Small Molecule Inhibitors of Malate Synthase against M. Tuberculosis

描述（由申请人提供）：本R21探索性提案在合理开发药物时使用了一种综合的靶向方法，可以有效缩短结核病的治疗时间和/或改善对多重耐药结核病的治疗。在结核病研究界存在着广泛的共识，即缩短药物治疗时间而不增加复发率的关键在于特异性靶向对结核分枝杆菌的非复制持久性（NRP）至关重要的蛋白质。其中一个目标是苹果酸合成酶（MS）。研究表明，MS对小鼠的持续生长至关重要，并在体外NRP模型中过度表达。最近在质谱蛋白结构解析方面的成功和质谱功能分析的可用性为将计算机辅助药物设计方法与药物化学相结合，发现新的治疗NRP结核病的药物开辟了一条道路。我们假设，使用计算机辅助药物设计和药物化学的结合来识别和设计MS抑制剂是可能的。这些化合物在低微摩尔范围内显示出对MS的活性，将在全细胞水平上进行筛选，以在基于Wayne低氧模型的微孔板试验中杀死复制和NRP结核分枝杆菌的能力，并对哺乳动物细胞毒性进行选择性筛选。这一迭代过程有望产生几种低微摩尔，铅或药物样ms抑制剂，这些铅将成为进一步开发的候选物，包括更广泛的药物化学努力和后续项目中NRP动物模型的评估。为了实现这一目标，我们的具体目标如下：(1)通过寻找MS天然底物的接近类似物来鉴定MS的铅或药物样低分子量抑制剂，并使用从头/合理药物设计鉴定新的抑制剂，通过计算机筛选虚拟聚焦组合文库和市售化合物数据库；综合最好的候选人；(2)测定候选药物对MS功能活性的抑制作用。(3)鉴定对活性生长和/或非复制持续性（NRP）结核分枝杆菌具有显著选择性活性的化合物。根据活性、细胞毒性和由此产生的选择性指标进行排序。公共卫生相关性：本建议旨在寻找新的苹果酸合成酶抑制剂，这是治疗非复制性持续性结核病的潜在新靶点。该项目首次汇集了与nrp相关的靶标、苹果酸合成酶、具有优化发现有用先导化合物机会的特性的化合物库、计算机辅助药物设计方法和药物化学。

DESCRIPTION (provided by applicant): This R21 exploratory proposal uses an integrated target-oriented approach in the rational development of drugs that may effectively shorten the duration of therapy in tuberculosis and/or improve the treatment of multi-drug resistant TB. There exists a broad consensus in the TB research community that the key to shortening the duration of drug therapy without an increase in relapse rates lies in the specific targeting of proteins that are essential to the non-replicating persistence (NRP) of Mycobacterium tuberculosis. One such target is malate synthase (MS). It has been shown that MS is crucial for persistent growth in mice and is over-expressed in vitro models of NRP. Recent success in the structure elucidation of the MS protein and the availability of functional assays for MS opens a way to apply computer-aided drug design methods in concert with medicinal chemistry efforts to discover new therapeutic agents for NRP TB. We hypothesize that it would be possible to identify and design inhibitors for MS using a combination of computer-aided drug design and medicinal chemistry. Those compounds demonstrating activity at low micromolar range against MS will be screened at the whole cell level for the ability to kill replicating and NRP M. tuberculosis in a microplate assay based on the Wayne low oxygen model and for selectivity with respect to mammalian cell toxicity. This iterative process is expected to yield several low micromolar, lead- or drug-like inhibitors of MS. Such leads would be candidates for further development including more extensive medicinal chemistry efforts and the evaluation in animal models of NRP in subsequent projects. To achieve this goal, our specific aims are as follow: (1) Identify lead- or drug-like low molecular weight inhibitors of MS using a search for close analogs of the natural substrates of MS and identification of new inhibitors using de novo/rational drug design, in silico screening of virtual focused combinatorial libraries and the databases of commercially available chemical compounds; to synthesize the best candidates; (2) Assay candidate drugs for their inhibition of MS functional activity. (3) Identify compounds demonstrating significant selective activity against actively growing and/or non-replicating, persistent (NRP) Mycobacterium tuberculosis. Prioritize on the basis of activity, cytotoxicity and resultant selectivity indices. PUBLIC HEALTH RELEVANCE: This proposal is intended to find new malate synthase inhibitors, a potential new target for non- replicating persistent tuberculosis. This project brings together for the first time an NRP-associated target, malate synthase, libraries of compounds with properties that optimize the chances of finding useful lead compounds, computer-aided drug design approach, and medicinal chemistry.