在C.白念珠菌蛋白质组仍有待进一步研究。我们已经证明， Ndu 1插入序列对于Ndu 1的活性是至关重要和必需的。在目标1中， ndu 1插入物将通过蛋白质生物化学研究进行研究。插入区在其他念珠菌中的作用 还将评估蛋白质，以了解插入物是否对功能普遍重要。 考虑到Ndu 1对体内生长和毒力的重要性，我们靶向Ndu 1及其插入区域， 抗真菌分子的发展。我们发现了一种重新利用的FDA批准的药物氯硝柳胺（NCL）， 开发了该分子的Eudragit EPO纳米颗粒制剂，以提高其溶解度和生物利用度。 NCL纳米粒（NCL-EPO-NP）可以抑制念珠菌在替代碳源中的生长， 并在体外拆除成熟生物膜，并在粘膜念珠菌病的生物膜模型中在体内根除它。的 NCL-NP的活性比单独的仿制药高6-10倍。在目标2中，我们进一步表征和 评价NCL-EPO-NP的药代动力学和口服生物分布、毒性特征和体内功效， 一种治疗口腔念珠菌病的口服方案，作为独立治疗或与其他抗真菌药物联合治疗。