MICA: Modes of action and resistance mechanisms towards anti-trypanosomal benzoxaboroles

Human African trypanosomiasis (HAT), often known as sleeping sickness, is a disease found primarily in rural Africa. It is caused by tiny single celled parasites called trypanosomes. These organisms are transmitted by tsetse flies which suck human blood and can inject parasites while doing so. The parasites initially proliferate in blood but then invade the central nervous system. The presence of parasites proliferating in the brain causes a progressive breakdown in neurological functioning, including alterations in sleep-wake patterns which lend the disease its common name. Drugs that are currently available for HAT are unsatisfactory for a number of reasons. One compound, melarsoprol, is based on arsenic and is so toxic that one in every twenty people treated with the drug die as a result of its administration. Another compound, called eflornithine, has to be injected into veins through a drip several times a day for a fortnight (or a week if given in combination with another drug called nifurtimox (which itself is a potential cancer-causing agent!). New drugs are urgently needed and two compounds are currently in clinical trials. One, called fexinidazole, is being developed by the Drugs for Neglected Diseases initiative (DNDi) and is currently in phase II clinical trials. The second compound, now in Phase I trials is SCYX-7158, one of the benzoxaborole class of compound developed by DNDi in conjunction with Anacor and Scynexis. Whilst fexinidazole is what is known as a nitroheterocyclic compound, whose activity depends on its being activated by an enzyme found inside trypanosomes, little is known about how benzoxaboroles work. They are a very novel class of compound. What is clear, however, is that of the hundreds of variations on this theme that have been produced, they appear not to all work in the same way, based on the time it takes to kill parasites and some differences in the appearance of parasites targeted by these compounds. Here we propose to use a variety of state-of-the-art technologies to determine exactly how different members of the benzoxaborole class work. We will find enzymatic targets of these drugs which will then enable chemists to develop even more potent inhibitors. Moreover, we will also learn about mechanisms of resistance to the drugs which will allow us to classify them into groups where cross resistance is unlikely to occur. Our ultimate aim is to learn as much as possible about this class of compound with a view to fuelling the pipeline of anti-trypanosomal drug development during a period dedicated to the elimination and possibly even eradication of this fatal disease.

Improvements in understanding how new chemical entities can be converted to effective drugs has underpinned development of new drugs for parasitic diseases, including human African trypanosomiasis. An effective series of compounds, the benzoxaborole class, was developed primarily through the implementation of pharmacokinetic and pharmacodynamic (PK/PD) standards. One of the series, SCYX-7158, has entered clinical trials. Less attention, however, was paid to mode of action against trypanosomes. Here we propose a systematic, multifaceted approach to understand the mode of action of the benzoxaborole series. This will be achieved by exploiting a sub-set of distinct members of the class that have demonstrated differential potency, time to kill and PK parameters. We will exploit several orthogonal approaches to answer the question of how these compounds interact with trypanosomes. To determine how the compounds interfere with cellular metabolism, each will be used over a range of concentrations and exposure times against bloodstream form trypanosomes, and metabolites extracted from lysates will be quantified to assess which pathways are impacted. Preliminary work suggests that SCYX-7158 perturbs S-adenosylmethionine metabolism, pointing to a methyltransferase target. The second powerful route to understanding drug mode of action involves selecting parasites for loss of function mutations, using RNA interference to elicit drug resistance. Again our preliminary data with selected compounds indicates that changes to cellular sumoylation and specific dehydrogenase enzymes stimulates resistance to the drugs. Finally we will also use a battery of microscopy based assays to determine alterations to cellular phenotype as cells die. Once data is collected across the series we will then use genetic approaches to validate the putative targets identified though these analyses.

Essentially within this program of research we aim to identify modes of action and drug targets for the benzoxaborole class in Trypanosoma brucei, the causative agent of human African trypanosomiasis. The consortium lead by the Drugs for Neglected Diseases initiative (DNDi), including Scynexis and Anacor (the company who holds the IP on the benzoxaborole class) will benefit from the information provided in terms of leading their own synthetic chemistry programs in developing improved drugs for HAT. Given the same class has also provided candidates for clinical consideration for other conditions including Leishmaniasis, Chagas' disease, malaria and veterinary trypanosomiasis, the information we obtain on mode of action will also be of use in considering how other compounds in the series might work against other protozoan pathogens, as well as in a more broad context in understanding the full potential of these compounds. These benefits will emerge during and shortly after the tenure of the award.

The ultimate beneficiaries from this work will be the victims of sleeping sickness in Africa. Bringing new compounds forward to treat patients, particularly orally available compounds such as the benzoxaboroles, will be a major advance since current drugs are unsatisfactory given the need for dosing by injection, difficulties in availability and manufacture, cost and a risk of resistance. Medical practitioners involved in administering chemotherapy will benefit though improved dosing regiment compared to existing drugs. National Control Programs in sleeping sickness endemic areas will benefit from having safe, oral, optimized drugs for use in control campaigns. The World Health Organisation, who currently lead policy on treatment regimens for HAT and who are now implementing a program to eliminate HAT will equally benefit from having optimized compounds available.

Staff involved in the work will also in from close collaboration with Anacor, and also with the platforms in place in Dundee and Glasgow for drug development. The ability to communicate these findings, to use accurate statistical approaches and the appreciation of data driven projects are additional skills that the research staff will either gain or have reinforced.