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In the interest of identification of new kinase-targeting chemotypes for target and pathway analysis and drug discovery in Trypanosomal brucei, a high-throughput screen of 42,444 focused inhibitors from the GlaxoSmithKline screening collection was performed against parasite cell cultures and counter-screened against human hepatocarcinoma (HepG2) cells. In this way, we have identified 797 sub-micromolar inhibitors of T. brucei growth that are at least 100-fold selective over HepG2 cells. Importantly, 242 of these hit compounds acted rapidly in inhibiting cellular growth, 137 showed rapid cidality. A variety of in silico and in vitro physicochemical and drug metabolism properties were assessed, and human kinase selectivity data were obtained, and, based on these data, we prioritized three compounds for pharmacokinetic assessment and demonstrated parasitological cure of a murine bloodstream infection of T. brucei rhodesiense with one of these compounds (NEU-1053). This work represents a successful implementation of a unique industrial-academic collaboration model aimed at identification of high quality inhibitors that will provide the parasitology community with chemical matter that can be utilized to develop kinase-targeting tool compounds. Furthermore these results are expected to provide rich starting points for discovery of kinase-targeting tool compounds for T. brucei, and new HAT therapeutics discovery programs.

BACKGROUND:Sleeping sickness is spread over 36 Sub-Saharan African countries. In West and Central Africa, the disease is caused by Trypanosoma brucei gambiense, which produces a chronic clinical manifestation. The Luba focus (Bioko Island, Equatorial Guinea) has not reported autochthonous sleeping sickness cases since 1995, but given the complexity of the epidemiological cycle, the elimination of the parasite in the environment is difficult to categorically ensure. METHODOLOGY/PRINCIPAL FINDINGS:The aim of this work is to assess, by a molecular approach (Polymerase Chain Reaction, PCR), the possible permanence of T. b. gambiense in the vector (Glossina spp.) and domestic fauna in order to improve our understanding of the epidemiological situation of the disease in an isolated focus considered to be under control. The results obtained show the absence of the parasite in peridomestic livestock but its presence, although at very low rate, in the vector. On the other hand, interesting entomological data highlight that an elevated concentration of tsetse flies was observed in two out of the ten villages considered to be in the focus. CONCLUSIONS:These findings demonstrate that even in conditions of apparent control, a complete parasite clearance is difficult to achieve. Further investigations must be focused on animal reservoirs which could allow the parasites to persist without leading to human cases. In Luba, where domestic livestock are scarcer than other foci in mainland Equatorial Guinea, the epidemiological significance of wild fauna should be assessed to establish their role in the maintenance of the infection.

Neglected diseases caused by kinetoplastid parasites are a health burden in tropical and subtropical countries. The need to create safe and effective medicines to improve treatment remains a priority. Microbial natural products are a source of chemical diversity that provides a valuable approach for identifying new drug candidates. We recently reported the discovery and bioassay-guided isolation of a novel family of macrolides with antiplasmodial activity. The novel family of four potent antimalarial macrolides, strasseriolides A-D, was isolated from cultures of Strasseria geniculata CF-247251, a fungal strain obtained from plant tissues. In the present study, we analyze these strasseriolides for activity against kinetoplastid protozoan parasites, namely, Trypanosoma brucei brucei, Leishmania donovani and Trypanosoma cruzi. Compounds exhibited mostly low activities against T. b. brucei, yet notable growth inhibition and selectivity were observed for strasseriolides C and D in the clinically relevant intracellular T. cruzi and L. donovani amastigotes with EC50 values in the low micromolar range. Compound C is fast-acting and active against both intracellular and trypomastigote forms of T. cruzi. While cell cycle defects were not identified, prominent morphological changes were visualized by differential interference contrast microscopy and smaller and rounded parasites were visualized upon exposure to strasseriolide C. Moreover, compound C lowers parasitaemia in vivo in acute models of infection of Chagas disease. Hence, strasseriolide C is a novel natural product active against different forms of T. cruzi in vitro and in vivo. The study provides an avenue for blocking infection of new cells, a strategy that could additionally contribute to avoid treatment failure.