A Novel Target for the Treatment and Prevention of Malaria

Plasmodium acetyl-coA synthetase identified as a promising new target for antimalarial treatment and prevention.

October 6, 2022

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Malaria is an infectious disease that spreads exclusively through bites of infected mosquitoes of the Anopheles genus. Parasites of the Plasmodium group cause the disease, with P. falciparum resulting in most human fatalities. In the human host, the parasite undergoes an exoerythrocytic (liver-stage) phase and an intraerythrocytic stage (asexual development). While current global malaria control efforts have limited the disease’s incidence in the past few decades, there are still more than 240 million new cases each year, resulting in over 600,000 deaths, mostly in young children in Africa. [1] Moreover, worryingly, the parasite is developing resistance to most approved drugs, including artemisinin-based combination therapies, the first-line treatment. [2] Therefore, new therapies are urgently needed, with a preference for compounds that kill Plasmodium in more than one stage of its life cycle – a precondition to be suitable for disease prevention as well as disease treatment.

 

A consortium to fight malaria

 

In light of the emerging resistance of the parasite, the Malaria Drug Accelerator (MalDA) consortium was assembled in 2012. [3] This consortium is a collaboration of currently 18 international research groups that seek to identify novel therapeutic targets against malaria and Plasmodium, initially using chemogenomic approaches. Literally millions of small molecules have so far been tested in phenotypic assays, which revealed a plethora of candidate compounds with antimalarial activity. However, most of these compounds target only a very limited range of common parasitic pathways, such as the degradation of hemoglobin, the functionality of mitochondria, and cellular homeostasis. Unfortunately, these are known to be susceptible to common resistance mechanisms, such as mutations in the Plasmodium chloroquine resistance transporter CQT, in an ATP-binding cassette transporter MDR1, or in a plasma membrane P-type ATPase, ATP4. The search for alternative processes affected by any of these compounds stepped into high gear and recently produced a new exciting target – Plasmodium’s essential enzyme acetyl-coenzyme A synthetase. Scientists of the MalDA consortium just published these findings in Cell Chemical Biology[4]


Two antimalarial compounds held the key to a novel drug target

 

In their search for novel target processes, the researchers focused on two of the many antimalarial compounds identified in their prior work, MMV019721 and MMV084978. They zoomed in on these two because both compounds were active against both the liver and the asexual blood life-cycle stages of Plasmodium, and because neither compound was cross-resistant with previously identified targets, suggesting a novel mode of action. To identify that target, a fairly straightforward approach was used: the scientists conducted in vitro evolution experiments to select for resistant parasites after either a continuous or a pulsed exposure to concentrations of each compound that exceeded their EC50 (the concentration at which a half-maximal response was achieved) by 3 to 6-fold. This way, they isolated mutants with increased resistance to the antimalarial compounds, and then performed whole genome sequencing (WGS) on dozens of these mutant clones of Plasmodium falciparum.

 

WGS analysis subsequently showed non-synonymous amino acid substitution mutations in the gene for the parasite’s acetyl-coenzyme A synthetase (PfAcAS) to have accumulated in the most resistant survivors. Notably, AcAS produces acetyl-CoA, a key molecule in cellular metabolism whose tightly controlled regulation is vitally important for survival. The mutations identified in the gene encoding this enzyme clustered around its active center. When similar PfAcAS mutations were introduced into a wild-type Plasmodium strain, elevated resistance resulted against both antimalarial compounds. Next, the scientists performed a metabolomic analysis of Plasmodium upon exposure to the two compounds and found a significant decrease in the levels of acetyl-CoA and related compounds, such as N-carbamoyl-L-aspartate, dihydroorotate, and orotate, consistent with an impeded function of PfAcAS.

 

Inhibition of Plasmodium acetyl-CoA synthetase kills the parasite

 

In order to prove direct interaction of the compounds with PfAcAS, full-length wild-type PfAcAS and two mutant varieties were purified and further studied in vitro. Single-inhibition measurements were performed upon exposure to the antimalarial compounds for ATP, acetate, and CoA (the three substrates needed for the reaction catalyzed by PfAcAS), at fixed saturating concentrations of two of the substrates, and variable concentrations of the third substrate. Surprisingly, one of the antimalarial compounds displayed linear competitive inhibition against CoA (and not the other two substrates), whereas the other showed linear mixed inhibition against acetate. The mutant PfAcAS enzymes, which displayed impeded substrate binding activity, were not as susceptible to the antimalarial compounds. Overall, the scientists had proven beyond a doubt that both compounds acted directly on PfAcAS.

 

But how exactly do these two antimalarial compounds kill the parasite? The scientists show that PfAcAS is located in the Plasmodium nucleus and that the compounds caused hypoacetylation of histones. Inhibition of PfAcAS may therefore result in disruption of acetyl-CoA recycling in the parasite’s nucleus which may subsequently cause a decrease in histone acetylation and dysregulate the epigenetic program of gene expression in Plasmodium. If the program of gene expression is disrupted, the parasite cannot survive.

 

New therapeutics targeting PfAcAS?

 

But humans also have acetyl-coenzyme A synthetase. So was the action of these antimalarial compounds against PfAcAS specific to the parasitic enzyme? Luckily, the answer was yes. When the researchers tested the inhibitory effect of the compounds on purified human acetyl-coenzyme A synthetase, IC50 values (i.e. the concentration at which the compounds were inhibiting 50% of the enzyme’s activity) were over 1000-fold higher for MMV019721, and 48-fold higher for MMV084978. Consequently, the scientists concluded that it should be possible to develop small-molecule inhibitors of PfAcAS as part of our expanding arsenal of therapeutics against malaria.

 

In their experiments, the researchers utilized Pall’s JumbosepTM Centrifugal Device to concentrate the wild-type and mutant PfAcAS enzyme, and our Macrosep® 10kDa Advance concentrator to increase the concentration of human AcAS. If you want to learn more about these and our other selective protein concentrators, please visit our website.

 

 

References

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