MOLECULAR DOCKING, LIGAND QUALITY AND ANTIPLASMODIAL EVALUATION OF BENZAMIDE, COUMARIN AND BENZODIAZEPINE ANALOGS

Abstract

Malaria chemotherapy is an essential strategy for malaria elimination but resistance has challenged existing antimalarials, including frontline artemisinin combination therapy (ACT); hence, new antimalarial drugs must be discovered and developed. This study investigated the antiplasmodial efficacy and cytotoxicity through in vitro models while also testing the antiplasmodial efficacy, and the in vivo acute toxicity of benzamide, coumarin and benzodiazepine analogss. This study also evaluated the ligand quality of the molecules and their possible Plasmodium falciparum protein targets. Three molecules, 4- amino-N-hydroxybenzamide (AHB), ethyl 2-oxo-2H-chromene-3-carboxylate (ECC), and 2,2,4-trimethyl-2-3-dihyro-1H-benzo[b][1,4] diazepine (BDZ) were screened for their in vitro antiplasmodial activities tested against P. falciparum 3D7 standard strain using the SYBR Green Dye I measuring IC50 and their cytotoxicities against MCF-7 breast cancer cells using the [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay. Their antiplasmodial efficacies were determined using Peter’s 4-day suppressive test against Plasmodium berghei in M. musculus while acute toxicities were investigated in the Mus musculus (mice). Ligand qualities were determined using ligand efficiency metrics, and molecular docking was conducted to determine the ligand interactions between ECC and the following enzymatic proteins, P. falciparum dihydroorotate dehydrogenase (PfDHOH) and P. falciparum purine nucleoside phosphorylase (PfPNP); and the molecular interaction between BDZ and PfDXR - Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase, P. falciparum falcipain-2 and P. falciparum plasmepsin X (PfPMX). AHB showed no cytotoxicity against MCF-7 at (CC50) = 277.7 μM, while ECC showed inhibition with CC50= 3.930 μM, and BDZ showed no cytotoxicity CC50= 7373 μM. The in vitro antiplasmodial activity showed potency at (AHB)IC50 = 0.0020 ± 0.008 μM, (ECC) IC50= 0.0010 ± 0.002 μM, (BDZ) IC50= 0.0036 ± 0.003 μM respectively. BDZ showed the highest selectivity index at > 200,000, suggesting that it exhibited the best safety/efficacy among the three compounds. AHB displayed LD50 = >5000 mg/kg while ECC and BDZ displayed LD50 = 3162.28 mg/kg. Histopathological examinations showed non-toxicity by the three analogs on the liver and kidney of M. musculus. The percentage suppression of AHB (80.53 ± 3.26 %) at 400 mg/kg, was comparable to the standard chloroquine (81.71 ± 1.82 %) at 100 mg/kg where the mean survival time for both exceeded 30 days. ECC and BDZ showed excellent efficacies (70.98 ± 20.89 % and 83.66 ± 11.67 %) at 200 mg/kg, comparable to chloroquine 80.97 ± 5.82 %. The chemosuppression values for AHB and BDZ were significant at P value < 0.05. The ligand quality of ECC and BDZ displayed good Ligand Efficiency compared to chloroquine and artemisinin and higher enzyme affinities, and ligand efficiency dependent lipophilicity than the standard drugs. ECC and BDZ displayed good characteristics. The docking studies displayed strong hydrophobic interactions between ECC, PfDHODH, and PfPNP, suggesting good potency. BDZ’s binding with PfDXR, Pffalcipain-2, and PfPMX also displayed potency derived from hydrophobic and hydrogen interactions. Conclusively, this study showed AHB, ECC and BDZ were non-toxic to mammalian cells rodents’ liver and kidneys. These molecules exhibited good antiplasmodial inhibitory potential against both P. falciparum in vitro and P. berghei in vivo. ECC and BDZ displayed high ligand efficiency and strong molecular interactions with their protein targets. Therefore, all three analogs can be moved for further optimization in drug development.