Harnessing evolutionary fitness in Plasmodium falciparum for drug discovery and suppressing resistance

Abstract

Drug resistance emerges in an ecological context where fitness costs restrict the diversity of escape pathways. These pathways are targets for drug discovery, and here we demonstrate that we can identify small-molecule inhibitors that differentially target resistant parasites. Combining wild-type and mutant-type inhibitors may prevent the emergence of competitively viable resistance. We tested this hypothesis with a clinically derived chloroquine-resistant (CQ r ) malaria parasite and with parasites derived by in vitro selection with Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors. We screened a chemical library against CQ s and CQ r lines and discovered a drug-like compound (IDI-3783) that was potent only in the CQ r line. Surprisingly, in vitro selection of Plasmodium falciparum resistant to IDI-3783 restored CQ sensitivity, thereby indicating that CQ might once again be useful as a malaria therapy. In parallel experiments, we selected P. falciparum lines resistant to structurally unrelated PfDHODH inhibitors (Genz-666136 and DSM74). Both selections yielded resistant lines with the same point mutation in PfDHODH:E182D. We discovered a compound (IDI-6273) more potent against E182D than wild-type parasites. Selection of the E182D mutant with IDI-6273 yielded a reversion to the wild-type protein sequence and phenotype although the nucleotide sequence was different. Importantly, selection with a combination of Genz-669178, a wild-type PfDHODH inhibitor, and IDI-6273, a mutant-selective PfDHODH inhibitor, did not yield resistant parasites. These two examples demonstrate that the compromise between resistance and evolutionary fitness can be exploited to design therapies that prevent the emergence and spread of resistant organisms.alaria is a vector-borne infectious disease transmitted by female Anopheles mosquitoes and caused by protozoan parasites of the genus Plasmodium. Malaria is widespread in tropical and subtropical regions, including parts of the Americas, Asia, and Africa. Malaria afflicts 350-500 million people each year, resulting in ∼800,000 deaths per year (1). The majority of malaria-related deaths occur in young children below the age of five in Sub-Saharan Africa (1). Resistance has emerged to nearly all antimalarial drugs, including the front-line artemisinin-based combination therapies (2). Widespread resistance necessitates both the discovery and development of new antimalarial therapies as well as strategies to protect current and future therapies from the threat of resistance. We explore here the possibility of blocking the emergence of resistance with a population biology trap: by identifying situations where resistance to one compound confers hypersensitivity to another, we can design combination therapies that not only kill the parasite, but also guide its evolution away from resistance.Drug resistance pathways in Plasmodium falciparum are sharply limited by tradeoffs among growth, transmissibility, and resistance. For example, there is a single dominant resistance-fitn...

Adaptive evolution of malaria parasites in French Guiana: Reversal of chloroquine resistance by acquisition of a mutation in pfcrt

“…Using the 106/1 76I line, QN single-step selection experiments generated lines with an additional PfCRT Q352K or Q352R mutation that had acquired QN resistance but simultaneously regained CQ susceptibility (20). Separately, in vitro selection for resistance to the IDI-3783 compound that is selectively active against CQR parasites resulted in acquisition of the Q352R mutation in PfCRT (and concomitantly, a loss of CQR), thus providing a conceptual framework for the development of combination therapies that would exploit this evolutionary trap (21). These mutations occur in pfcrt exon 10, which corresponds to amino acid substitutions in transmembrane domain 9.…”

Antimalarial drug resistance: linking Plasmodium falciparum parasite biology to the clinic

“…One approach to reducing the risk of resistance to DSM265 emerging would be to pair this compound with another agent that has a similar pharmacokinetic profile and against which resistance would require a separate set of parasite mutations, thus decreasing the probability of dual drug resistance 108 . A complementary approach could be to pre-emptively block DSM265-resistance pathways by choosing as a partner another DHODH inhibitor against which DSM265-resistant parasites would be hypersensitive 109 . A similar phenomenon, whereby resistance to one class of inhibitors causes the mutated target to become hypersensitive to another inhibitor, has also been described for some PfATP4 mutants 110 .…”

“…Regarding the latter, an interesting approach has been the design of CQ-like compounds that couple targeting heme with a chemosensitizing component that counteracts mutant PfCRT-mediated CQ resistance 134,135 . Another strategy is to combine drugs that generate opposing selection pressures on the same target, as illustrated above with the example of DHODH, in which case a gain of resistance to one inhibitor can cause parasite hypersensitivity to another 109 . As mentioned in Box 1, detailed knowledge about PfCRT and PfMDR1 has shown that certain mutations conferring resistance to one drug can sensitize parasites to other agents, and has laid the theoretical groundwork for clinical trials (NCT02612545 and NCT02453308) with the triple ACTs AL plus AQ or DHA-PPQ plus mefloquine to test whether these combinations can successfully eliminate resistant and sensitive infections, and, potentially, block the emergence of parasites resistant to all three agents.…”

Malaria Genomics in the Era of Eradication

“…To circumvent the emergence of drug resistance, specific genomic loci can be identified (e.g., dhodh and pfcrt [Lukens et al 2014]) that exist in two alternate allelic states (Fig. 3).…”