Identification of inhibitors that dually target the new permeability pathway and dihydroorotate dehydrogenase in the blood stage of Plasmodium falciparum.

Benjamin K Dickerman,Paul R Gilson,Malcolm J Mcconville,Brendan S Crabb,Simon A Cobbold,Brendan Elsworth,Catherine Q Nie

doi:10.1038/srep37502

Benjamin K Dickerman, Paul R Gilson + Show 5 more

Open Access

https://doi.org/10.1038/srep37502

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Abstract

Plasmodium parasites are responsible for the devastating disease malaria that affects hundreds of millions of people each year. Blood stage parasites establish new permeability pathways (NPPs) in infected red blood cell membranes to facilitate the uptake of nutrients and removal of parasite waste products. Pharmacological inhibition of the NPPs is expected to lead to nutrient starvation and accumulation of toxic metabolites resulting in parasite death. Here, we have screened a curated library of antimalarial compounds, the MMV Malaria Box, identifying two compounds that inhibit NPP function. Unexpectedly, metabolic profiling suggested that both compounds also inhibit dihydroorotate dehydrogense (DHODH), which is required for pyrimidine synthesis and is a validated drug target in its own right. Expression of yeast DHODH, which bypasses the need for the parasite DHODH, increased parasite resistance to these compounds. These studies identify two potential candidates for therapeutic development that simultaneously target two essential pathways in Plasmodium, NPP and DHODH.

Highlights

IntroductionSorbitol enters the infected erythrocytes via the NPPs, leading to osmotic lysis of the erythrocytes, release of haemoglobin and a decrease in the turbidity of the cell suspension, which can be readily measured
Sorbitol enters the infected erythrocytes via the NPPs, leading to osmotic lysis of the erythrocytes, release of haemoglobin and a decrease in the turbidity of the cell suspension, which can be readily measured. While this approach identified several compounds that potently blocked parasite NPPs with K0.5 of less than 100 nM13, a three day parasite proliferation assay revealed the EC50 for growth were many fold higher at 2–15 μM
To identify more potent NPP inhibitors we took advantage of the Malaria Box library which contains 400 compounds derived from several High-throughput screening (HTS) that have collectively identified >20,000 compounds that were highly inhibitory to parasite growth14,28,29

Summary

Introduction

Sorbitol enters the infected erythrocytes via the NPPs, leading to osmotic lysis of the erythrocytes, release of haemoglobin and a decrease in the turbidity of the cell suspension, which can be readily measured While this approach identified several compounds that potently blocked parasite NPPs with K0.5 (drug concentration at which sorbitol uptake is inhibited by 50%) of less than 100 nM13, a three day parasite proliferation assay revealed the EC50 for growth were many fold higher at 2–15 μM. Further experiments revealed that in addition to inhibiting NPP, both compounds inhibited the essential mitochondrial enzyme, dihydroorotate dehydrogenase (DHODH), which is indirectly targeted by another front line antimalarial, atovaquone These compounds represent strong candidates for drug development as pleiotropic inhibitory effects are expected to hinder development of drug resistance

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