In Silico Drug Target Identification and Experimental Validation Against Novel Targets in Nigerian Isolates of Trypanosoma brucei gambiense: A Structural Genomics-Driven Approach

Human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense remains a neglected tropical disease threat in Nigeria's Northwest and North Central states, with current treatment drugs (fexinidazole, acoziborole) facing access limitations and emerging efficacy concerns in immunocompromised patients. Structural genomics-driven drug target identification using genome sequences of locally circulating T. b. gambiense strains offers a rational approach to pipeline diversification. This dissertation applies a structural genomics-driven workflow to identify and experimentally validate novel drug targets in T. b. gambiense isolates from Zamfara and Kogi States, Nigeria. Whole-genome sequencing of 12 clinical T. b. gambiense isolates was performed on Oxford Nanopore and Illumina platforms, with hybrid assemblies constructed using Unicycler. Protein structure prediction employed AlphaFold2 for all predicted proteins lacking homologous solved structures. Novel druggable target candidates were identified through automated druggability scoring using DoGSiteScorer and selectivity analysis against human proteome structural analogs. Top five targets were expressed in E. coli, and high-throughput screening was performed against the MMV Pandemic Response Box compound library. Target validation used CRISPR-Cas9 conditional knockdown in bloodstream form parasites. Three structurally novel T. brucei proteins were experimentally confirmed as essential by conditional knockdown, including a trypanosome-specific RNA helicase with no human structural homolog. Virtual screening identified three compounds from the MMV library with IC50 below 1 micromolar against the RNA helicase target in cell-free enzymatic assays. These compounds demonstrated selective trypanosomal toxicity over human cell lines at therapeutic index values above 20. This work delivers the first structurally informed druggable target set from Nigerian T. b. gambiense strains. Keywords: Trypanosoma brucei gambiense, structural genomics, drug target, AlphaFold2, HAT.