ABSTRACT New drugs with different modes of action than SCYX-7158, the current clinical candidate, are needed for chemotherapy of human African trypanosomiasis (HAT) caused by T. brucei spp. Starting with the FDA- approved drug lapatinib (EC50 = 1600 nM; selectivity index (SI) = 4 (compared to human HepG2 cells), we have synthesized over 540 analogs in a medicinal chemistry campaign focused on optimizing SI, toxicity profile, metabolism, physicochemical properties, aqueous solubility (Aq. Sol.) and CNS penetrance. Our new lead NEU4438 is orally bioavailable and extends the life of trypanosome-infected mice 2.4 (P = 0.008; Kaplan- Meir analysis), accompanied by a 109-fold reduction in parasitemia. A quinolinimine, NEU-4438 has excellent potency (GI50: 0.013 μM), selectivity (SI: >2000), physicochemical properties (aq. sol.: 880 μM; LogD7.4: 0.9; cLogP: 2.52) and in vitro ADME (human PPB%: 15; HLM Clint: 21.8 µl/min/mg protein; RH Clint: 13.1 µ//min/106 cells). Six NEU4438-related advanced hits are available for evaluation as possible leads, should NEU4438 fail to cure HAT in murine models of the disease. We will further our work by optimizing the quinolinimine scaffold using singleton and parallel medicinal chemistry approaches after constructing a virtual library, shaped by drug-like expectations in silico. Compounds will be synthesized and screened through a funnel established to select those with the best selectivity index, solubility, and physicochemical properties. Advanced hits will be evaluated in mice for safe safety, tissue exposure, and brain penetrance, after which those with the best features will be tested for efficacy in mice models of HAT to select lead drugs (exemplified by NEU4438), that may be progressed into preclinical candidates. Towards identification of the targets of the drug lead, NEU4438- binding proteins will be identified using photoaffinity probes. Concurrently, molecular modes of action will be studied by NEU4438 perturbation of the trypanosome proteome, followed by tests of hypotheses formulated from the proteomics data. In order to identify physiological targets, the phenotypes observed after knockdown of genes encoding NEU4438-binding proteins will be expected to “phenocopy” the effect of adding NEU4438 to T. brucei. Our achievements put us on track to deliver two new preclinical candidates, and to identify their physiological targets as well as modes of action in the next funding period.