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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.

侵袭性真菌感染是免疫功能低下患者死亡的主要原因。翻译分子了解临床益处很困难，因为真菌病原体及其宿主具有相似的真核生物特征生理。因此，目前的抗真菌药物临床疗效有限，对宿主的杀真菌作用较差，有些病例是有毒的，并且由于出现耐药性而越来越无效。在过去的二十年里，通过遗传和药理学方法，我们确定钙调磷酸酶 (CN) 是关键侵袭性真菌病的决定因素和抗真菌药物开发的潜在目标。 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，并且不具有真菌选择性。我们的总体目标是利用我们的病原真菌 CN-FK506-FKBP12 复杂的 X 射线结构来构建- 使用补充药物指导设计广谱非免疫抑制 FK506 类似物化学和组合生物合成开发真菌特异性抗真菌药物的新范例。我们的中央假设是基于结构的方法使用分子建模、核磁共振动力学、针对真菌 CN 特异性靶向的分子动力学 (MD) 模拟将改善治疗效果干涉。为了更广泛的治疗视角，我们将重点关注主要的和新难治的临床真菌病原体：烟曲霉、白色念珠菌、耳念珠菌、新型隐球菌、根霉米霉。在目标 1 中，我们将合成在 C21 和 C22 处进行化学修饰的 FK506/FK520 类似物位置，以及通过组合生物合成和合成在其他结构相关位置（C9，C31）策略。 C21和C22残基将通过单步使用不同的起始分子进行修饰反应。与此同时，我们将采用组合生物合成方法来生产修饰的 FK506 类似物通过对链霉菌（FK506 的天然生产者）进行基因操作。具有高亲和力的类似物将筛选形成真菌 CN-FKBP12 复合物的抗真菌活性。在目标 2 中，我们将定义选择性真菌 CN 抑制的决定因素与我们的致病真菌 CN 三元复合 X 射线结构，耦合使用基于 NMR 的抑制剂结合动力学和 MD 模拟来选择性地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。蛋白质定量图谱配体相互作用与基因突变分析一起，将能够设计优化且更具选择性的最大限度地减少哺乳动物免疫抑制并增强抗真菌活性的类似物。在目标 3 中，我们将测试类似物具有额外的体外抗真菌活性，并通过原代鼠 T 确定对哺乳动物 CN 的活性细胞活化测定。表现出有希望的抗真菌活性和减少免疫抑制的类似物将是在侵袭性念珠菌病、曲霉菌病和隐球菌病小鼠模型中测试了疗效。这部作品利用我们的结构生物学和核磁共振动力学经验来设计和合成新型真菌- 特定的钙调神经磷酸酶抑制剂作为一种独特的抗真菌方法，对耐药菌株也有活性。

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.

钙调神经素靶标参与应力存活和真菌毒力。

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