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

当前基于高通量筛选的抗结核药物（包括结核病药物）药物发现算法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活性的化合物（前药）在高通量筛选中无法检测到。同样，在体外ADME评估过程中LC-MS发现代谢不稳定的化合物不会进行体内评估，因为假设代谢物是无活性的（2型化合物）。要证明这一假设，需要鉴定代谢物的结构、合成和直接测试代谢物。在开发阶段，这需要大量的时间和人力。我们将建立快速检测肝酶衍生代谢产物抗结核（TB）活性的体外检测方法。这些结核病活性代谢物测定（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物生成系统（MGS）和快速抗结核检测。酶促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蓝还原或发光细胞内ATP测定快速活力评估相容。HepaRG和MCL-5细胞上清液也将被评估为MGS。为了区分活性亲本化合物（3型）和代谢稳定的活性代谢物，活性亲本化合物（1型）将与TAMA数据一起进行亲本化合物稳定性的LC-MS分析。优化后的TAMA将用于10万化合物的HTS中，以鉴定前药和潜在的3型化合物。通过LC-MS和随后的数据分析确定所有命中的稳定性将区分3型和1型化合物。选定的代谢物将通过LC-MS/MS和NMR鉴定，合成并直接评估结核分枝杆菌作为概念证明。MGS可以增加复合图书馆的多样性，并考虑重新访问先前筛选的图书馆。通过这种方法在早期检测活性代谢物的能力

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.