Conflicting Requirements of Plasmodium falciparum Dihydrofolate Reductase Mutations Conferring Resistance to Pyrimethamine-WR99210 Combination

Abstract

Plasmodium falciparum strains bearing quadruple mutations of dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) at codons 51, 59, 108, and 164 are highly resistant to pyrimethamine (PYR), a diaminopyrimidine, but sensitive to WR99210 (WR), a cycloguanil analog, suggesting different enzyme-inhibitor binding interactions. A combination of these inhibitors to delay the onset of antifolate resistance is proposed. Using error-prone PCR, libraries of random mutants of wild-type PfDHFR and PfDHFR-TS were generated and used to transform Escherichia coli, and transformants were then selected for PYR or WR resistance. Mutants highly resistant to either PYR or WR were also generated from libraries obtained from further random mutagenesis of quadruple mutants (QM) with mutations in PfDHFR or PfDHFR-TS. For reversion mutants carrying altered residues I51N, N108S, and L164I, a further mutation of D54N was required to achieve resistance against WR, but these mutants regained sensitivity to PYR. When a combination of PYR and WR was used, fewer resistant mutants were generated from both mutant libraries using the QM gene templates. The effectiveness of the drug combination in reducing the appearance of resistance mutations is likely due to conflicting requirements for mutations conferring resistance to the two drugs. Thus, a combination of inhibitors from these two drug classes should be effective in impeding the emergence of P. falciparum resistance to antifolates.

Scalable, Continuous Evolution of Genes at Mutation Rates above Genomic Error Thresholds

“…Pf DHFR resistance to the antimalarial drug, pyrimethamine, occurs in the wild primarily through four active site mutations (N51I, C59R, S108N, and I164L), but the broader resistance landscape remains largely unknown. Laboratory evolution and landscape-mapping studies have mostly been limited to the quadruple mutant fitness peak (qm-wild) and suggest that resistance reproducibly arises from the crucial S108N mutation, followed by step-wise paths to qm-wild (Chusacultanachai et al, 2002; Hankins et al, 2001; Japrung et al, 2007; Lozovsky et al, 2009; Sirawaraporn et al, 1997; Wooden et al, 1997). We asked whether high-throughput directed evolution of Pf DHFR resistance to pyrimethamine would reveal a more complex landscape with additional fitness peaks, including ones that forgo S108N.…”

“…By encoding Plasmodium falciparum DHFR (Wooden et al, 1997) on OrthoRep, we were able to evolve highly resistant Pf DHFRs in 90 independent replicates through the simple serial passaging of cells in the presence of the Pf DHFR inhibitor, pyrimethamine. Prevailing analyses of Pf DHFR resistance focus on a single fitness peak observed widely in the field (Chusacultanachai et al, 2002; Hankins et al, 2001; Japrung et al, 2007; Lozovsky et al, 2009; Sirawaraporn et al, 1997), but our experiment reveals a more complex landscape including other genotypes of similar fitness. We find that a highly adaptive first-step mutation constrains path choice, leading to convergence, but also that rare mutations direct trajectories to alternative outcomes or a suboptimal fitness trap, illustrating the balance between fate and chance in drug resistance.…”

Blind prediction of HIV integrase binding from the SAMPL4 challenge

“…Combinations of two different classes of inhibitors that act on the same enzyme have been shown to inhibit broad panels of many different multi-drug-resistant mutants and to also decrease the probability of the emergence of new drug-resistant mutants, as exemplified by the combination of an active site inhibitor and an allosteric inhibitor of Bcr-Abl, a kinase target for cancer chemotherapy[25, 58]. This also appears to be the case for HIV treatment.…”

Assessment of in vitro sensitivity of Plasmodium vivax fresh isolates

“…Median (95% CI) IC 50 of WR99210 in P. vivax isolates collected in the present study was similar to our previous observation in the same area [15] . The relatively poor in vitro susceptibility of P. vivax to WR99210 could be explained by the slow action of this drug and/or the innate resistance as well as the presence of p-aminobenzoic acid and folate in the media used which acted as competitive antagonists of antifolate activity [16] . The observed in vitro IC 50 values of WR99210 therefore, may not reflect the actual in vivo sensitivity of P. vivax as the medium used was supplemented with folic acid.…”