Better understand P. falciparum resistance to sulfadoxine to better prevent: new insights from the field to the atom – SULPAR

Malaria is a public health problem, particularly for populations at risk, namely pregnant women and children. In addition to protective measures against mosquito vectors (impregnated nets), sulfadoxine-pyrimethamine (SP) remains the only available and effective prophylactic drug combination recommended as intermittent preventive treatment in pregnant women (IPTp) by WHO in sub-Saharan Africa. Moreover, SP plus amodiaquine is also widely used for seasonal malaria chemoprophylaxis in children under 5 years old.
This combination therapy blocks folate synthesis by inhibiting dihydropteroate synthetase (DHPS) and dihydrofolate reductase (DHFR) parasitic enzymes. Drug pressure has selected for parasites that have accumulated mutations in the genes encoding these enzymes, making them less susceptible or resistant to SP.
In central Africa, P. falciparum isolates carrying 5 mutations in the pfdhps gene have recently been identified: 4 mutations (S436A, A437G, A581G, A613S) already well described and a 5th new mutation, I431V. This pfdhps vagKgs mutant allele is almost always associated to the pfdhfr triple mutant (N51I, C59R, S108N), all together forming an octuple CirnI/vagKgs mutant. In a recent epidemiological study, we showed that the prevalence of infections with this octuple mutant was very high in the north of Cameroon (˜ 50%), in Chad, and in Nigeria. A study we conducted in Cameroon in 2020 showed an overrepresentation of infections with P. falciparum parasites carrying this octuple mutant in women receiving IPTp-SP, suggesting selection by SP.
The main objectives of our project are i) to improve the knowledge of this new PfDHPS I431V mutation and associated mutants and ii) to identify alternatives to sulfadoxine (SDX). So, we propose a broad interdisciplinary collaboration between biologists, physicists and field practitioners to develop a 3-level approach:
- At human level, we will evaluate the impact of infections due to the octuple mutant on pregnancy outcomes in women receiving IPTp-SP. This evaluation will be done through two approaches. A retrospective study of data and samples from the RECIPAL cohort (prospective and longitudinal) will investigate possible correlations between infections with octuple mutants and pregnancy outcomes. Then, a large cross-sectional survey will be conducted in Benin, including P. falciparum-infected women at delivery. This study will compare pregnancy outcomes (low birth weight, premature birth, maternal anemia) between women under IPTp-SP, infected by an octuple mutant and those infected by a non-octuple parasite.
- At biological level, two methodologies will be performed. First, the production via E. coli will allow the evaluation of pharmacokinetic parameters (Km, kcat) and inhibition constants (Ki) of different PfDHPS mutants. Secondly, murine P. berghei parasites edited with CRISPR/Cas9 technology will allow us to study in vivo the phenotypic characteristics of these mutant PfDHFR/PfDHPS genotypes (susceptibility to sulfadoxine, fitness of asexual and sexual stages, and transmission).
- At atomic level, modeling the structure/activity relationship of the PfDHPS enzyme with its substrate (pABA) and the inhibitor (SDX) will improve our understanding of the role of mutations at the active site, and the mechanisms involved in the evolution of resistance. An innovative approach of identifying "hot spots"will make possible to anticipate the emergence of new resistance genotypes. Finally, this approach will allow the screening of larges chemical libraries in order to identify new inhibitory structures against PfDHPS or to reposition drugs.
The very different but complementary approaches of this consortium will successfully address the important question of the impact of the octuple PfDHFR/PfDHPS mutants on SP prophylaxis but also to find alternatives to SDX.