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: 8423679
• 负责人: Scott G. Franzblau
• 金额: $ 23.48万
• 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 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8423679
• 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; 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; screening; 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.

描述（由申请人提供）：早期药物发现中抗结核肝脏代谢物的体外检测摘要当前基于高通量筛选的抗菌药物（包括结核病药物）药物发现算法未能考虑到早期活性代谢物的可能性在药物发现过程中。在高通量筛选中未检测到仅在肝脏代谢后才具有活性的化合物（前药）。同样，在体外 ADME 评估过程中通过 LC-MS 发现代谢不稳定的化合物不会进入体内评估，假设代谢物将无活性（2 型化合物）。证明这一假设需要鉴定代谢物结构、合成并直接测试代谢物。在“从命中到领先”阶段，这太费时费力了。我们将建立体外测定法来快速检测肝酶衍生代谢物的抗结核（TB）活性。这些结核活性代谢物测定（TAMA）应该不需要代谢物识别和合成来确认活性代谢物的存在或不存在。我们将把肝酶代谢物生成系统 (MGS) 与快速抗结核检测结合起来。酶 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 与结核分枝杆菌生长和通过荧光测定 Alamar Blue 还原或发光细胞内 ATP 测量进行快速活力评估兼容。 HepaRG 和 MCL-5 细胞上清液也将作为 MGS 进行评估。为了区分活性母体化合物（类型 3）的活性代谢物与代谢稳定的活性母体化合物（类型 1），将对母体化合物稳定性进行并发 LC-MS 分析，并与 TAMA 数据一起进行分析。优化的 TAMA 将用于 100K 化合物的 HTS 中，以鉴定前药和潜在的 3 型化合物。通过 LC-MS 确定所有命中的稳定性以及随后的数据分析将区分 3 型化合物和 1 型化合物。选定的代谢物将通过 LC-MS/MS 和 NMR 进行鉴定、合成并直接针对结核分枝杆菌进行评估，作为概念证明。 MGS 可以增加化合物库的多样性，并考虑重新访问先前筛选的库。通过这种方法能够在早期检测活性代谢物 药物发现过程将允许一些代谢稳定性较差的活性（3 型）化合物的进一步发展，否则仅使用基于 LC-MS 的稳定性测定将不会优先考虑这些化合物。