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

描述(申请人提供)：抗结核肝脏代谢物在早期药物发现中的体外检测摘要目前基于高通量筛选的抗菌药物发现算法，包括那些治疗结核病的药物，未能考虑到药物发现过程早期活性代谢物的可能性。只有在肝脏代谢后才有效的化合物(前体药物)在高通量筛查中检测不到。同样，在体外ADME评估中被LC-MS发现代谢不稳定的化合物不会进入体内评估，假设代谢物将是不活跃的(类型2化合物)。要证明这一假设，需要鉴定代谢物结构，直接合成和测试代谢物。在主打阶段，这是太耗费时间和劳动了。我们将建立体外检测方法来快速检测肝酶衍生代谢物的抗结核活性。这些TB活性代谢物分析(TAMA)应该不需要代谢物ID和合成来确认活性代谢物的存在或不存在。我们将结合肝酶代谢产物生成系统(MGS)和快速抗结核检测。酶MGS包括肝微粒体、S9组分、重组CYP450酶和这些系统的组合，可用于一锅分析。初步数据表明，酶促MGS与结核分枝杆菌的生长和通过荧光检测阿拉马蓝还原或细胞内发光的ATP测量快速评估活性是相容的。HepaRG和MCL-5细胞上清液也将被评估为MGS。为了区分活性母体化合物(类型3)的活性代谢物和代谢稳定性，活性母体化合物(类型1)将同时进行母体化合物稳定性的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，以识别前药物和潜在的类型3化合物。用LC-MS测定所有HITS的稳定性和随后的数据分析将区分类型3和类型1的化合物。选定的代谢物将通过LC-MS/MS和核磁共振进行鉴定，合成并作为概念验证直接针对结核分枝杆菌进行评估。MGS可以增加化合物文库的多样性，并考虑重新访问以前筛选过的文库。通过这种方法在早期检测活性代谢物的能力 药物发现过程将允许一些代谢稳定性较差的活性(类型3)化合物的进一步发展，否则将仅使用基于LC-MS的稳定性分析来排除这些化合物。

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.