Human African trypanosomiasis (HAT) and leishmaniasis are identified by the World Health Organization (WHO) as neglected tropical diseases (NTDs), together with Chagas disease and Buruli ulcer. These NTDs mostly affect people in remote or rural area, and there are very limited control and therapeutic options. The investment on research and development against NTDs is insufficient.
Human African trypanosomiasis (HAT) is a vector-borne parasitic disease caused by Trypanosoma brucei subspecies. Transmitted by the tsetse fly, the disease mainly affects rural populations in sub-Saharan Africa and is fatal if untreated. New drugs are needed against HAT that are safe, affordable, easy to administer, active against first and second stage disease, and effective against both subspecies of T. brucei.
From medicinal chemistry investigation in Karl Werbovetz group, several N1-substituted 1,2-dihydroquinoline-6-ols were discovered displaying nanomolar IC50 values in vitro against T. b. rhodesiense and selectivity indexes (SI) up to >18,000. OSU-40 (1-benzyl-1,2-dihydro-2,2,4–trimethylquinolin-6-yl acetate) is selectively potent against T. b. rhodesiense in vitro (IC50 = 14 nM, selectivity index = 1700), and has been proposed to cause the formation of reactive oxygen species (ROS) in African trypanosomes. In the present study, we sought to provide further support for the hypothesis that OSU-40 kills trypanosomes through oxidative stress. Inducible RNAi interference (RNAi) was applied to down-regulate key enzymes in parasite antioxidant defense, including trypanothione synthetase (TbTryS) and a superoxide dismutase (TbSODB). Both TbTryS RNAi-induced and TbSODB RNAi-induced cells showed impaired growth and increased sensitivity towards OSU-40 by 2.4-fold and 3.4-fold respectively. Decreased expression of key parasite antioxidant enzymes was thus associated with an increased sensitivity to OSU-40, consistent with the hypothesis that OSU-40 acts through oxidative stress. Finally, the dose-dependent formation of free radicals was observed after incubation of T. brucei with OSU-40 utilizing electron spin resonance (ESR) spectroscopy. These data support the notion that the mode of antitrypanosomal action for this class of compounds is to induce oxidative stress.
The ultimate goal of our work with antitrypanosomal dihydroquinolines is to develop a new drug against HAT. A series of studies were carried out with representative dihydroquinolines to evaluate their drug-like properties to aid the development of these compounds as prospective new HAT treatments. The kinetic solubility assay confirmed that the salt forms (OSU-36•HCl and OSU-95•HCl) showed improved solubility compared to the esters (OSU-40 and OSU-75), respectively. The liver cytotoxicity assay showed that dihydroquinolines were highly selective on trypanosomes over human liver cells, suggesting the compounds were not particularly more toxic to liver cells. Clonal OSU-40 resistant T. brucei parasites were developed through 22-month selection under increasing OSU-40 pressure. These clonal cells showed dramatically lowered sensitivity to OSU-40 and OSU-36•HCl and, to our surprise, higher sensitivity to both nifurtimox and H2O2. Whole genome sequencing will be performed to determine differences between the parental line and resistant line.
Leishmaniasis is a parasitic disease caused by more than 20 species of the protozoan Leishmania. Canine leishmaniaisis caused by Leishmania infantum is one of the major zoonotic diseases that lead to death in dogs. Drugs applied in therapy for canine leishmaniasis are not satisfactory. Studies on new combination therapy and canine vaccines have been reported targeting canine leishmaniasis, but discovery and development of novel drugs against this disease caused by Leishmania infantum has not been reported.
High throughput screening assays on L. infantum and L. donovani were designed, developed and validated in a rapid and simple 384-well format with a satisfactory robustness (Z’ = 0.71 and 0.86, respectively). From the screening of a 427-member library, six compounds (2, 14, 17, 19, 20 and 21) exhibited IC50 values that are comparable to pentamidine (lower than 5 μM) against L. infantum and/or L. donovani. Comparison of L. infantum screen to L. donovani screen indicates that screen against L. donovani is not an ideal approach to identify hits on L. infantum because of a high false positive rate. The antileishmanial screening assay against L. infantum developed in this study is a good initiation point leading to new drug candidates to the discovery pipeline for canine leishmaniasis.