The neglected diseases of African sleeping sickness, Chagas’ disease, and leishmaniasis are caused by the protozoan trypanosomatid parasites Trypanosoma and Leishmania, and are major world-wide causes of disease and death. There is a compelling need for the development of new drug treatments because existing treatments are inadequate due to high levels of toxicity and growing drug resistance within the parasites. Glycolysis is an attractive target because many of the trypanosomatid glycolytic enzymes are sequestered within the glycosome, giving them unique regulatory properties. Trypanosomatid pyruvate kinase (PYK) is particularly attractive because, although a cytosolic enzyme, is allosterically regulated by fructose-2,6-bisphosphate whilst the allosteric PYK in mammalians is regulated by fructose-1,6-bisphosophate. X-ray crystallography has been used in this study to solve three structures of pyruvate kinase from Leishmania mexicana in order to determine the mechanism of allosteric activation of PYK. Knowledge of the conformational changes that PYK undergoes during enzyme activation showed that the best place for the design of inhibitors of PYK from L. mexicana is the effector site and the surrounding area. This study has used a structure-based approach to drug design, encompassing virtual screening and combinatorial chemistry to identify novel inhibitors of PYK from L. mexicana. The most potent inhibitor of PYK from L. mexicana was MN92, synthesised through combinatorial chemistry, which had an IC₅₀ of 71μM and a K_iPEP of 45μM. This compound is believed to bind to the PYK effector site and is suitable for further studies of ligand optimisation.