Identification of a non-competitive inhibitor of Plasmodium falciparum aspartate transcarbamoylase

Abstract

Aspartate transcarbamoylase catalyzes the second step of de-novo pyrimidine biosynthesis. As malarial parasites lack pyrimidine salvage machinery and rely on de-novo production for growth and proliferation, this pathway is a target for drug discovery. Previously, an apo crystal structure of aspartate transcarbamoylase from Plasmodium falciparum (PfATC) in its T-state has been reported. Here we present crystal structures of PfATC in the liganded R-state as well as in complex with the novel inhibitor, 2,3-napthalenediol, identified by high-throughput screening. Our data shows that 2,3-napthalediol binds in close proximity to the active site, implying an allosteric mechanism of inhibition. Furthermore, we report biophysical characterization of 2,3-napthalenediol. These data provide a promising starting point for structure based drug design targeting PfATC and malarial de-novo pyrimidine biosynthesis.

Novel Highlight in Malarial Drug Discovery: Aspartate Transcarbamoylase

“…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.…”

“…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.…”

“…To identify the initial hits for PfATCase, we performed a Differential Scanning Fluorimetry (DSF) based screening against our in-house small fragment library (Lunev et al, 2018). 2,3-naphthalenediol showed a significant increase in the thermal stability of PfATCase, indicating it binds and stabilizes the enzyme in vitro.…”

Molecular Target Validation of Aspartate Transcarbamoylase from Plasmodium falciparum by Torin 2

“…The kinetic properties of PfATC and their mutants were investigated as previously reported using the malachite green method and Ceriotti's colorimetric method. 18,19 Pull-Down Assay. Equal volumes of the lysate containing soluble recombinant His-tagged mutant ATC and the lysate containing Strep-tagged WT-ATC were mixed and incubated on ice for 2 h. The mix of lysates was further separated into two fractions, H (His-tagged PfATC-R109A/K138A) and S (Strep-tagged WT-PfATC).…”

“…The structure of PfATC has already been characterized by us in both the apo T-state 18 and the ligand-bound R-state. 19 Furthermore, on the basis of mutagenic studies of EcATC summarized by Lipscom and Kantrowitz 20 and structural information obtained by us, a double mutant of PfATC with significantly reduced catalytic activity was designed, and the in vitro activities of the wild-type (WT) enzyme and this double mutant (PfATC-R109A/K138A) were previously reported. 18 In this article, the crystal structure of the double mutant PfATC-R109A/K138A (hereafter called RK) is reported.…”