UA2239 Inhibits Malaria cGMP-PKG Pathway PDF

A potent acyclic nucleoside phosphonate inhibits the malaria cGMP-PKG dependent egress pathway via a unique mechanism of action Marie Ali1, Rea Dura1, Thomas Cheviet2, Marc-Antoine Guery1, Antoine Claessens1, Emma Colard-Itté3, Catherine Lavazec3, Laurence Berry1, Suzanne Peyrottes2, Kai Wengelnik1...

A potent acyclic nucleoside phosphonate inhibits the malaria cGMP-PKG dependent egress pathway via a unique mechanism of action Marie Ali1, Rea Dura1, Thomas Cheviet2, Marc-Antoine Guery1, Antoine Claessens1, Emma Colard-Itté3, Catherine Lavazec3, Laurence Berry1, Suzanne Peyrottes2, Kai Wengelnik1, Sharon Wein1*, Rachel Cerdan1* 1 LPHI, University Montpellier, CNRS, Inserm, Montpellier, France. 2 IBMM, University Montpellier, CNRS, Montpellier France 3 Université Paris Cité, CNRS, Inserm, Institut Cochin, F-75014 Paris, France *co Abstract The urgent need for new antimalarial therapies arises from the alarming spread of malaria parasite resistance to existing drugs. A promising candidate, UA2239, an acyclic nucleoside phosphonate and purine analog, demonstrates robust inhibition of Plasmodium growth both in vitro and in animal model by irreversibly impeding egress of both merozoites and gametes from erythrocytes. The generation of UA2239- resistant P. falciparum lines identified several mutations in cGMP-dependent protein kinase (PKG) of the essential cGMP-PKG dependent egress process. The resistance phenotype was validated by site directed mutagenesis. However, PKG is not the targets of UA2239 because the compound does not inhibit the activity of recombinant PfPKG. Instead, UA2239 decreases the level of cGMP in the parasite, making guanylate cyclase (PfGC) the most likely primary target. UA2239 therefore impairs the essential cGMP-dependent egress pathway, inhibits parasite growth and transmission, and introduces a new mechanism of action by targeting cyclic-GMP synthesis. Abbreviations: C2: compound 2 CQ: chloroquine DHA: dihydroartemisinin GC : guanylate cyclase GER parasites: genome-edited resistant parasites 1 hpi: hours post invasion iRBC: infected red blood cell NAG: N-acetylglucosamine NPP: new permeation pathways PDE: phosphodiesterase PKG: cGMP-dependent protein kinase RBC: red blood cell SEM: standard error of the mean SNP: single nucleotide polymorphism Main text: 5000 mots et 10 figures+tables Method : 3000 mots max Introduction Malaria is caused by parasites of the genus Plasmodium with the most fatal cases occurring after P. falciparum infection. Despite the efforts and progress made in recent decades1, the number of deaths due to malaria is still estimated at 608,000 in 2022. After a significant increase at the beginning of the COVID-19 pandemic, malaria cases and deaths have been falling slowly for the last three years, but remain higher than before the pandemic. Among the means of combating malaria, the very first vaccines are currently administered in a limited number of African countries2 while few dozen vaccine candidates are under development3,4. Nevertheless, chemotherapy remains indispensable for the fight against malaria. Current treatments are based on artemisinin combination therapy1. The emergence of artemisinin-resistant strains in Asia and more recently in Africa, in addition to already established resistance to all commercially available drugs, worsens the situation and emphasizes the urgency of finding alternative options. Effective treatments are therefore necessary to bolster the therapeutic arsenal. Development of new chemical entities with novel mechanisms of action is a priority to face the increasing resistance of parasites to available medicines. Future antimalarial treatments are expected to target at least two stages of the complex life cycle of the parasite, in the human host or during the transmission by mosquitoes5. In humans, P. falciparum multiplies initially in liver cells and after release of merozoites into the blood stream replicates asexually in erythrocytes in 48-hour cycles of invasion- growth-multiplication resulting in the formation of up to 32 daughter cells that egress actively from the infected red blood cell (iRBC) and invade new red blood cells6. A fraction of parasites differentiates into sexual forms (gametocytes) that differentiate and mate in the mosquito gut upon a blood meal7. After several weeks of growth and multiplication, parasites settle in the salivary glands ready to be injected in a new human host. Recently, we synthetized a novel class of nucleoside analogs, acyclo-nucleoside phosphonates (ANPs) that show remarkable antimalarial properties8. The best activities were observed with the chemical series of purine analogues8. The lead 2 compound UA2239, a guanosine monophosphate analog has strong activity in vitro on P. falciparum with a concentration to inhibit 50% of the parasite growth (IC50) of 74 nM for the 3D7 strain and for the chloroquine-resistant strains FcM29 and W2. UA2239 is also active in vivo in P. berghei-infected mice with an efficient dose to inhibit 50% of the parasite growth (ED50) of 0.5 mg/kg after intraperitoneal administration. UA2239 has no activity on mammalian K562 cells resulting in a high selectivity index (SI > 10,000). Nucleoside and nucleotide analogues are widely used as therapeutic agents in the clinical treatment of viral infections and of cancer due to their anti-proliferative properties9. Their mechanism of action usually involves their conversion into poly- phosphorylated derivatives and their interaction with cellular or viral polymerases (as substrate and/or competitive inhibitor)9. Certain ANPs have been developed to inhibit enzymes of the purine salvage pathway in Plasmodium and show antimalarial activities10. Surprisingly, initial phenotypic characterization of the mode of action of UA2239 on P. falciparum revealed no effect on parasite intraerythrocytic development including the formation of merozoites. This contradicts the hypothesis of inhibition of DNA synthesis and suggests a disruption in merozoite egress11. Merozoite egress is governed by the cGMP-PKG dependent pathway. The signaling cascade leading to merozoite egress involves the activation of PKG and a subsequent calcium signal essential for egress (Fig. 1c) (refs). The activation of PKG requires elevated levels of cGMP as a result of a balanced activity of its production from GTP by guanylate cyclase (GC), and its hydrolysis to GMP by phosphodiesterases (PDE) (ref). Inhibition of PfPKG lead to an impairment of egress, the parasites are blocked in the red blood cell. Inhibition of PfGC should show the same effect. On the contrary, inhibition of PfPDE will induce the egress of immature merozoites, that will in turn won’t be able to invade new red blood cells (ref). PKG have been extensively tartget. Compound 2 (C2, also known as ML1)25 belongs to the class of imidazopyridines and targets the catalytic domain of the PfPKG. A medicinal chemistry programme led to the development of a more potent C2 analog (ML10) (Baker 2017). Zaprinast and BIPPO are described as inhibitors of PfPDEs and their effects lead to a premature egress of parasites. Thus UA2239 and C2 belong to different chemical families. In this work, we studied the pharmacological properties of UA2239 and show that the compound is active against at least two stages of the parasite life cycle inhibiting the intraerythrocytic cycle and gametogenesis. UA2239 does not inhibit DNA synthesis but irreversibly blocks parasite egress. UA2239-resistant parasite lines could be generated and carry mutations in cGMP-dependent protein kinase (PfPKG) but these lines show an important fitness cost. Unexpectedly, we showed that the likely target of UA2239 is the guanylate cyclase, PfPGC and not PfPKG. The compound thus shows a novel mode of action blocking the parasite at multiple stages of its life-cycle and opens the 3 way for the development of new antimalarial compounds targeting the synthé