Imidazolopiperazines Kill both Rings and Dormant Rings in Wild-Type and K13 Artemisinin-Resistant Plasmodium falciparum In Vitro

Abstract

Artemisinin (ART) resistance has spread through Southeast Asia, posing a serious threat to the control and elimination of malaria. ART resistance has been associated with mutations in the Plasmodium falciparum kelch-13 (Pfk13) propeller domain. Phenotypically, ART resistance is defined as delayed parasite clearance in patients due to the reduced susceptibility of early ring-stage parasites to the active metabolite of ART dihydroartemisinin (DHA). Early rings can enter a state of quiescence upon DHA exposure and resume growth in its absence. These quiescent rings are referred to as dormant rings or DHA-pretreated rings (here called dormant rings). The imidazolopiperazines (IPZ) are a novel class of antimalarial drugs that have demonstrated efficacy in early clinical trials. Here, we characterized the stage of action of the IPZ GNF179 and evaluated its activity against rings and dormant rings in wild-type and ART-resistant parasites. Unlike DHA, GNF179 does not induce dormancy. We show that GNF179 is more rapidly cidal against schizonts than against ring and trophozoite stages. However, with 12 h of exposure, the compound effectively kills rings and dormant rings of both susceptible and ART-resistant parasites within 72 h. We further demonstrate that in combination with ART, GNF179 effectively prevents recrudescence of dormant rings, including those bearing pfk13 propeller mutations.

Keywords: GNF179; dormant rings; drug susceptibility; imidazolopiperazines; malaria; rings; triple therapy.

FIG 1

IPZ rapidly kill schizonts and slowly but potently kill ring-stage parasites without inducing dormancy. (A) Drug treatment schematic; (B) 24-h readout of 3D7 asexual blood-stage IC₅₀s of GNF179 and ART using MitoTracker orange viability dye; (C) 72-h readout of 3D7 asexual blood-stage IC₅₀s of GNF179 and ART using MitoTracker orange viability dye; (D) killing kinetic images of GNF179 (100 nM) after 6 and 24 h of exposure to Dd2 WT ring parasites and live parasites (green-blue) and dead parasites (blue only) using high-content imaging (HCI). Blue corresponds to DNA (DAPI stain), green corresponds to functional mitochondria (MitoTracker orange stain), and red corresponds to red blood cells (wheat germ agglutinin [WGA] conjugated to Alexa Fluor 647 stain). Drug treatment was applied on 3- to 6-h-old ring-stage parasites.

FIG 2

IPZ kill both ring and dormant ring parasites regardless of the K13 genotype. (A and B) IC₉₀ activity against rings and dormant rings of clinical isolates of ART-resistant parasites bearing K13KDU691 (1,400 nM) (A) and GNF179 (100 nM) (B) after 72 h, detected using SYBR green. The growth controls for ring and dormant rings were DMSO-treated rings and DMSO-treated dormant rings.

FIG 3

IPZ, but not PI4K inhibitors, rescue K13-mediated ART drug resistance. (A and B) Dd2 WT and Dd2 K13 mutants (C580Y, R539T, and I543T) (A) and seven clinical isolate ART-resistant parasites (B) were examined for sensitivity to DHA only or DHA (700 nM) combined either with KDU691 (1,400 nM) or GNF179 (100 nM). The growth control was DMSO-treated rings.