Design, synthesis and biological evaluation of novel inhibitors of rhodesain, cysteine protease of Trypanosoma brucei rhodesiense, as antitrypanosomal agents

Human African Trypanosomiasis (HAT), also known as sleeping sickness, is an endemic parasitic disease of sub-Saharan Africa, caused by two subspecies of protozoa of Trypanosoma genus: T. brucei gambiense and T. b. rhodesiense. This vector-borne disease is transmitted by the bite of tsetse fly (Glossina genus), with an estimated number of actual cases of 30000 each year. T. b. gambiense is widespread in central and western Africa, where induces a chronic form of the disease (95% of HAT cases). On the contrary, T. b. rhodesiense causes a rapid-onset, higher mortality rate, acute form of trypanosomiasis and it is very common in southern and eastern Africa. Both parasitic subspecies produce two main stages of HAT: stage 1, or hemolymphatic stage, begins after the tsetse fly bite and can persist several weeks, causing non-specific symptoms like rash, fever, muscle aches and fatigue. If left untreated, stage 1 HAT evolves into the stage 2 (the late or neurological stage) during which the parasite crosses the blood brain barrier (BBB), leading to serious mental deterioration, sleep disturbances, coma and death. Because of the high degree of antigenic variation, due to the turnover of the variant surface glycoproteins (VSGs) of Trypanosoma coat, it was not possible to develop an effective vaccine. Current chemotherapy includes only four drugs: suramin and pentamidine effective only in the stage 1, and melarsoprol and eflornithine useful to treat the neurological stage. At present, eflornithine is administered in combination with nifurtimox, a trypanocidal orally active nitrofuran, as an option for melarsoprol-refractory stage 2 HAT, caused by T. b. rhodesiense, or as first-line treatment of the second-stage gambiense Trypanosomiasis. Considering that many cases of drug-resistance were recorded and the impossibility to develop an effective vaccine, there is a clear need to identify new effective drugs, ideally directed against novel targets. In this context, rhodesain, the major cysteine protease of T. b. rhodesiense, is an essential enzyme for parasite survival, involved in crossing the BBB, in the degradation of host immunoglobulins and in the turnover of VSGs. Therefore, starting from previously identified lead compounds, we focused our efforts to develop six series (A-F) of novel peptidyl and peptidomimetics inhibitors, the latter containing a 1,4-benzodiazepine (BDZ) scaffold, targeting rhodesain. We also tried to improve the drug-like properties of the inhibitors and we carried out docking studies, to better understand the role of the different portions of the molecules in the interaction with the target enzyme.