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

描述（由申请人提供）：在早期药物发现中体外检测抗TB肝脏代谢物摘要当前用于抗微生物剂（包括用于结核病的抗微生物剂）的基于高通量筛选的药物发现的算法未能考虑药物发现过程早期活性代谢物的可能性。仅在肝脏中代谢后才有活性的化合物（前药）在高通量筛选中未检测到。类似地，在体外ADME评价期间通过LC-MS发现代谢不稳定的化合物不进行体内评价，假设代谢物无活性（2型化合物）。证明这一假设需要鉴定代谢物结构、合成和直接测试代谢物。这是太多的时间和劳动密集型在击中领先阶段。我们将建立体外试验，以快速检测肝酶衍生代谢物的抗结核（TB）活性。这些TB-活性代谢物测定（TAMA）应满足代谢物ID和合成的需要，以确认是否存在活性代谢物。我们将把联合收割机肝酶代谢物生成系统（MGS）与快速抗结核检测相结合。酶MGS包括肝微粒体、S9组分、重组CYP 450酶以及这些系统的组合，可用于一锅法测定。初步数据表明酶促MGS与M相容。通过荧光测定法测定Alcohol Blue还原或发光细胞内ATP测量，对结核菌生长和快速生存力进行评估。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（3型）的活性代谢物与代谢稳定的活性母体化合物（1型），将进行母体化合物稳定性的并行LC-MS分析，并与TAMA数据一起分析。优化的TAMA将用于100 K化合物的HTS中，以鉴定前药和潜在的3型化合物。通过LC-MS测定所有命中的稳定性和随后的数据分析将区分3型和1型化合物。将通过LC-MS/MS和NMR鉴别选定的代谢物，合成并直接针对M进行评价。肺结核作为概念的证明。MGS可以增加化合物文库的多样性，并考虑重新访问先前筛选的文库。通过这种方法在早期检测活性代谢物的能力是非常重要的。 药物发现过程将允许一些代谢稳定性差的活性（3型）化合物的进一步发展，否则仅使用基于LC-MS的稳定性测定将降低这些化合物的优先级。 公共卫生相关性：目前的药物发现实践并不是为了检测由肝酶产生的维持或获得生物活性的代谢物，但这些代谢物有可能成为有价值的药物。为了促进结核病新药的发现，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对结核分枝杆菌有活性的肝脏代谢物。

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.