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

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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.