A potent series targeting the malarial cGMP-dependent protein kinase clears infection and blocks transmission

Abstract

To combat drug resistance, new chemical entities are urgently required for use in next generation anti-malarial combinations. We report here the results of a medicinal chemistry programme focused on an imidazopyridine series targeting the Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG). The most potent compound (ML10) has an IC50 of 160 pM in a PfPKG kinase assay and inhibits P. falciparum blood stage proliferation in vitro with an EC50 of 2.1 nM. Oral dosing renders blood stage parasitaemia undetectable in vivo using a P. falciparum SCID mouse model. The series targets both merozoite egress and erythrocyte invasion, but crucially, also blocks transmission of mature P. falciparum gametocytes to Anopheles stephensi mosquitoes. A co-crystal structure of PvPKG bound to ML10, reveals intimate molecular contacts that explain the high levels of potency and selectivity we have measured. The properties of this series warrant consideration for further development to produce an antimalarial drug.

Use of a highly specific kinase inhibitor for rapid, simple and precise synchronization ofPlasmodium falciparumandPlasmodium knowlesiasexual stage parasites

“…With this assay, an EC 50 of 4.2 nM (±SD 2.7 nM) was obtained (Figure 1A, Table 1, Supp. Figure 1A), very similar to the EC 50 of 2.1 nM (±SD 0.2 nM) determined previously using the hypoxanthine incorporation assay [46]. The EC 50 of Compound 2 in the same SYBR Green 72-hour growth inhibition assay was 100 nM (±SD 35 nM), also similar to previously published values using the hypoxanthine incorporation assay (Figure 1B, Supp.…”

“…To verify the specificity of the inhibitors for PKG in the SYBR Green assay, we determined in parallel the EC 50 for a 3D7-derived transgenic line that harbors a gatekeeper mutation (T618Q) in the PKG sequence [40, 46]. In this mutant, access to the ATP-binding pocket is blocked, preventing binding of ATP-competitive PKG inhibitors that utilise the gatekeeper pocket, including ML10, Compound 2 and Compound 1, however, this mutation has no detectable effect on the endogenous activity of PKG or parasite growth [41, 46, 48]. The EC 50 values of ML10 and Compound 2 in this strain were several orders of magnitude higher than that of 3D7 (Figures 1C and 1D), confirming that ML10 is indeed a highly specific inhibitor of PKG, as previously described [46].…”

“…The EC 50 of Compound 2 in the same SYBR Green 72-hour growth inhibition assay was 100 nM (±SD 35 nM), also similar to previously published values using the hypoxanthine incorporation assay (Figure 1B, Supp. Figure 1B) [46]. To verify the specificity of the inhibitors for PKG in the SYBR Green assay, we determined in parallel the EC 50 for a 3D7-derived transgenic line that harbors a gatekeeper mutation (T618Q) in the PKG sequence [40, 46].…”

“…parasites. We show that the PKG inhibitor ML10, a derivative of the previously used imidazopyridine PKG inhibitor Compound 2 [46], reversibly arrests P. falciparum and P. knowlesi at a very late schizont stage at low nanomolar concentrations. Whereas we detected no significant difference in the EC 50 of ML10 among four different P. falciparum laboratory isolates, the EC 50 of ML10 was markedly influenced by the supplement used in the medium; human and horse serum increase the EC 50 compared with medium containing AlbuMax, likely owing to binding of the compound to serum components.…”

“…However, a detailed description of the use of PKG inhibitors has not been published. Recently a highly specific and potent derivative of Compound 2, referred to as ML10, with a single digit nM EC 50 and an approximately 75-fold higher specificity has been described [46]. To demonstrate that ML10 is a valuable tool for the synchronization of Plasmodium spp.…”