Computational studies of resistance mechanisms in Malaria

Abstract

The threat of multidrug resistance hangs over the world as it attempts to eradicate Malaria. The disease is primarily caused by Plasmodium falciparum, an intracellular obligate parasite that continues to evolve resistance to almost all antimalarial therapeutics available to treat it. As such, there is a dire need for the development of innovative strategies and technologies to address the challenge of multidrug resistant P. falciparum. This cannot be done rationally if one does not first possess an understanding of the underlying molecular mechanisms of resistance. In this thesis we use extensive biophysical simulations to investigate the mechanisms of two important multidrug resistance proteins and consider its relevance to drug design strategies to mitigate resistance evolution. Mutations in the P. falciparum chloroquine resistance transporter (PfCRT) can modulate the parasite's susceptibility to many previously and currently used antimalarials. This is achieved through the protein gaining the ability to transport these compounds, shifting drug concentrations around the parasite. However, drug transporting isoforms are often associated with fitness defects. In the case of chloroquine, this has resulted in resistance alleles declining in frequency within some populations after the drug stopped being used. We determined how the antimalarial chloroquine and a range of natural peptide substrates bind to PfCRT, and how chloroquine resistance associated mutations affect this and lead to changes in parasite fitness. The work makes suggestions for how fitness costs might be exploited in the design of future antimalarials. We then studied how multidrug transport is achieved by PfCRT and how it might be inhibited. We used molecular dynamics simulations in conjunction with the enhanced-sampling technique metadynamics to determine compound binding sites. The simulations suggest a polyspecific mechanism is at work, and that several binding sites exist within the PfCRT cavity. We suggest that this may lead to the evolution of the selective transport of antimalarials with collateral sensitivities, challenging this as a basis for triple artemisinin combination therapy partner selection. PfATP4 is an important emerging drug target, with at least 30 unique pharmacophores targeting it. However, a series of resistance associated mutations have been reported to those compounds. We constructed the most complete dataset of PfATP4 inhibitors and resistance associated mutations to date. We combined this data with high throughput docking to make plausible hypotheses on drug binding locations, including for the compounds cipargamin and SJ733. The binding sites suggest that competitive inhibition of this protein is challenging, and higher resistance rates may be the result. This thesis provides timely insights into the substrate and drug binding dynamics of PfCRT and PfATP4. We conclude with discussion of the place of structural and computational methods in the rationalization, and ultimately, prediction of drug resistance. Show more