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Abstract
Human African trypanosomiasis is a fatal disease caused by Trypanosoma brucei rhodesiense and T. b. gambiense which establish acute and chronic infection, respectively. This disease is a threat to ~70 million people with more than 97% cases manifesting as chronic HAT. Current chemotherapy has undesirable properties, necessitating the development of safer orally bioavailable leads with different mode(s) of action than the existing drugs. Lapatinib, an approved EGFR inhibitor, was used as a starting point for synthesis of new hits against T. brucei. These hits were selected based on their physicochemical properties, potency, and selectivity against whole trypanosomes. We developed delayed cytocidality assay to prioritize compounds. These selection criteria produced NEU-4438, that increased survival of T. brucei Lister427 infected mice by 4.5-fold. NEU-4438 reduced parasite load in T. brucei AnTat1.1 infected mice by greater than 100-fold, establishing this drug as a lead for chronic HAT drug discovery.
To investigate the mode(s) of action of NEU-4438, we compared it with those of SCYX-7158 (a clinical candidate). Global drug-perturbome proteomics using physiologically equivalent concentrations (i.e. TbDCC25) of NEU-4438 and SCYX-7158 generated data that was used to develop hypotheses regarding modes of action. Tests of these hypotheses revealed that the two compounds have different biological actions. NEU-4438 prevented trypanosome entry into S-phase. In contrast, SCYX-7158 blocked protein translation and prevented endocytosis of haptoglobin-hemoglobin. These findings support development of NEU-4438 as a drug against HAT due to its distinct modes of action compared to the current clinical candidate.
In a parallel drug discovery undertaking, Curaxin CBL0137 cured chronic HAT mouse model. Related carbazoles CBL0174 and CBL0187, had high intestinal permeability, desired solubility and metabolic profiles. The compounds possessed equivalent in vitro anti-proliferative activity and delayed cytocidality (DCC) as CBL0137. Further, their plasma Cmax exceeded ten-times the 50% anti-proliferative concentrations, yet CBL0174 and CBL0187 failed to control parasite burden in vivo. In studies to understand possible reasons for failure of CBL0174 and CBL0187, we found that anti-proliferative activity of CBL0187 plummets by 400% with increase in serum in the culture medium. Further, unlike CBL0137, free plasma drug concentration of CBL0174 and CBL0187 remains below 24 h-DCC99, indicating that drug ‘effective plasma exposure’ was below the threshold for irreversible cidality of T. brucei. These new lessons will be used for selection of hits for testing possible leads in anti-HAT drug development.