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

• 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
• 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

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 由于真菌病原体及其宿主具有相似的真核生物特性， physiology.因此，目前的抗真菌药物具有有限的临床功效，在宿主中的杀真菌效果差， 有些病例有毒，并且由于出现耐药性而变得越来越无效。在过去的二十年里， 通过遗传和药理学方法，我们确定钙调神经磷酸酶（CN）磷酸酶是关键 侵袭性真菌疾病的决定因素和抗真菌药物开发的潜在靶点。CN抑制剂 FK 506显著抑制真菌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 总体目标是利用我们的致病真菌CN-FK 506-FKBP 12复合物的X射线结构， 使用互补药物的广谱非免疫抑制性FK 506类似物的指导设计 化学和组合生物合成来开发真菌特异性抗真菌剂的新范例。我们的中央 假设是，基于结构的方法来设计抗真菌药物，使用分子建模，NMR动力学， 和分子动力学（MD）模拟真菌CN的特异性靶向将导致改善的治疗 干预为了更广泛的治疗前景，我们将重点关注主要和新发现的临床真菌感染。 病原体：烟曲霉、白色念珠菌、耳念珠菌、新型隐球菌和根霉 米。在目标1中，我们将合成在C21和C22处进行化学修饰的FK 506/FK 520类似物 位置，以及其他结构相关位置（C9，C31）通过组合生物合成和合成 战略布局C21和C22残基将使用不同的起始分子通过单步骤修饰， 反应.同时，我们将采用组合生物合成方法来生产修饰的FK 506类似物 通过对FK 506天然生产者链霉菌的遗传操作。具有高亲和力的类似物 以形成真菌CN-FKBP 12复合物，将筛选抗真菌活性。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性真菌CN三元复合物的X射线结构，耦合 利用基于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CN-FK 506-FKBP 12复合物形成。蛋白质定量图谱 配体相互作用，以及遗传突变分析，将使设计优化和更具选择性， 最小化哺乳动物免疫抑制和增强抗真菌活性的类似物。在目标3中，我们将测试 类似物的额外的体外抗真菌活性，并确定通过原代鼠T细胞对哺乳动物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被 在侵袭性念珠菌病、曲霉病和隐球菌病的小鼠模型中测试其功效。这项工作 利用我们的结构生物学和NMR动力学经验，设计和合成新型真菌- 特异性钙调磷酸酶抑制剂作为一种独特的抗真菌方法，也对耐药菌株有效。

项目成果

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.)
• 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

Dual action antifungal small molecule modulates multidrug efflux and TOR signaling.

• DOI: 10.1038/nchembio.2165
• 发表时间: 2016-10
• 期刊: NATURE CHEMICAL BIOLOGY
• 影响因子: 14.8
• 作者: Shekhar-Guturja, Tanvi;Gunaherath, G. M. Kamal B.;Wijeratne, E. M. Kithsiri;Lambert, Jean-Philippe;Averette, Anna F.;Lee, Soo Chan;Kim, Taeyup;Bahn, Yong-Sun;Tripodi, Farida;Ammar, Ron;Doehl, Katja;Niewola-Staszkowska, Karolina;Schmitt, Lutz;Loewith, Robbie J.;Roth, Frederick P.;Sanglard, Dominique;Andes, David;Nislow, Corey;Coccetti, Paola;Gingras, Anne-Claude;Heitman, Joseph;Gunatilaka, A. A. Leslie;Cowen, Leah E.
• 通讯作者: Cowen, Leah E.