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: 9324801
• 负责人: JOSEPH HEITMAN
• 金额: $ 59.34万
• 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 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9324801
• 关键词: Antifungal Agents; Area; Aspergillosis; Aspergillus fumigatus; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Calcineurin; Calcineurin inhibitor; Calmodulin; Candida albicans; Candidiasis; Cause of Death; Cell division; Chemicals; Chemistry; Clinical; Collaborations; Collection; Complex; Coupled; Crystallization; Crystallography; DNA; Development; Disease; Docking; Drug Targeting; Drug effect disorder; Enzyme Inhibitor Drugs; Enzymes; FDA approved; FK506; Foundations; Generations; Genetic; Genetic Transcription; Growth; HIV Envelope Protein gp41; Health; Human; Immune system; Immunity; Immunocompromised Host; Immunophilins; Immunosuppression; Immunosuppressive Agents; In Vitro; Industrial fungicide; Knowledge; Lead; Maps; Medical; Modification; Molds; Molecular; Molecular Target; Mutagenesis; Mycoses; NMR Spectroscopy; PPP3CA gene; PPP3CB gene; Pathogenesis; Pathogenicity; Patients; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Phenotype; Physiology; Production; Protein Region; Proteins; Research; Resistance; Resistance development; Screening Result; Signal Transduction; Site; Site-Directed Mutagenesis; Specificity; Spices; Structural Biologist; Structure; Surface; Tacrolimus Binding Protein 1A; Techniques; Testing; The science of Mycology; Therapeutic; Titrations; Translating; Treatment Efficacy; Work; Yeasts; analog; base; calcineurin phosphatase; clinical efficacy; cross reactivity; drug development; experimental study; fungus; immunogenic; improved; in vitro testing; inhibitor/antagonist; mouse model; novel; novel strategies; pathogen; public health relevance; segregation; structural biology

DESCRIPTION (provided by applicant): Invasive fungal infections are a leading cause of death in immunocompromised patients. Current antifungals have limited clinical efficacy, are poorly fungicidal, are in some cases toxic, and are increasingly ineffective due to emerging resistance. We have established that the conserved phosphatase calcineurin is broadly required for invasive fungal disease. The FDA-approved calcineurin inhibitor FK506 is active in vitro against major invasive fungal pathogens, but also suppresses host immunity. Our approach seeks to overcome the fungal versus human specificity barrier to significantly advance antifungal treatment. The objective of this study is to utilize a structural biology-based strategy to define fungal-mammalian calcineurin structural differences, validate fungal-specific targets, and generate and optimize novel FK506 analogs to treat invasive fungal diseases. Our central hypothesis is that by employing a structural biological approach using both crystallography and NMR spectroscopy that we will define novel targetable fungal-specific areas in the calcineurin complex critical for fungal pathogenesis. For maximum clinical breadth, we will focus on the two major clinical pathogens: the yeast Candida albicans and the mold Aspergillus fumigatus. Our preliminary studies document proof of principle non-immunosuppressive FK506 analogs with robust antifungal activity. We hypothesize that structures of the calcineurin A and B complex, coupled with calmodulin and the immunophilin complex (FKBP12-FK506), will reveal novel fungal-specific targets for inhibition. We have recently solved the structure for C. albicans, and will now solve structures for the calcineurin heterodimer alone and complexed with FKBP12-FK506/analogs from A. fumigatus. The multiple molecular views will allow identification of sites that are distinct between human and fungal calcineurin complexes that can be exploited for targeted inhibitor development. Protein regions that are dynamic or resist crystallization will be structurally characterized by NMR. Putative inhibitory domains will be validated via genetic and biochemical assays, utilizing site-directed mutagenesis of key contact and surface residues to examine structural stability, fungal phenotype, and drug action/resistance. Non-immunosuppressive fungal-specific FK506 analogs will be generated by Amplyx Pharmaceuticals based on the SAR results of our first iteration. This will guide the production of second generation analogs optimized for retention of antifungal activity and abrogation of immunosuppression by capitalizing on the unique structural differences between the host and fungal enzymes. Medicinal chemistry and inhibitor docking experiments will be conducted to alter analogs based on screening results. Lead compounds will be tested using an iterative approach both in vitro and in murine models of invasive candidiasis and invasive aspergillosis. We will capitalize on structural biology as a new approach to targeting calcineurin by defining fungal-specific features with no mammalian counterpart to generate novel antifungal therapeutics.

描述（由申请人提供）：侵袭性真菌感染是免疫功能低下患者死亡的主要原因。目前的抗真菌药物临床疗效有限，杀真菌效果差，在某些情况下是有毒的，并且由于出现耐药性而越来越无效。我们已经确定保守的磷酸酶钙调磷酸酶在侵袭性真菌疾病中广泛需要。fda批准的钙调磷酸酶抑制剂FK506在体外对主要侵袭性真菌病原体有活性，但也抑制宿主免疫。我们的方法旨在克服真菌与人类特异性屏障，以显著推进抗真菌治疗。本研究的目的是利用结构生物学为基础的策略

项目成果

Forging the ring: from fungal septins' divergent roles in morphology, septation and virulence to factors contributing to their assembly into higher order structures.

锻造环：从真菌脓毒症在形态、分隔和毒力方面的不同作用，到有助于其组装成更高阶结构的因素。

• DOI: 10.1099/mic.0.000359
• 发表时间: 2016
• 期刊: Microbiology (Reading, England)
• 作者: Vargas-Muñiz,JoseM;Juvvadi,PraveenR;Steinbach,WilliamJ
• 通讯作者: Steinbach,WilliamJ

FKBP12 dimerization mutations effect FK506 binding and differentially alter calcineurin inhibition in the human pathogen Aspergillus fumigatus.

FKBP12 二聚化突变影响 FK506 结合并差异改变人类病原体烟曲霉中钙调神经磷酸酶的抑制作用。

• DOI: 10.1016/j.bbrc.2020.03.062
• 发表时间: 2020
• 期刊: Biochemical and biophysical research communications
• 影响因子: 3.1
• 作者: Juvvadi,PraveenR;Bobay,BenjaminG;Gobeil,SophieMC;Cole,DChristopher;Venters,RonaldA;Heitman,Joseph;Spicer,LeonardD;Steinbach,WilliamJ
• 通讯作者: Steinbach,WilliamJ

An Antifungal Combination Matrix Identifies a Rich Pool of Adjuvant Molecules that Enhance Drug Activity against Diverse Fungal Pathogens.

• DOI: 10.1016/j.celrep.2015.10.018
• 发表时间: 2015-11-17
• 期刊: Cell reports
• 影响因子: 8.8
• 作者: Robbins N;Spitzer M;Yu T;Cerone RP;Averette AK;Bahn YS;Heitman J;Sheppard DC;Tyers M;Wright GD
• 通讯作者: Wright GD

Characterization of the FKBP12-Encoding Genes in Aspergillus fumigatus.

• DOI: 10.1371/journal.pone.0137869
• 发表时间: 2015
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Falloon K;Juvvadi PR;Richards AD;Vargas-Muñiz JM;Renshaw H;Steinbach WJ
• 通讯作者: Steinbach WJ