Mechanism of small molecule inhibition of Plasmodium falciparum myosin A informs antimalarial drug design

Abstract

Malaria is responsible for more than a half million deaths per year. The Plasmodium parasites responsible continue to develop resistance to all known agents, despite treatment with different antimalarial combinations. The atypical Myosin A motor (PfMyoA) is part of a core macromolecular complex called the glideosome, essential for Plasmodium parasite mobility and therefore an attractive drug target. Here, we characterize the interaction of a small molecule (KNX-002) with PfMyoA. KNX-002 inhibits PfMyoA ATPase activity in vitro and blocks asexual blood stage growth of merozoites, one of three motile Plasmodium life-cycle stages. Combining biochemical assays, X-ray crystallography and molecular dynamics, we demonstrate that KNX-002 targets a novel pocket in PfMyoA, sequestering it in a post-rigor state detached from actin. KNX-002 binding affects Mg2+ coordination near ATP, preventing ATP hydrolysis and thus inhibiting motor activity. This first-in-class small-molecule inhibitor of PfMyoA paves the way for developing a new generation of antimalarial treatments.

The class XIV myosin ofToxoplasma gondii, TgMyoA, is druggable in an animal model of infection

“…The T130A mutation is located distal from the predicted KNX-002 binding pocket [52], suggesting an allosteric mechanism of conferring resistance of the motor to the compound. The T130A mutation is within the transducer subdomain, which plays a critical role in the communication pathway between the nucleotide-binding pocket and the actin-binding domain that is closer to where KNX-002 binds.…”

“…Our preliminary SAR data (Figure 3) are consistent with a key role for the pyrazole, cyclopropyl, and p -OMe aryl units within KNX-002, with a generally good correlation between the results of the recombinant myosin ATPase and parasite growth inhibition assays. Subsequent studies, inspired both by our SAR data and the recently reported structure of the PfMyoA-KNX-002 complex [52], should explore the effect on biological activity of: (i) alternative replacements for the thiophene group and (ii) a switch in the positions of the NH and CH 2 groups, amongst other options. Trivedi et al recently reported identification of an analog of KNX-002, named KNX-115, which shows >10-fold improved potency against PfMyoA [53].…”

“…Excitingly, KNX-002 and a recently discovered analog named KNX-115 were shown to inhibit PfMyoA, the TgMyoA homolog of the malaria parasite, P. falciparum [54, 55], and to block the growth of blood stage P. falciparum in culture and the invasion of hepatocytes by liver stage parasites [54, 55]. Structure-function studies revealed that the compound sequesters PfMyoA in a state of low affinity for actin [54], consistent with a decrease in duty ratio we observe in wild-type TgMyoA treated with KNX-002 (Suppl. Figure 13).…”

“…Subsequent studies, inspired both by our SAR data and the recently reported structure of the PfMyoA-KNX-002 complex [54], could explore the effect on biological activity of: (i) alternative replacements for the thiophene group and (ii) a switch in the positions of the NH and CH2 groups, amongst other options. Encouragingly, Trivedi et al recently identified an analog of KNX-002 that shows >20-fold improved potency against recombinant PfMyoA [55].…”

“…Excitingly, KNX-002 and a recently discovered analog named KNX-115 were shown to inhibit PfMyoA, the TgMyoA homolog of the malaria parasite, P. falciparum [54,55], and to block the growth of blood stage P. falciparum in culture and the invasion of hepatocytes by liver stage parasites [54,55].…”