Novel Highlight in Malarial Drug Discovery: Aspartate Transcarbamoylase

Abstract

Malaria remains one of the most prominent and dangerous tropical diseases. While artemisinin and analogs have been used as first-line drugs for the past decades, due to the high mutational rate and rapid adaptation to the environment of the parasite, it remains urgent to develop new antimalarials. The pyrimidine biosynthesis pathway plays an important role in cell growth and proliferation. Unlike human host cells, the malarial parasite lacks a functional pyrimidine salvage pathway, meaning that RNA and DNA synthesis is highly dependent on the de novo synthesis pathway. Thus, direct or indirect blockage of the pyrimidine biosynthesis pathway can be lethal to the parasite. Aspartate transcarbamoylase (ATCase), catalyzes the second step of the pyrimidine biosynthesis pathway, the condensation of L-aspartate and carbamoyl phosphate to form N-carbamoyl aspartate and inorganic phosphate, and has been demonstrated to be a promising target both for anti-malaria and anti-cancer drug development. This is highlighted by the discovery that at least one of the targets of Torin2 - a potent, yet unselective, antimalarial - is the activity of the parasite transcarbamoylase. Additionally, the recent discovery of an allosteric pocket of the human homology raises the intriguing possibility of species selective ATCase inhibitors. We recently exploited the available crystal structures of the malarial aspartate transcarbamoylase to perform a fragment-based screening to identify hits. In this review, we summarize studies on the structure of Plasmodium falciparum ATCase by focusing on an allosteric pocket that supports the catalytic mechanisms.

Keywords: Plasmodium falciparum; X-ray structure; allosteric pocket; anti-malarials; aspartate transcarbamoylase; pyrimidine biosynthesis.

Figure 1

The de novo pyrimidine biosynthesis pathway. Enzymes in de no pathway are italicized and colored with light orange. Enzymes: CPS II, carbamoyl phosphate synthetase II; ATCase, aspartate transcarbamoylase; DHO, dihydroorotase; DHODH, dihydroorotate dehydrogenase; OPRT, orotate phosphoribosyl transferase; ODC, orotidine 5’-monophosphate decarboxylase.

Figure 2

The reaction catalyzed by ATCase and feedback regulation mechanism in de novo pyrimidine biosynthesis pathway, and inhibitors against ATCase. (A) Aspartate Transcarbamoylase (ATCase) combines L-aspartate and carbamoyl phosphate into carbamoyl aspartate through an enzyme stabilized transition state and inhibition feedback by CTP. The ATC inhibitor PALA closely resembles this transition state intermediate. CTP (a product of the pyrimidine biosynthesis pathway) provides feedback inhibition of ATC activity. (B) Structures of PALA analogues as E. coli ATCase inhibitors. (C) Structures of T-state inhibitors against E. coli ATCase that prevent the allosteric transition. (D) Structures of allosteric inhibitors of huATCase ( Lei et al., 2020).

Figure 3

The structure of PfATCase compare to huATCase and catalytic subunit of E.coli ATCase. (A) A ribbon diagram of the crystal structure of the truncated PfATCase indicating an overall trimeric assembly (Lunev et al., 2016), three active sites formed at the oligomeric interfaces are labeled with stars. (B) structural alignment of the monomeric PfATCase structure (blue, PDB code: 5ILQ) with human ATCase (grey, PDB code: 5G1O) and the catalytic chain of E.coli ATCase (magenta, PDB code: 1ZA1) (C). The structural alignments were carried out with Pymol (Schrödinger and DeLano, 2020). (D) multiple protein sequence alignment of the human, P. falciparum and E. coli ATCases using Tcoffee (Armougom et al., 2006; Di Tommaso et al., 2011).

Figure 4

Crystal structure of the 2,3-naphthalenediol-PfATCase complex. (A) structural alignment of the 2,3-naphthalenediol-bounded PfATCase [PDB ID: 6FBA; blue (Lunev et al., 2018)] with citrate-bound PfATCase [PDB ID: 5ILN; grey (Lunev et al., 2018)], RMSD=0.478 Å, providing a structural model of PfATCase in the T-state compared with the R-state. The location of the active site is shown for orientation. The conformational change in the loop128-142 in both cases is highlighted in red. (B) shows the structural rearrangements of 2,3-naphthalenediol binding site, the traditional active site is highlighted in yellow for orientation, 2Fo-Fc electron density of 2,3-naphthalenediol is shown in blue mesh at a contour of 1.0σ. (C) shows the binding site of 2,3-naphthalenediol and the polar contacts between 2,3-naphthalenediol and surrounding residues. (D) magnified view of the newly discovered allosteric binding site and active binding site. The structural alignments were carried out with Pymol (Schrödinger and DeLano, 2020).