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. 2020 Jul 16;27(7):806-816.e8.
doi: 10.1016/j.chembiol.2020.04.001. Epub 2020 Apr 30.

Inhibition of Resistance-Refractory P. falciparum Kinase PKG Delivers Prophylactic, Blood Stage, and Transmission-Blocking Antiplasmodial Activity

Manu Vanaerschot  1 , James M Murithi  1 , Charisse Flerida A Pasaje  2 , Sonja Ghidelli-Disse  3 , Louis Dwomoh  4 , Megan Bird  5 , Natasha Spottiswoode  1 , Nimisha Mittal  6 , Lauren B Arendse  7 , Edward S Owen  8 , Kathryn J Wicht  1 , Giulia Siciliano  9 , Markus Bösche  3 , Tomas Yeo  1 , T R Santha Kumar  1 , Sachel Mok  1 , Emma F Carpenter  10 , Marla J Giddins  11 , Olalla Sanz  12 , Sabine Ottilie  6 , Pietro Alano  9 , Kelly Chibale  7 , Manuel Llinás  13 , Anne-Catrin Uhlemann  11 , Michael Delves  14 , Andrew B Tobin  4 , Christian Doerig  15 , Elizabeth A Winzeler  6 , Marcus C S Lee  10 , Jacquin C Niles  2 , David A Fidock  16
Affiliations

Inhibition of Resistance-Refractory P. falciparum Kinase PKG Delivers Prophylactic, Blood Stage, and Transmission-Blocking Antiplasmodial Activity

Manu Vanaerschot et al. Cell Chem Biol. .

Abstract

The search for antimalarial chemotypes with modes of action unrelated to existing drugs has intensified with the recent failure of first-line therapies across Southeast Asia. Here, we show that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by Plasmodium sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies, validated with conditional knockdown parasites, molecular docking, and recombinant kinase assays, identified cGMP-dependent protein kinase (PKG) as the primary target of MMV030084. PKG is known to play essential roles in Plasmodium invasion of and egress from host cells, matching MMV030084's activity profile. Resistance selections and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under pressure. These studies highlight PKG as a resistance-refractory antimalarial target throughout the Plasmodium life cycle and promote MMV030084 as a promising Plasmodium PKG-targeting chemotype.

Keywords: Plasmodium falciparum; cGMP-dependent protein kinase (PKG); chemoproteomics; conditional knockdown; kinase; malaria drug discovery; phosphoproteomics; resistance; target identification.

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Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
MMV030084 Has Prophylactic, Anti-ABS, and Transmission-Blocking Potential (A) Structure of MMV030084, a trisubstituted imidazole. (B) Overall activity profile of MMV030084. (C) MMV030084 inhibited invasion of P. berghei ANKA parasites into HepG2 liver cells. (D) Compound dose-response assays with ABS parasite stages, highly synchronized to within 3-h periods of development, indicated that MMV030084 is highly active only against synchronized schizonts. Minimal activity was observed against rings and trophozoites. (E) Microscopy studies confirmed that MMV030084 is active against schizonts, showing the profile of an egress inhibitor. (F) Male gamete formation is inhibited by MMV030084 in the presence of compound, but not when compound is washed out before gamete formation.
Figure 2
Figure 2
Identification and Validation of PKG as the Primary Target of MMV030084 (A) MMV030084-exposure of schizonts affected the phosphorylation status of several peptides in various pathways and provided evidence indicating that MMV030084 likely targets a kinase that is central to Pf parasite metabolism. (B) PKG, CDPK1, and AAT emerged as candidate targets in competitive chemoproteomic studies using bead-bound compounds on treated and untreated (control) parasite lysates (Table S1 (E1-3)). (C) Exposing MMV030084-treated parasite lysates to a panel of bead-immobilized kinase inhibitors (Kinobeads) or bead-immobilized MMV030084 (‘084-beads) identified the lowest Kdapp values for PKG, CDPK1, and AAT. Results shown are the mean of two repeated experiments, except for AAT, which was detected in only one of the experiments (Table S1 (E1-3)). (D) cKD of PKG resulted in the largest growth defect compared with control conditions, with cKDs of CDPK1 and AAT affecting parasite growth to a lesser extent. Error bars indicate the SEM based on two independently repeated experiments with technical duplicates. p values are based on unpaired t tests (Table S1 (F)). (E) cKD of PKG increased parasite susceptibility to MMV030084, while cKD of CDPK1 or AAT caused no or minor differences in MMV030084 susceptibility versus control conditions. Results shown are based on four independently repeated experiments with technical duplicates (Table S1 (G)). (F) MMV030084 and its analogs dock well into the crystal structure of PKG (Table S1 (H)). H-bonding residues are highlighted in the docking graphs. (G) MMV030084 potently inhibited the kinase activity of recombinant PfPKG in vitro. IC50 data are based on three independently repeated experiments with technical duplicates. MMV’084, MMV030084; TCMDC‘154, TCMDC-141154; ctrl, control; PKG, cGMP-dependent protein kinase (PF3D7_1436600); CDPK1, calcium-dependent protein kinase 1 (PF3D7_0217500); AAT, putative amino acid transporter (PF3D7_1231400); Kdapp, apparent dissociation constant; cKD, conditional knockdown; SEM, standard error of the mean. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Figure 3
Figure 3
Resistance Selections with Cultured Pf ABS Parasites Identify TKL3, PP1, and URP as Low-Level Resistance Mediators for MMV030084 (A) Outline of the MMV030084 resistance selection attempts that identified TKL3, PP1, and URP as candidate resistance mediators. (B) Susceptibility of the selected and confirmatory gene-edited clones to MMV030084. Error bars indicate the SEM based on ≥4 independently repeated experiments with technical duplicates. p values are based on Mann-Whitney U tests (Table S1 (I)). (C) Parasite growth is only minimally inhibited by cKD of TKL3 and URP, while cKD of PP1 renders ABS parasites effectively non-viable (Table S1 (F)). Error bars indicate the SEM based on two independently repeated experiments with technical duplicates. p values are based on unpaired t tests. (D) cKD of TKL3 decreased parasite susceptibility to MMV030084, whereas cKD of PP1 and URP showed no effect. Error bars indicate the SEM based on two independently repeated experiments with technical duplicates. p values are based on unpaired t tests (Table S1 (G)). Sel., selected; Ed., CRISPR/Cas9 edited; aTc, anhydrotetracycline; TKL3, tyrosine kinase-like protein 1 (PF3D7_1349300); PP1, protein phosphatase 1 (PF3D7_1414400); URP, ubiquitin regulatory protein (PF3D7_0808300); cKD, conditional knockdown; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
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