One Step Ahead of Drug-Resistant Malaria

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Malaria is a disease caused by Plasmodium spp., which are unicellular eukaryotes. These parasites are transmitted by Anopheles spp. mosquitoes when they take a blood meal of a vertebrate host. Plasmodium sporozoites pass into the host and invade liver cells during the pre-erythrocytic (hepatic) phase. Infected liver cells then release merozoites. Merozoites can infect red blood cells (erythrocytic phase) and develop into trophozoites and then schizonts to release more merozoites. Other merozoites become dormant as hypnozoites which remain in the liver. Some trophozoites mature into gametocytes which can then be transmitted to a mosquito vector, and the cycle continues (Fikadu and Ashenafi 2023). The cGMP-dependent protein kinase (PKG, or PfPKG in P. falciparum), is essential for pre-erythrocytic stages in the Plasmodium spp. life cycle, as it affects sporozoite infection of liver cells, development of merosomes, and exit of merosomes from liver cells.

Figure. Activity of RUPB-61 in primary hepatocytes against human-infective and simian-infective sporozoites (A) Effect of RUPB-61 and KDU-691 on P. falciparum infection of primary human hepatocytes, normalized to vehicle control. Infection was measured by quantifying the number of infected cells. Data shown are the mean of two biological replicates, each performed in technical duplicates. (B) Effect of RUPB-61 and KDU-691 on the number of P. cynomolgi hypnozoites formed in primary simian hepatocytes, normalized to vehicle control. Data shown are mean of three biological replicates, each performed in technical duplicates or triplicates. (C) Effect of RUPB-61 and KDU-691 on the number of P. cynomolgi schizonts in primary simian hepatocytes, normalized to vehicle control. Data shown are mean of three biological replicates, each performed in technical duplicates or triplicates. — **Figure**. Activity of RUPB-61 in primary hepatocytes against human-infective and simian-infective sporozoites **(A)** Effect of RUPB-61 and KDU-691 on *P. falciparum* infection of primary human hepatocytes, normalized to vehicle control. Infection was measured by quantifying the number of infected cells. Data shown are the mean of two biological replicates, each performed in technical duplicates. **(B)** Effect of **RUPB-61** and **KDU-691** on the number of *P. cynomolgi* hypnozoites formed in primary simian hepatocytes, normalized to vehicle control. Data shown are mean of three biological replicates, each performed in technical duplicates or triplicates. **(C)** Effect of **RUPB-61** and **KDU-691** on the number of *P. cynomolgi* schizonts in primary simian hepatocytes, normalized to vehicle control. Data shown are mean of three biological replicates, each performed in technical duplicates or triplicates.

The P. falciparum cGMP-dependent protein kinase (PfPKG) is the primary cellular target of RUBP-61. It was previously established that RUBP-61 is active against P. falciparum asexual erythrocytic stages and P. berghei sporozoites in HepG2 cells (Gilleran et al. 2024). In the present study, it prevented the formation of hypnozoites and schizonts by P. cynomolgi in primary human hepatocytes. A single oral dose of RUBP-61 appears to completely block P. berghei sporozoites from infecting mice liver cells in vivo.

RUBP-61 remains effective against South American and Southeast Asian field Plasmodium falciparum isolates resistant to chloroquine, mefloquine, cycloguanil, sulfadoxine, and pyrimethamine. PfPKG has what is known as a “gatekeeper” residue, a Threonine at position 618, providing access to a pocket that PKG inhibitors require access to in order to be effective. It is predicted that replacement of Thr618 with bulkier amino acids would lead to RUBP-61 resistance, but based on an isolate database, occurrence of non-synonymous mutations at this site is extremely rare.

RUBP-61 has some activity against the human kinome, but X-ray crystallography and biochemical modeling appear to be reliable tools for guiding optimization. This biochemical modeling also suggests that Plasmodium falciparum with the gatekeeper mutation Thr618Ser should remain sensitive to RUBP-61.

Malaria continues to be a major health threat, especially in tropical and subtropical regions of the Global South, and antimalarial resistance, insecticide resistance, limitation of chemoprotective agents, and the climate crisis make the disease increasingly difficult to control. Existing pre-erythrocytic stage-targeting drugs, which are instrumental for preventing new infections, have limitations including serious side effects, limited efficacy in malaria-endemic regions, high cost, and facile evolution of resistance. RUBP-61 has potential as a starting point for a new pre-erythrocytic stage-targeting drug. X-ray crystallography and biochemical structural modeling of PfPKG interactions is prospectively validated as accurate, which will assist with optimization of RUBP-61 and derivatives as chemopreventive agents

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Fikadu M, Ashenafi E. Malaria: An Overview. Infect Drug Resist. 2023 May 29;16:3339-3347. doi: 10.2147/IDR.S405668. PMID: 37274361; PMCID: PMC10237628.

Gilleran JA, Ashraf K, Delvillar M, Eck T, Fondekar R, Miller EB, Hutchinson A, Dong A, Seitova A, De Souza ML, Augeri D, Halabelian L, Siekierka J, Rotella DP, Gordon J, Childers WE, Grier MC, Staker BL, Roberge JY, Bhanot P. Structure-Activity Relationship of a Pyrrole Based Series of PfPKG Inhibitors as Anti-Malarials. J Med Chem. 2024 Mar 14;67(5):3467-3503. doi: 10.1021/acs.jmedchem.3c01795. Epub 2024 Feb 19. Erratum in: J Med Chem. 2024 Jul 25;67(14):12459. doi: 10.1021/acs.jmedchem.4c01411. PMID: 38372781.