Structure-based therapeutic development against the Apicomplexan parasites

Abstract

Apicomplexa are a large group of pathogenic parasites, which cause major infectious diseases including malaria and toxoplasmosis. Malaria is a mosquito-borne disease caused by Plasmodium Spp. It infects several hundred million people worldwide each year. Toxoplasma gondii has become one of the most prevalent parasites with one-third of the world population at risk of latent toxoplasmosis. Rising drug-resistance in apicomplexan parasites has become a huge challenge. New drugs with better potency and safety profiles against drug-resistant parasites and effective preventive vaccines are urgently required. Cytochrome bc1 is a catalytic complex in mitochondrial electron transport chain and a validated drug target for apicomplexan parasites. Atovaquone eliminates parasites by inhibiting Qo site of cytochrome bc1, and widely-used for both malaria and toxoplasmosis treatments. Mutations in the Qo site of apicomplexan cytochrome bc1 has contributed to the rapid emergence of atovaquone-resistance. To overcome this problem, 4(1H)-quinolone family compounds targeting the Qi site of cytochrome bc1 have been developed. The complexes of bovine cytochrome bc1 (the surrogate for the human host) with potent 4(1H)-quinolones determined by X-ray crystallography and cryo-EM are discussed in this thesis and show the compounds bound solely to the Qi site. Currently, there is no cytochrome bc1 structure from any of the apicomplexan parasites available. In-silico docking of 4(1H)-quinolones performed with a parasite homology model demonstrated possible binding poses of lead compounds. The greater differential for the Qi site’s primary structure combined with crystallographic and computational modelling provides new insight to support the development of new compounds with selective potency against parasite and lower toxicity. Cryo-EM is confirmed to be the first proof of principle for structural insight on apicomplexan cytochrome bc1 that has never been achieved by crystallography due to challenges in protein purification from parasites and complex nature of the protein. In order to prevent the emergence of future drug resistance, the second target for 4(1H)-quinolones was also considered. Type II NADH:ubiquinone oxidoreductase (NDH2) is an enzyme in apicomplexan electron transport chain that also inhibited by 4(1H)-quinolones. Recombinant NDH2 purification and crystallisation are described in this thesis. Vaccine has been considered as an effective tool for malaria prevention. Nowadays, only one licenced malaria vaccine is available. Finding new vaccine candidates is highly active research. 19 kDa fragment merozoite surface protein 1 (MSP119) is a protein on parasite’s membrane that is antibody target for vaccine development. Rusticyanin, a copper protein from Thiobacillus ferrooxidans, has been reported to have antimalarial activity through an interaction with MSP119. In this work, interactions between MSP119 and rusticyanin were characterised. Crystallisation of MSP119-rusticyanin complex was attempted but only rusticyanin crystals were obtained. The residues 1-28 of rusticyanin cannot be seen in electron density that may be disordered in view of the interaction with MSP119.