Resistance to Some But Not Other Dimeric Lindenane Sesquiterpenoid Esters Is Mediated by Mutations in a Plasmodium falciparum Esterase

Abstract

Unique lindenane sesquiterpenoid dimers from Chloranthecae spp. were recently identified with promising in vitro antiplasmodial activity and potentially novel mechanisms of action. To gain mechanistic insights to this new class of natural products, in vitro selection of Plasmodium falciparum resistance to the most active antiplasmodial compound, chlorajaponilide C, was explored. In all selected resistant clones, the half-maximal effective concentration (EC₅₀) of chlorajaponilide C increased >250-fold, and whole genome sequencing revealed mutations in the recently discovered P. falciparum prodrug activation and resistance esterase (PfPARE). Chlorajaponilide C was highly potent (mean EC₅₀ = 1.6 nM, n = 34) against fresh Ugandan P. falciparum isolates. The analysis of the structure-resistance relationships revealed that in vitro potency of a subset of lindenane sesquiterpenoid dimers was not mediated by PfPARE mutations. Thus, chlorajaponilide C, but not some related compounds, required parasite esterase activity for in vitro potency, and those compounds serve as the foundation for development of potent and selective antimalarials.

Keywords: Malaria Box; Pathogen Box; PfPARE; Plasmodium; lindenane sesquiterpenoid dimers; structure-resistance relationship study.

Figure 1.

Ex vivo sensitivity to chlorajaponilide C was assessed in fresh Ugandan clinical isolates (n = 31) of P. falciparum, and dihydroartemisinin (DHA) was used as the control. Each point represents a single assessment for a clinical isolate. The geometric mean and 95% confidence intervals are shown in red.

Figure 2.

In vitro selection of P. falciparum resistance to chlorajaponilide C. (A) Schematic selection of resistance to chlorajaponilide C against the P. falciparum 3D7-WT strain. Parasites were treated with 0.003% DMSO (control) or 15 nM chlorajaponilide C for 14 days (single selection). (B) Dose response curves for chlorajaponilide C in the parent 3D7-WT and resistant 3D7-R1–4 strains. The reported values represent averages and the SEM of three independent assays.

Figure 3.

Structures of antimalarial lindenane sesquiterpenoid dimers and in vitro potency against P. falciparum 3D7-WT and 3D7-R1 strains. The reported values represent averages and the SEM of three independent assays. *: Compounds exhibiting significantly reduced in vitro potency against 3D7-R1 (p < 0.0001 determined by one-way analysis of variance). a: Data reported previously. N.D. signifies “not determined”.

Figure 4.

Screening of open-access compound libraries to assess antimalarial cross-resistance against 3D7-R1. (A) Scatter plot of percentage of growth results comparing 3D7-WT vs 3D7-R1 screening of the Malaria Box and Pathogen Box compound repository at 1 μM. Chlorajaponilide C (20 nM, red dot) and artemisinin (200 nM, yellow dot) were used as controls. (B) Structures of MMV011438 and MMV011576 and dose response curves of MMV011438 and MMV011576 in 3D7-WT and 3D7-R1 strains. The reported values represent averages and the SEM of three independent assays.

Figure 5.

PfPARE mediates resistance to lindenane sesquiterpenoid dimers. (A) Dose response curves of chlorajaponilide C and MMV011438 in P. falciparum 3D7-WT and 3D7 strains harboring an active-site S179T allelic replacement in PfPARE (3D7-PARE-S179T). (B) Dose response curves of chlorajaponilide C and fortunilide A in P. falciparum resistant strains harboring different point mutations in PfPARE as indicated. Resistance to both compounds was reversed by expression of wildtype PfPARE in the 3D7-R1 strain (3D7-R1-WT-PARE). The reported values represent averages and the SEM of three independent assays.

Figure 6.

Lindenane sesquiterpenoid dimers are cytocidal. Parasite growth was measured at 72 h by the SYBR Green I assay in cultures where compounds were washed out at the indicated time points, and parasites were returned to culture to complete 72 h incubations. A representative Giemsa-stained smear before the compounds were washed out is shown. The reported LC₅₀ values represent averages and the SEM of three independent assays.