In vitro detection of anti-TB liver metabolites in early drug discovery

早期药物发现中抗结核肝脏代谢物的体外检测

• 批准号: 8301448
• 负责人: Scott G. Franzblau
• 金额: $ 19.48万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-15 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8301448
• 关键词: 2-cyclopentyl-5-(5-isoquinolylsulfonyl)-6-nitro-1H-benzo(D)imidazole; Accounting; AlamarBlue; Algorithms; Antitubercular Agents; Biological; Biological Assay; Cells; Coupling; Data; Data Analyses; Detection; Development; Diversity Library; Enzymes; Evaluation; Generations; Growth; In Vitro; Incubated; Laboratories; Lead; Libraries; Liver; Liver Microsomes; Mass Spectrum Analysis; Measurement; Measures; Mediating; Metabolic; Metabolism; Methodology; Methods; Mycobacterium tuberculosis; Organic solvent product; Parents; Pharmaceutical Preparations; Process; Prodrugs; Reaction; Recombinants; Screening procedure; Staging; Structure; System; Testing; Time; Tuberculosis; Validation; abstracting; antimicrobial drug; assay development; base; design; drug discovery; high throughput screening; in vitro Assay; in vivo; novel; particle; rapid growth; small molecule libraries; tuberculosis drugs

DESCRIPTION (provided by applicant): In vitro detection of anti-TB liver metabolites in early drug discovery Abstract Current algorithms for high throughput screening-based drug discovery for antimicrobial agents, including those for tuberculosis, fail to account for the possibility of active metabolites early in the drug discovery process. Compounds that would only be active after metabolism in the liver (prodrugs) are not detected in high throughput screens. Similarly, compounds that are found to be metabolically unstable by LC-MS during in vitro ADME evaluation are not progressed to in vivo evaluation on the assumption that metabolites will be inactive (type 2 compounds). Proving that assumption would require identification of the metabolite structure, synthesis and testing the metabolite directly. This is too time and labor intensive at the hit to lead stage. We will establish in vitro assays to rapidly detect the anti-tuberculosis (TB) activity of liver enzyme-derived metabolites. These TB-active metabolite assays (TAMA) should obviate the need for metabolite ID and synthesis to confirm the presence or absence of an active metabolite. We will combine liver enzyme metabolite generation systems (MGS) with rapid anti-TB assays. Enzymatic MGS includes liver microsomes, S9 fractions, recombinant CYP450 enzymes and combinations of these systems and can be used in a one-pot assay. Preliminary data suggests enzymatic MGS are compatible with M. tuberculosis growth and rapid viability assessment by fluorometric determination of Alamar Blue reduction or luminescent intracellular ATP measurement. HepaRG and MCL-5 cell supernatants will also be assessed as MGS. To distinguish active metabolites of active parent compounds (type 3) from metabolically stable, active parent compounds (type 1) concurrent LC-MS analysis of parent compound stability will be performed and analyzed together with the TAMA data. An optimized TAMA will be used in a HTS of 100K compounds to identify prodrugs and potential type 3 compounds. Determination of stability of all hits by LC-MS and subsequent data analysis will differentiate type 3 from type 1 compounds. Selected metabolites will be identified by LC-MS/MS and NMR, synthesized and evaluated directly against M. tuberculosis as proof of concept. The MGS can increase compound library diversity and give consideration to revisiting previously screened libraries. The ability to detect active metabolites by this method early in the drug discovery process will allow for the further progression of some active (type 3) compounds with poor metabolic stability that otherwise would be deprioritized using only LC-MS based stability assays. PUBLIC HEALTH RELEVANCE: Current drug discovery practices are not designed to detect metabolites produced by liver enzymes that maintain or attain biological activity, yet these have the potential to become valuable drugs. In an effort to facilitate the discovery of new drugs for tuberculosis we are developing new laboratory tests that will detect, at an early stage of the drug discovery process, liver metabolites that are active against Mycobacterium tuberculosis.

描述（由申请人提供）：早期药物发现中抗TB肝脏代谢产物的体外检测抽象的当前算法，用于基于高吞吐量筛查的抗菌剂的药物发现，包括结核病的药物，无法解释药物发现过程中活性代谢的可能性。在高吞吐量筛选中未检测到只有在肝脏中新陈代谢后才活跃的化合物。同样，由于代谢产物不活跃的假设，LC-MS在体外评估过程中发现在代谢上不稳定的化合物并未进行体内评估（2型化合物）。证明假设将需要鉴定代谢物结构，合成和直接测试代谢产物。这是时间和劳动范围内的时间，在铅阶段的热门阶段。我们将建立体外测定，以快速检测肝衍生代谢产物的抗结核活性（TB）活性。这些TB活性代谢物测定（TAMA）应消除对代谢物ID的需求，并合成以确认存在或不存在活性代谢物。我们将将肝酶代谢物生成系统（MGS）与快速抗TB分析相结合。酶促MGS包括肝微粒体，S9级分，重组CYP450酶以及这些系统的组合，可用于一锅测定。初步数据表明，酶促MGS与结核分枝杆菌的生长和通过荧光测定Alamar Blue还原或发光细胞内ATP测量通过荧光测定来评估。 HEPARG和MCL-5细胞上清液也将作为MG评估。为了区分活性母体化合物（类型3）的活性代谢产物与代谢稳定的活性父化合物（类型1）同时进行父级化合物稳定性的LC-MS分析，并将与TAMA数据一起进行分析。优化的TAMA将在100K化合物的HTS中使用，以识别前药和潜在的3型化合物。通过LC-MS确定所有命中的稳定性和随后的数据分析将使3型与1型化合物区分开。选定的代谢产物将通过LC-MS/MS和NMR鉴定，并直接与结核分枝杆菌合成并评估为概念证明。 MGS可以增加复合图书馆的多样性，并考虑重新审视先前筛选的库。早期通过这种方法检测活性代谢物的能力 药物发现过程将允许某些活性（3型）化合物的代谢稳定性差的进一步进展，否则仅使用基于LC-MS的稳定性测定法会剥夺这些化合物。 公共卫生相关性：当前的药物发现实践并非旨在检测维持或获得生物学活性的肝酶产生的代谢产物，但这些可能会成为有价值的药物。为了促进发现结核病的新药物，我们正在开发新的实验室测试，这些测试将在药物发现过程的早期阶段检测到活跃于结核分枝杆菌的肝脏代谢产物。