Malaria is an infectious disease that remains a clear and present threat to human health. It has been estimated that the disease is responsible for more than half a million deaths annually. In 2016, nearly half of the world’s population was at risk of malaria and according to the latest World Malaria Report 2021 (WHO, 2021), there were an estimated 241 million cases of malaria in 2020 in 85 malaria endemic countries, increasing from 227 million in 2019, and malaria deaths increased by 12% compared with 2019, to an estimated 627 000. Children under 5 years accounted for the majority of malaria deaths in areas of high malaria transmission (77% in 2020). To date there are more than 200 known species of the genus Plasmodium, but just five of them cause human malaria, comprising P. vivax, P. ovale, P. malariae, P. knowlesi and P. falciparum (Majori, 2012), the most virulent malaria parasite. A potent vaccine is currently not available and therefore disease control depends mostly on drugs (Greenwood et al., 2008). Malaria is presently undergoing resurgence and the control of P. falciparum has become a major challenge in global health. Due to the high mutational rate of the parasite and its resulting rapid adaptation to environmental changes, both drug resistance and the geographic distribution of the disease are increasing (Piel et al., 2010). The inevitable emergence of antimalarial drug resistance (López et al., 2010) forces continuous efforts towards the discovery and development of new antimalarial drugs. There is therefore an urgent need for novel chemotherapeutic targets. Highly attractive avenues for the antimalarial drug discovery are metabolic pathways. The pyrimidine-biosynthesis pathway of Plasmodium falciparum is a promising target for antimalarial drug discovery as we reported previously (Lunev et al., 2016; Lunev et al., 2018). Active proliferation during the intraerythrocytic stage of P. falciparum requires a supply of purines and pyrimidines for parasite growth to support the production of DNA and parasite replication. As the malarial parasite lacks a purine biosynthetic pathway (De Koning et al., 2005; Hyde, 2007), as well as a functional pyrimidine-import pathway (Reyes et al., 1982; Rathod and Reyes, 1983; Gardner et al., 2002), the parasite relies solely on the de novo synthesis pathway to produce pyrimidines, and therefore the de novo pyrimidine biosynthesis pathway has been demonstrated to be a promising target for antimalarial drug discovery (Downie et al., 2008; Vaidya and Mather, 2009; Rodrigues et al., 2010; Belen Cassera et al., 2011).

The de novo synthesis of pyrimidines process in general contains six sequential enzymatic steps ( Figure 1 ) in P. falciparum and starts with the Carbamoyl phosphate synthetase II (CPS II) which is responsible for the formation of carbamoyl phosphate in the cytosol from bicarbonate, glutamine and ATP (Müller et al., 2010; Müller et al., 2010). The Aspartate transcarbamoylase (PF3D7_1344800, ATCase), the second enzyme in the pathway, catalyzes the condensation of aspartate and carbamoyl phosphate to form N-carbamoyl-l-aspartate and inorganic phosphate. The third step is the intramolecular condensation catalyzed by dihyroorotase (DHOase) to the product dihydroorotate. Then dihydroorotate dehydrogenase oxidizes dihydroorotate to orotate. Subsequently orotate and 5-phosphoribosyl-1-pyrophosphate (PRPP) are combined to produce orotidine-5’-monophosphate (OMP) by orotate phosphoribosyl transferase (OPRTase), the final product, molecular uridine monophosphate (UMP), is yielded by decarboxylation of OMP catalyzed by OMP decarboxylase (O’Donovan and Neuhard, 1970; Jones, 1980).

In the following review, we summarize the current knowledge of the PfATCase, by highlighting the potential of an allosteric pocket and describing how these structures provide insights into the ATCase catalytic mode of action. We will also review current studies on drug discovery against other ATCases, not solely against the human malaria parasite by Torin 2 (Bosch et al., 2020).

Aspartate transcarbamoylase catalyzes the second step in de novo pyrimidine biosynthesis, where the condensation of L-aspartate (L-Asp) and carbamoyl phosphate (CP) to form N-carbamoyl aspartate (CA) and phosphate (Pi) ( Figure 2A ). The Escherichia coli aspartate transcarbamoylase holoenzyme represents the canonical form and is composed of six catalytic and six regulator subunits in which three regulatory pairs coordinate two catalytic trimers (G., 1964; Gerhart and Schachman, 1965; Weber, 1968; Ke et al., 1984; Stevens et al., 1990). The E. coli enzyme has been extensively studied and is now a textbook example that regulates pyrimidine biosynthesis pathway through its catalytic and regulatory mechanisms and has been fully characterized by Lipscomb and Kantrowitz (2012). The E. coli ATCase is inhibited by the final products of the pyrimidine biosynthesis pathway (CTP) and by a combination of CTP and UTP. The catalytic reaction by the canonical ATCase is sequential (Wang et al., 2005); CP binds first inducing a conformational changes and creating a binding site for L-aspartate. Similarly, N-carbamoyl aspartate leaves the active site before phosphate.

The P. falciparum ATCase is a homotrimer with three active sites in which each of the three active sites is formed at adjacent oligomeric interface ( Figure 3A ), following the canonical example. Each catalytic site is composed of two functional domains - the aspartate domain, which is mainly responsible for the binding of the substrate L-aspartate, and the carbamoyl phosphate domain, which is mainly responsible for the binding of the substrate carbamoyl phosphate. Apart from the catalytic site, the E. coli ATCase also contains regulatory sites, which are targets for the binding of the allosteric effectors – ATP and CTP (Lipscomb and Kantrowitz, 2012). These regulatory sites on the E. coli ATCase, are located approximately 60 Å from the closest active site. However, no homologs of the ATCase allosteric chain have been reported in the plasmodial genome.

PfATCase is a 43.3 kDa polypeptide with 375 amino acids. In previous studies we determined the crystal structure of truncate aspartate transcarbamoylase from P. falciparum (Lunev et al., 2016). Superposition of the PfATCase structure with catalytic chain of E. coli ATCase (PDB code: 1ZA1) and huATCase (PDB code: 5G1O) ( Figures 3B–D ), showed a high degree of homology with the catalytic domain of E. coli ATCase and huATCase. Similarly to E. coli ATCase, PfATCase is a homo-trimer in which each of the three active site is formed at the oligomeric interface. Each active site comprises residues from two adjacent subunits in the trimer with a high degree of evolutionary conservation.

As reported the active site of ATCase exists in two distinct states, which are known as the T state and R state. These two states differ in substrate affinity and activity, with the T state, active site present in an open conformation with lower affinity and lower activity for substrate than the R state (Lipscomb and Kantrowitz, 2012; Ruiz-Ramos et al., 2016). We have recently elucidated several structures (Lunev et al., 2016; Lunev et al., 2018) of the plasmodial ATCase (PfATCase) which provide insight into the conformational changes present in the transition between T and R states in the plasmodial enzyme. While the parasite lacks an ATCase regulatory element, an understanding of this transition may allow for the discovery of drugs that can provide a similar allosteric inhibition impact to that shown by the CTP feedback inhibition of the human and E. coli enzymes.

The structures reported by ourselves and others (Lipscomb and Kantrowitz, 2012; Lunev et al., 2016; Ruiz-Ramos et al., 2016) show that the conformation of ATCase can be induced from the T state to the R state when both aspartate and carbamoyl phosphate are present at the active site as well as N-phosphonacetyl-L-aspartate (PALA Figure 2A ) – an analog of the transition state of the reaction catalyzed by ATCase. Additionally, the enzyme can also change from T state to R state by binding carbamoyl phosphate (CP) and succinate, an analog of aspartate.

Previously, we determined the high-resolution of X-ray crystal structure of the 2, 3-napthalenediol-PfATCase complex ( Figure 4A ). 2, 3-napthalenediol was identified buried in cavity between two adjacent monomers, which is close to the traditional substrates binding site ( Figure 4B ) and is accommodated in a hydrophobic region buried under the 128-142 loop. Analysis the binding site of 2, 3-napthalenediol reveals the hydroxyl groups of 2, 3-napthalenediol forms polar contact with the side chain of Glu140 and a water bridge with Pro333 and Leu334’s carbonyl main chain oxygens ( Figure 4C ). The structural alignment of the 2, 3-napthalenediol-PfATCase complex structure with apo-PfATCase structure did not significantly affect the structure of PfATCase compared to un-ligand crystal structure. Which strongly suggests that the 2, 3-napthalenediol-PfATCase complex (PDB ID: 6FBA) is still in T state. In addition, both the X-ray and DSF results determined that 2, 3-napthalenediol can stabilize the PfATCase.

Furthermore, the comparison of PfATCase complexed with 2, 3-naphthalenediol, citrate-liganded crystal structures (PDB ID: 5ILN) suggest an allosteric mode of inhibition, as 2, 3-naphthalenediol binds in a cavity between adjacent subunits of the trimer. The 128-142 loop of the citrate-bound PfATCase loop showed a significant shift compared to 2, 3-naphthalenediol-bound structure (11.2 Å between the alpha-carbons of Thr134 in the two structures) ( Figure 4D ), indicating that 2, 3-naphthalenediol could hold PfATCase in its low affinity, low activity T-state, preventing the 128-142 loop pushing the Asp domain and CP domain towards each other to form the carbamoyl aspartate and phosphate.

The superposition of the PfATCase with human ATCase and catalytic subunit of E.coli ATCase showed high level of sequence and secondary structure conservation ( Figures 3B–D ). Tcoffee (Armougom et al., 2006; Di Tommaso et al., 2011) analysis against human ATCase and the catalytic subunit of E.coli ATCase showed that of 12 active site residues 9 (75%) were absolutely conserved, and the residues which we found to have polar contact with 2, 3-naphthalenediol showed 2 of 3 (66.7%) are absolutely conversed.

Malaria is an infectious disease that remains a clear and present threat to human health. Though several anti-malarial medications are available, the spread of multidrug-resistant severely limits their efficacy. There is pressing need for academic research to discover new targets and drugs for the treatment of severe malaria. For intracellular proliferation, P. falciparum requires biosynthesis of pyrimidines for parasite growth to support the production of DNA and parasite replication. ATCase supports the second step of the de novo biosynthesis pathway and, as the malaria parasite lacks a functional pyrimidine-import pathway, the de novo pyrimidine biosynthesis pathway has been demonstrated to be a major target for antimalarial drug development. While PALA has been long available as a strong ATCase inhibitor, it represents a suboptimal starting point for the development of an anti-malarial for several reasons. Firstly, PALA has been shown to be relatively poor inhibitor of the plasmodial ATCase (Bosch et al., 2020). Secondly, while PALA is a strong in vitro inhibitor of canonical ATCases it appears to be limited in development towards a selective inhibitor as it is a mimic of the enzyme transition state. This transition state is likely to be absolutely conserved across species, suggesting that inhibitors of the transition state of the plasmodial homolog may show significant side effects through their inhibition of the human homologs. Finally, the transition state intermediate mimicked by PALA is by nature rather highly charged, which possibly accounts for its relatively poor performance in cell-based assays, as this high charge state is not compatible with efficient transfer across cell membranes. A situation that would be made more challenging when considering the additional membranes that must be traversed to access the parasite ATCase. A potential mechanism to address these limitations and leverage the sensitivity of the parasite to inhibition of de novo pyrimidine biosynthesis is to identify allosteric sites of inhibition. Such sites would allow for more selectivity between species and would not be constrained by the limitations imposed by the heavily charged transition state. Our studies have focused on the structure of PfATCase, and identified an allosteric pocket through the determination of a co-crystal structure of PfATCase with 2,3-naphthalenediol. By comparing the crystal structure of the 2.3-naphthalenediol:ATCase complex structure as an exemplary of the low-affinity low-activity T-state with a high-affinity high activity R-state represented by the structure of PfATCase in complex with citrate, there exists the intriguing potential for the development of an allosteric inhibitor of PfATCase represented by the 2,3-napthalenediol binding site. The further understanding of the mechanism of inhibition of 2,3-napthalenediol would provide an opportunity for further drug development. Such a compound would strengthen the case of PfATCase as a drug target and would be an invaluable addition to the antimalarial “toolbox”.

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