Targeting the intersubunit cavity of Plasmodium falciparum glutathione reductase by a novel natural inhibitor: Computational and experimental evidence

• Compounds screened targeting homodimer cavity of Plasmodium falciparum glutathione reductase (PfGR).
• Two natural compounds (TTB and EPB) identified targeting PfGR.
• Long MD simulations for 30 ns delineated stability of TTB while CTC got knocked out of the cavity.
• A closely related structure of TTB, named MDPI 21618 resulted in good IC50 values against PfGR and hGR.
• TTB also resulted in inhibition of chloroquine-sensitive and chloroquine-resistant strains.

Glutathione reductase (GR), a homodimeric FAD-dependent disulfide reductase, is essential for redox homeostasis of the malaria parasite Plasmodium falciparum and has been proposed as an antimalarial drug target. In this study we performed a virtual screening against PfGR, using the structures of about 170,000 natural compounds. Analysis of the two top-scoring molecules, TTB and EPB, indicated that these ligands are likely to interact with the homodimer intersubunit cavity of PfGR with high binding energy scores of −9.67 and −9.60 kcal/mol, respectively. Both compounds had a lower affinity for human GR due to differences in structure and electrostatic properties. In order to assess the putative interactions in motion, molecular dynamics simulations were carried out for 30 ns, resulting in TTB being more dynamically and structurally favored than EPB. A closely related compound MDPI 21618 was tested on recombinant PfGR and hGR, resulting in IC50 values of 11.3 ± 2.5 μM and 10.2 ± 1.7 μM, respectively. Kinetic characterization of MDPI 21618 on PfGR revealed a mixed-type inhibition with respect to glutathione disulfide (Ki = 9.7 ± 2.3 μM) and an uncompetitive inhibition with respect to NADPH. Furthermore, MDPI 21618 was found to inhibit the growth of the chloroquine-sensitive P. falciparum strain 3D7 with an IC50 of 3.2 ± 1.9 μM and the chloroquine-resistant Dd2 strain with an IC50 of 3.2 + 1.6 μM. In drug combination assays with chloroquine, artemisinin, or mefloquine MDPI 21618 showed an antagonistic action, which might suggest partially overlapping routes of action. This study further substantiates research on PfGR as a potential antimalarial drug target.