Fast-Killing Tyrosine Amide ((S)-SW228703) with Blood- and Liver-Stage Antimalarial Activity Associated with the Cyclic Amine Resistance Locus (Pf CARL)

Abstract

Current malaria treatments are threatened by drug resistance, and new drugs are urgently needed. In a phenotypic screen for new antimalarials, we identified (S)-SW228703 ((S)-SW703), a tyrosine amide with asexual blood and liver stage activity and a fast-killing profile. Resistance to (S)-SW703 is associated with mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL) and P. falciparum acetyl CoA transporter (PfACT), similarly to several other compounds that share features such as fast activity and liver-stage activity. Compounds with these resistance mechanisms are thought to act in the ER, though their targets are unknown. The tyramine of (S)-SW703 is shared with some reported PfCARL-associated compounds; however, we observed that strict S-stereochemistry was required for the activity of (S)-SW703, suggesting differences in the mechanism of action or binding mode. (S)-SW703 provides a new chemical series with broad activity for multiple life-cycle stages and a fast-killing mechanism of action, available for lead optimization to generate new treatments for malaria.

Keywords: PfACT; PfCARL; Plasmodium; malaria; resistance; tyrosine amide.

Figure 1.. (S)-SW703 is a potent, enantiomerically specific, and fast-killing inhibitor of P. falciparum asexual blood-stages.

(A) Structure of (S)-SW703. (B) P. falciparum (strain Dd2) growth inhibition (asexual intraerythrocytic parasites) by (S)-SW703 and its R-enantiomer; representative data from one study (3 technical replicates) shown; mean ± standard deviation. Table 1 shows the average data from at least three independent studies per compound. (C) To assess rate of kill, parasites were incubated with (S)-SW703 or benchmark compounds for 0, 24, or 48 hours at 10X EC₅₀. Compounds were washed off and treated parasites were assessed for re-invasion of labeled erythrocytes. Representative assay from two independent experiments (3 biological replicates each) is shown; mean ± standard deviation. (D) Liver-stage activity was assessed using P. berghei as described; representative data from one of two independent studies (8 technical replicates each) shown; mean ± standard deviation The average EC₅₀ across the two independent studies was EC₅₀ = 0.57 ± 0.21 μM.

Figure 2.. PfCARL and PfACT mutations are associated with resistance to (S)-SW703.

(A-B) Representative (S)-SW703 (A) and KAF156 (B) EC₅₀ curves for parent strains and representative resistant clones; one clone for each PfCARL or PfACT mutation: PfCARL^Q821H, flask 2 clone F3; PfCARL^L830I, flask 1 clone B4; PfCARL^S1057T, flask 3 clone D11; PfCARL^L1073I, C2 clone beta; PfACT^Q116K, flask 4 clone F2. Representative data from a single experiment are shown; mean ± standard deviation from three technical replicates. Averaged data from independent experiments (n ≥ 3) are presented in Table 2. (C) EC₅₀ values of all clones for each PfCARL or PfACT mutation. Dd2 is the parent strain for PfCARL^Q821H, PfCARL^L830I, PfCARL^S1057T, and PfACT^Q116K clones; Dd2-Polδ mutant is the parent strain for PfCARL^L1073I. Datapoints represent mean EC₅₀ values for each individual clone. Bars and error bars represent mean and standard deviation derived from these datapoints. (D) Cartoon model of PfCARL with locations of known mutations. Black dots represent previously reported mutations;^{, , –} colored dots represent mutations identified in this study (Q821H, salmon pink; L830I, sky blue; S1057T, forest green; 1073I, plum purple). Note that in some cases, black dots represent multiple mutations at the same site. Mutations are labeled according to their amino acid positions; mutations that are predicted to lie within transmembrane helices (TMHMM) are labeled above or below the helix.

Figure 3.

Structural comparison between (S)-SW703 and compounds reported to share the PfCARL/PfACT resistance phenotype.^{, , –}