Structural Biological Development of Fungal-Specific Calcineurin Inhibitors

真菌特异性钙调神经磷酸酶抑制剂的结构生物学发展

基本信息

批准号：
10248016
负责人：
JOSEPH HEITMAN
金额：
$ 66.92万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-04 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10248016
关键词：
Affinity Anabolism Animal Model Animals Antifungal Agents Aspergillosis Aspergillus fumigatus Binding Biologic Development Biological Biological Assay Calcineurin Calcineurin inhibitor Calorimetry Candida albicans Candida auris Candidiasis Cause of Death Chemicals Chemistry Clinical Collaborations Complex Coupled Cryptococcosis Cryptococcus neoformans Crystallization DNA Sequence Alteration Data Collection Development Disease Drug resistance Enzymes Exhibits FK506 Foundations Genetic Human Immune system Immunocompromised Host Immunosuppression In Vitro Industrial fungicide Infection Interdisciplinary Study Isotope Labeling Knowledge Lead Libraries Ligands Medical Methods Modeling Modification Molecular Mus Mutation Analysis Mycoses NMR Spectroscopy Oral Pathogenesis Pathogenicity Patients Pharmaceutical Chemistry Pharmaceutical Preparations Pharmacology Physiologic pulse Physiology Positioning Attribute Predisposition Proteins Reaction Resistance Rhizopus Roentgen Rays Signal Pathway Standardization Streptomyces Structure Structure-Activity Relationship T-Cell Activation Tacrolimus Binding Protein 1A Testing Therapeutic Intervention Titrations Translating Treatment Efficacy Work analog base biophysical properties calcineurin phosphatase clinical efficacy clinically relevant combinatorial design drug development effective therapy efficacy testing experience genetic manipulation improved in vitro Assay in vivo inhibitor/antagonist iterative design molecular dynamics molecular modeling mouse model novel pathogenic fungus protein structure simulation small molecule structural biology

项目摘要

Invasive fungal infections are a leading cause of death in immunocompromised patients. Translating molecular understanding into clinical benefit is difficult because fungal pathogens and their hosts have similar eukaryotic physiology. As a result, current antifungals have limited clinical efficacy, are poorly fungicidal in the host, are in some cases toxic, and are increasingly ineffective due to emerging drug resistance. Over the last two decades, through genetic and pharmacologic approaches we established that calcineurin (CN) phosphatase is a key determinant for invasive fungal disease and a potential target for antifungal drug development. The CN inhibitor FK506 significantly inhibits fungal CN but is also immunosuppressive in the host and not fungal-selective. Our overall objective is to leverage our pathogenic fungal CN-FK506-FKBP12 complex X-ray structures for structure- guided design of broad-spectrum non-immunosuppressive FK506 analogs using complementary medicinal chemistry and combinatorial biosynthesis to develop a novel paradigm of fungal-specific antifungals. Our central hypothesis is that a structure-based approach to design antifungals using molecular modeling, NMR dynamics, and molecular dynamic (MD) simulations for specific targeting of fungal CN will lead to improved therapeutic intervention. For a broader treatment perspective, we will focus on the major and newly recalcitrant clinical fungal pathogens: Aspergillus fumigatus, Candida albicans, Candida auris, Cryptococcus neoformans, and Rhizopus oryzae. In Aim 1, we will synthesize FK506/FK520 analogs with chemical modifications at the C21 and C22 positions, and at other structurally relevant positions (C9, C31) by combinatorial biosynthetic and synthetic strategies. The C21 and C22 residues will be modified using different starter molecules through single-step reactions. In parallel, we will employ combinatorial biosynthetic approach to produce modified FK506 analogs through genetic manipulation of Streptomyces species, the natural producer of FK506. Analogs with high affinity to form fungal CN-FKBP12 complexes will be screened for antifungal activity. In Aim 2, we will define selective determinants of fungal CN inhibition with our pathogenic fungal CN ternary complex X-ray structures, coupled with NMR-based inhibitor binding dynamics and MD simulations to selectively define the inhibitor interactions that differentiate fungal and human CN-FK506-FKBP12 complex formation. Quantitative mapping of protein ligand interactions, together with genetic mutational analyses, will enable design of optimized and more selective analogs that minimize mammalian immunosuppression and enhance antifungal activity. In Aim 3, we will test analogs for additional in vitro antifungal activity and define activity on mammalian CN through primary murine T cell activation assays. Analogs that exhibit promising antifungal activity and reduced immunosuppression will be tested for efficacy in murine models of invasive candidiasis, aspergillosis, and cryptococcosis. This work capitalizes on our structural biology and NMR dynamics experience to design and synthesize novel fungal- specific calcineurin inhibitors as a unique antifungal approach that are also active against drug-resistant isolates.

侵入性真菌感染是免疫功能低下患者死亡的主要原因。翻译分子对临床益处的了解很困难，因为真菌病原体及其宿主具有类似的真核。生理。结果，当前的抗真菌剂的临床疗效有限，宿主中的杀真菌性很差某些情况有毒，并且由于新兴的耐药性而越来越无效。在过去的二十年中，通过遗传和药理学方法，我们确定钙调神经磷酸酶（CN）磷酸酶是关键侵入性真菌疾病的决定因素和抗真菌药物开发的潜在靶标。 CN抑制剂 FK506显着抑制真菌CN，但在宿主中也具有免疫抑制作用，而不是真菌选择性。我们的总体目的是利用我们的致病真菌CN-FK506-FKBP12复合X射线结构用于结构 - 宽光谱非免疫抑制FK506类似物的指导设计使用互补药化学和组合生物合成，以开发一种新型真菌特异性抗真菌性的范式。我们的中心假设是一种基于结构的方法，使用分子建模，NMR动力学设计抗真菌剂和分子动力（MD）模拟用于真菌CN的特异性靶向将导致治疗性改善干涉。为了更广泛的治疗观点，我们将重点关注主要和新的顽固临床真菌病原体：曲霉菌，白色念珠菌，念珠菌，念珠菌，新虫和根瘤菌 oryzae。在AIM 1中，我们将在C21和C22的化学修饰中合成FK506/FK520类似物位置，以及在结构相关的其他位置（C9，C31），合并生物合成和合成策略。 C21和C22残基将通过单步分子进行修饰反应。同时，我们将采用组合生物合成方法来产生改良的FK506类似物通过对链霉菌物种的基因操纵，FK506的天然生产国。高亲和力的类似物为了形成真菌CN-FKBP12复合物，将筛选抗真菌活性。在AIM 2中，我们将定义选择性通过我们的致病真菌CN三元复合物X射线结构抑制真菌CN的决定因素，耦合使用基于NMR的抑制剂结合动力学和MD模拟，以选择性定义抑制剂相互作用区分真菌和人类CN-FK506-FKBP12复合物的形成。蛋白质的定量图配体相互作用以及基因突变分析将使设计优化和更有选择性最小化哺乳动物免疫抑制并增强抗真菌活性的类似物。在AIM 3中，我们将测试类似物，用于额外的体外抗真菌活性，并通过原代鼠T定义哺乳动物CN的活性细胞激活测定。表现出有希望的抗真菌活性和免疫抑制降低的类似物将是在浸润性念珠菌病，曲霉病和隐球菌病的鼠模型中测试了功效。这项工作利用我们的结构生物学和NMR动态经验来设计和综合新型真菌 - 特定的钙调蛋白抑制剂作为一种独特的抗真菌方法，对药物抗药性分离株也具有活性。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Structures of Pathogenic Fungal FKBP12s Reveal Possible Self-Catalysis Function.

DOI：
10.1128/mbio.00492-16
发表时间：
2016-04-26
期刊：
mBio
影响因子：
6.4
作者：
Tonthat NK;Juvvadi PR;Zhang H;Lee SC;Venters R;Spicer L;Steinbach WJ;Heitman J;Schumacher MA
通讯作者：
Schumacher MA

Leveraging Fungal and Human Calcineurin-Inhibitor Structures, Biophysical Data, and Dynamics To Design Selective and Nonimmunosuppressive FK506 Analogs.

DOI：
10.1128/mbio.03000-21
发表时间：
2021-12-21
期刊：
mBio
影响因子：
6.4
作者：
Gobeil SM;Bobay BG;Juvvadi PR;Cole DC;Heitman J;Steinbach WJ;Venters RA;Spicer LD
通讯作者：
Spicer LD

Had1 Is Required for Cell Wall Integrity and Fungal Virulence in Cryptococcus neoformans.

DOI：
10.1534/g3.117.300444
发表时间：
2018-02-02
期刊：
G3 (Bethesda, Md.)
影响因子：
0
作者：
Jung WH;Son YE;Oh SH;Fu C;Kim HS;Kwak JH;Cardenas ME;Heitman J;Park HS
通讯作者：
Park HS

Calcineurin Targets Involved in Stress Survival and Fungal Virulence.

DOI：
10.1371/journal.ppat.1005873
发表时间：
2016-09
期刊：
PLoS pathogens
影响因子：
6.7
作者：
Park HS;Chow EW;Fu C;Soderblom EJ;Moseley MA;Heitman J;Cardenas ME
通讯作者：
Cardenas ME

Metal Chelation as a Powerful Strategy to Probe Cellular Circuitry Governing Fungal Drug Resistance and Morphogenesis.

DOI：
10.1371/journal.pgen.1006350
发表时间：
2016-10
期刊：
PLoS genetics
影响因子：
4.5
作者：
Polvi EJ;Averette AF;Lee SC;Kim T;Bahn YS;Veri AO;Robbins N;Heitman J;Cowen LE
通讯作者：
Cowen LE