Construction of potent and specific inhibitors of M. tuberculosis redox enzymes using fragment screening methods

结核分枝杆菌(Mtb)是一种人类病原体，可导致结核病(TB)。世界卫生组织(WHO)最近的报告显示，世界上三分之一的人口感染了结核病，由于结核分枝杆菌在全球范围内的传播，以及几种结核分枝杆菌菌株对曾经构成有效结核病治疗基础的抗生素产生抗药性，世界卫生组织宣布进入“全球紧急状态”。例如，现在有许多结核分枝杆菌菌株对主要药物利福平和异烟肼--指的是多药耐药(MDR)结核病--具有耐药性。其他菌株对许多其他药物(广泛耐药，或XDR TB)具有耐药性，一些菌株对所有主要结核病药物(完全耐药，或TDR TB)具有耐药性。结核分枝杆菌对药物产生抗药性是一种自然现象，由细菌DNA的随机突变引起，这些突变会导致蛋白质结构的变化，从而防止它们被特定的抗生素灭活。今天面临的结核分枝杆菌耐药性问题是由于多年来对几种结核病药物的耐药性逐渐发展和传播造成的。在同一时期(约20世纪60年代至90年代)，新的结核病药物开发很少，导致有效的结核病抗生素缺乏。虽然新药现在正在流水线中，但有效的结核病药物仍然短缺，还有其他复杂的因素--其中包括结核病患者更容易感染艾滋病毒/艾滋病的问题，反之亦然。为了解决这个问题，我们的计划是推进一条新的路线，以开发能够抑制一组酶(称为P450)的活性的新药，这些酶对结核分枝杆菌的生存和它们在人类宿主中持续感染的能力是必不可少的。虽然以前的结核病药物通常是通过对大型复杂化学品库进行广泛筛选来选择的，但我们将使用一种相对较新的方法来开发针对结核分枝杆菌的新的有效药物。片段筛选的过程涉及使用相对较小的化学物库，通常相当小。这些化学物库与建议的酶靶标(P450)混合，在不同的“片段”与P450结合的情况下，将通过包括改变蛋白质热稳定性的方法来检测这一点。在知道选定的片段与P450结合后，将使用X射线结晶学技术确定蛋白质的三维结构，这也将准确地揭示片段与P450结合的位置。这一信息对于片段筛选过程的进一步发展是必不可少的，因为一旦知道P450内许多化学片段的结合位置，就可以开发一种策略，使用化学方法连接结合在相邻位置的片段，以便制造对特定P450酶显示出更好选择性的更大的化合物。通常需要循环的化学“改进”，但这些可以导致非常有效的抗生素药物。我们的计划是在结核分枝杆菌P450上使用这个片段筛选和开发程序，这对结核分枝杆菌的生存至关重要(一种称为CYP121A1的P450)，对于它利用人的胆固醇在肺中生存的能力(Cyps 124A1、125A和142A1)，以及它感染和传播的能力(称为毒力，CyP128A1)。针对每一种Mtb P450蛋白开发的抗生素药物将优化与其目标的紧密结合，然后通过直接针对细菌病原体测试抑制剂来验证它们终止Mtb生长/杀死细菌的能力。进一步的重要研究将关注结核分枝杆菌对这些抑制剂治疗的反应--特别是寻找细菌产生的化学物质的变化，以了解抑制剂如何影响结核分枝杆菌，并提高抑制剂的有效性。

Mycobacterium tuberculosis (Mtb) is a human pathogen that causes the debilitating disease tuberculosis (TB). Recent reports from the World Health Organization (WHO) indicated that one third of the world's population is infected with TB, and the WHO declared a "global emergency" due to the worldwide spread of Mtb and since several Mtb strains are resistant to antibiotics that once formed the basis of effective TB treatments. For instance, there are now many Mtb strains that are resistant to the leading drugs rifampicin and isoniazid - referred to a multidrug resistant (MDR) TB. Other strains are resistant to many other drugs (extensively drug resistant, or XDR TB), and some are resistant to all major TB drugs (totally drug resistant, or TDR TB). The development of resistance to drugs by Mtb is a natural phenomenon caused by random mutations of bacterial DNA that result in changes to structures of proteins that prevent them being inactivated by particular antibiotics. The issues of Mtb drug resistance faced today arise from the gradual development and spread of resistance to several TB drugs over many years. In the same time period (~1960s to 1990s) there were very few new TB drugs developed, which resulted in a dearth of effective TB antibiotics. While new drugs are now coming through the pipeline, there is still a shortage of effective TB drugs and there are other complicating factors - among which is the issue of TB patients being more susceptible to infection by HIV/AIDS and vice versa. To address this issue, our plan is to progress a novel route to the development of new drugs that can inhibit the activities of a group of enzymes (called P450s) which are known to be essential to the survival of Mtb bacteria and to their ability to sustain infection in their human host. While previous TB drugs have usually been selected by extensive screening of large libraries of complex chemicals, we will use instead a relatively new approach to development of new and effective drugs against Mtb. The process of fragment screening involves using relatively small libraries of chemicals of generally quite small size. These libraries of chemicals are mixed with the proposed enzyme targets (the P450s) and in cases where different "fragments" bind to the P450s, this will be detected by methods including changes to the thermal stability of the proteins. With knowledge that selected fragment bind the P450s, the three dimensional structures of the proteins will be determined using the technique of X-ray crystallography, which will also reveal exactly where fragments bind within the P450s. This information is essential for the further development of the fragment screening process, since once the binding positions of a number of the chemical fragments inside the P450s are known a strategy can be developed to use chemistry approaches to link fragments bound in adjacent positions in order to make larger compounds that show much better selectivity for the particular P450 enzyme. Cycles of chemical "improvement" are usually needed, but these can lead to very effective antibiotic drugs. Our plan is to use this fragment screening and development programme on Mtb P450s essential for survival of Mtb (a P450 called CYP121A1), for its ability to survive in the lungs by using human cholesterol (CYPs 124A1, 125A and 142A1), and for its ability to infect and spread (called virulence, CYP128A1). The antibiotic drugs developed against each of these Mtb P450 proteins will be optimized for tight binding to their targets, and then their ability to terminate growth of Mtb/kill the bacterium will be verified by testing the inhibitors directly against the bacterial pathogen. Further important studies will look at the responses of Mtb to treatment with these inhibitors - in particular looking for changes in the profile of chemicals the bacteria produces in order to understand how the inhibitors affect the Mtb and to enable improvements in inhibitor effectiveness.