Development of a fluorescence-based assay to detect fungus-derived proteolytic ac

开发基于荧光的检测方法来检测真菌来源的蛋白水解酶

• 批准号: 8011475
• 负责人: Amit Galande
• 金额: $ 19.84万
• 依托单位: SRI INTERNATIONAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8011475
• 关键词: Animal Model; Animals; Antibodies; Antifungal Agents; Antifungal Therapy; Antigens; Aspergillosis; Aspergillus; Aspergillus fumigatus; Biological; Biological Assay; Body Fluids; Breathing; Bronchoalveolar Lavage; Bronchoalveolar Lavage Fluid; Buffers; Cell Wall; Characteristics; Clinical; Complex; Contracts; Detection; Development; Diagnosis; Diagnostic; Diagnostic Procedure; Diagnostic tests; Disease; Early Diagnosis; Environment; Enzymes; Fluorescence; Foundations; Fungal DNA; Genome; Goals; Human; Immunocompromised Host; In Vitro; Infection; Laboratory Finding; Libraries; Life; Lung; Measurable; Methodology; Methods; Molds; Molecular Target; Monitor; Nutrient; Organism; Outcome; Patients; Peptide Hydrolases; Phase; Polysaccharides; Population; Property; Reaction; Reproduction spores; Sampling; Serum; Source; Specimen; Symptoms; Technology; Testing; Tissues; Translating; Validation; Virulence; Work; assay development; base; combinatorial; diagnostic accuracy; effective therapy; fluorophore; fungus; immunosuppressed; improved; in vivo; mortality; novel; novel diagnostics; outcome forecast; pathogen; protein aminoacid sequence; rapid diagnosis; success; tool

DESCRIPTION (provided by applicant): Aspergillus fumigatus is the most prevalent airborne fungal pathogen, causing severe and usually fatal invasive infections in immunocompromised hosts. Success of anti-fungal therapy relies heavily on accuracy of diagnosis, but rapid and reliable detection of A. fumigatus remains difficult owing to the non-specific clinical symptoms and lack of distinct molecular targets. This proposal aims to utilize thermostable proteases secreted by A. fumigatus into the host environment as a target for the development of a novel fluorescence-based diagnostic assay for invasive aspergillosis. The applicant hypothesizes that fungal proteolytic activity can be detected in serum and BAL samples during invasive aspergillosis using a panel of Internally Quenched Fluorogenic Probes (IQFPs). Aim 1 of the R21 phase will use a commercially available library of 360 IQFPs to optimize assay conditions for maximal detection of fungal proteolytic activity secreted in vitro. These conditions will then be used to test the feasibility of the assay for the detection of A. fumigatus in serum and BAL samples from animal models of infection (Source: IAAM contract). In Aim 2, a panel of eight IQFPs with most promising diagnostic properties will be selected for targeted optimization in order to maximize sensitivity and selectivity. The R33 phase we will then evaluate the efficacy of this IQFP-based diagnostic platform for the detection of A. fumigatus proteases in clinical samples from human patients (Source: AsTeC contract). In addition, a comprehensive combinatorial library of 640,000 IQFPs will be assembled in order to expand the diagnostic applications to include the monitoring of antifungal therapy and the detection of different pathogenic moulds.

描述（由申请方提供）：烟曲霉是最常见的空气传播真菌病原体，可在免疫功能低下的宿主中引起严重且通常致命的侵入性感染。抗真菌治疗的成功在很大程度上依赖于诊断的准确性，但快速可靠的检测A。由于非特异性的临床症状和缺乏明确的分子靶点，烟曲霉仍然很困难。该提议旨在利用由A.烟曲霉进入宿主环境作为一种新的基于荧光的侵袭性曲霉病诊断试验的发展目标。申请方假设，在侵袭性曲霉病期间，使用一组内部淬灭荧光探针（IQFP）可在血清和BAL样本中检测到真菌蛋白水解活性。R21阶段的目标1将使用360个IQFP的市售文库来优化测定条件，以最大限度地检测体外分泌的真菌蛋白水解活性。然后将这些条件用于测试用于检测A的测定的可行性。感染动物模型的血清和BAL样品中的烟曲霉菌（来源：IAAM合同）。在目标2中，将选择具有最有前途的诊断特性的八个IQFP小组进行有针对性的优化，以最大限度地提高灵敏度和选择性。在R33阶段，我们将评估这种基于IQFP的诊断平台检测A.在来自人类患者的临床样品中的烟曲霉蛋白酶（来源：AsTeC合同）。此外，将组装一个包含640，000个IQFP的综合组合库，以扩大诊断应用，包括监测抗真菌治疗和检测不同的致病性霉菌。

项目成果

Proteolytic fingerprinting of complex biological samples using combinatorial libraries of fluorogenic probes.

使用荧光探针组合文库对复杂生物样品进行蛋白水解指纹分析。

• DOI: 10.1002/0471140864.ps2122s70
• 发表时间: 2012
• 期刊: Current protocols in protein science
• 作者: Jambunathan,Kalyani;Watson,DouglasS;Kodukula,Krishna;Galande,AmitK
• 通讯作者: Galande,AmitK

Comparative analysis of the substrate preferences of two post-proline cleaving endopeptidases, prolyl oligopeptidase and fibroblast activation protein α.

• DOI: 10.1016/j.febslet.2012.06.015
• 发表时间: 2012-07-30
• 期刊: FEBS letters
• 影响因子: 3.5
• 作者: Jambunathan K;Watson DS;Endsley AN;Kodukula K;Galande AK
• 通讯作者: Galande AK

Substrate specifity profiling of the Aspergillus fumigatus proteolytic secretome reveals consensus motifs with predominance of Ile/Leu and Phe/Tyr.

烟曲霉蛋白水解分泌组的底物特异性分析揭示了以 Ile/Leu 和 Phe/Tyr 为主的共有基序。

• DOI: 10.1371/journal.pone.0021001
• 发表时间: 2011
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Watson,DouglasS;Feng,Xizhi;Askew,DavidS;Jambunathan,Kalyani;Kodukula,Krishna;Galande,AmitK
• 通讯作者: Galande,AmitK