Structural Genomics of Plasmodium falciparum Drug Resistance: Applying Cryo-EM to Characterise Artemisinin Resistance Mechanisms in Nigerian Isolates

Artemisinin-based combination therapies (ACTs) are the cornerstone of malaria treatment globally, but emerging artemisinin partial resistance in sub-Saharan Africa, including Nigeria, threatens this critical intervention. Mutations in the P. falciparum Kelch13 (PfKelch13) propeller domain disrupt parasite ubiquitin-proteasome processes, leading to delayed parasite clearance. While genetic markers are established, the structural consequences of African PfKelch13 variants on protein conformation and drug binding remain poorly characterised. This dissertation applies cryo-electron microscopy (cryo-EM) to characterise the structural basis of artemisinin partial resistance mechanisms in P. falciparum isolates from Rivers and Borno States in Nigeria. PfKelch13 protein complexes were expressed in a baculovirus expression system, purified to homogeneity, and subjected to single-particle cryo-EM analysis at 3.2 angstrom resolution. Structural models for wild-type and three variant proteins (F446I, A675V, and a novel Nigerian variant) were generated and compared. Molecular dynamics simulations were conducted to assess variant-induced conformational changes affecting drug binding pocket geometry. The F446I variant induced measurable perturbations in the propeller beta-blade architecture that reduced dihydroartemisinin docking affinity in silico. This structural genomics approach provides mechanistic insight beyond sequence-level analysis and informs next-generation antimalarial drug design targeting the PfKelch13 pathway. Keywords: Plasmodium falciparum, artemisinin resistance, cryo-EM, PfKelch13, structural genomics