ABSTRACT
The ability of Plasmodium falciparum to access and utilise vital nutrients, including riboflavin (vitamin B2), is essential for its growth and proliferation, positioning riboflavin metabolism as a promising target for antimalarial intervention. Two riboflavin analogues, roseoflavin and 8-aminoriboflavin, were found to inhibit malaria parasite proliferation by targeting riboflavin metabolism and the utilization of riboflavin-derived cofactors, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), with roseoflavin also showing activity in vivo in a mouse model of malaria. Eight additional riboflavin analogues were tested, but none proved to be more effective than roseoflavin or 8-aminoriboflavin, and the compounds that were active appeared to be off target.

To investigate the mechanisms underlying the antimalarial effects of roseoflavin, we generated P. falciparum strains resistant to roseoflavin through in vitro evolution over 27 weeks. These roseoflavin-resistant parasites showed a 4-fold increase in resistance to roseoflavin and were also cross-resistant to 8-aminoriboflavin. Whole-genome sequencing of the resistant parasites identified a missense mutation (T2015A) leading to an amino acid substitution (L672H) in the gene encoding a putative flavokinase (PfFK), the enzyme responsible for converting riboflavin into FMN. To confirm that this mutation was responsible for the resistance phenotype, we introduced the T2015A mutation into the native PfFK gene through single-crossover recombination. These parasites exhibited indistinguishable IC50 values, for both roseoflavin and 8-aminoriboflavin, to those observed in the resistant parasites that had been generated via in vitro evolution, validating the role of the mutation in conferring resistance. Functional studies revealed that the L672H mutation in PfFK reduced the binding affinity of the enzyme to roseoflavin, providing an explanation for the resistance. Additionally, the mutant PfFK is unable to phosphorylate 8-aminoriboflavin, but its activity could still be antagonised by increasing the extracellular riboflavin concentration, consistent with the hypothesis that both roseoflavin and 8-aminoriboflavin inhibit parasite growth by blocking FMN production and generating toxic flavin cofactor analogues.

In addition to targeting riboflavin metabolism, the concept of flavin-deficient erythrocytes (FDE) as a protective mechanism against malaria was explored. Previous studies from the 1980s and 1990s hypothesised that individuals with FDE might be partially protected against malaria. Screening individuals from Ferrara, Italy, and Huye, Rwanda, we identified populations with FDE (23% and 13%, respectively), none of whom had a dietary riboflavin deficiency. Importantly, FDE from individuals in Ferrara, as well as those depleted of flavins through riboflavin starvation in vitro, were found to inhibit the intraerythrocytic proliferation of P. falciparum. These FDE were more susceptible to oxidative stress, which may explain their inhibitory effect on parasite growth. Genetic analysis of individuals from Huye revealed mutations in the FAD synthase gene, suggesting a genetic basis for flavin deficiency in these populations. These findings provide further evidence that targeting flavin metabolism, either by using riboflavin analogues or by exploiting a natural erythrocyte flavin deficiency, could serve as an approach to a novel treatment and/or prophylactic strategy against malaria.