In Vitro and in Silico Pharmacologic Evaluation of the Antiretroviral Activity of Croton Species

Abstract

Introduction The Human Immunodeficiency Virus (HIV) affects the body's defence mechanisms and leads to many opportunistic infections. Globally, more than half a million individuals lost their lives in 2020 due to this disease. Antiretroviral drugs have played a significant role in improving the quality of life of HIV-infected individuals. However, the side effects of these drugs, coupled with the virus's resistance to the various regimens, necessitate the search for potential new and effective antiretroviral medication. Objective The objective of this study was to evaluate the cytotoxicity and anti-HIV activity of crude and solvent fractions of Croton macrostachyus, Croton megalocarpus, and Croton dichogamus and to isolate pure compounds with potential antiretroviral activity. Methods Stem bark and leaves of C. macrostachyus, C. megalocarpus, and twigs of C. dichogamus are extracted using 1:1 v/v dichloromethane and methanol, followed by liquid-liquid partitioning using four different solvents. The crude and solvent fractions were tested for inhibition of laboratory-adapted HIV-1IIIB strain replication in the Human T-lymphocytic MT-4 cell line. In addition, using bioassay-guided fractionation, pure compounds with anti-HIV activity were isolated. Finally, the chemical structure of the new compounds was elucidated and mode of action predicted using in silico approach. Results The highest anti-HIV activity was observed from the ethyl acetate fractions of the bark of C. megalocarpus (IC50 = 0.47 ± 0.12 μg/mL; selectivity index =346.3), the hexane fraction of the leaves of C. macrostachyus (IC50 = 0.02 ±0.01μg/mL; SI of 9752.7) and the methanol fraction of C. dicohgamus (IC50 =0.06 + 0.01 μg/ml; SI of 318.5). In addition, nine compounds were isolated from the ethyl acetate fraction of the bark of C. megalocarpus (ermiasolide A, B and C, 4H-α,7H-α,10α-eudesm-11-en-5β-ol, ermiasoid, lupeol, 11-acetoxy crotocascarin L, pinoresinol, and crotocascarin K), five compounds from the hexane fraction of the leaves of C. macrostachyus (2-methoxy benzyl benzoate, betulin, lupenone, lupeol acetate, lupeol) and four compounds from methanol fraction of the twigs of C. dichogamus (dihydroconiferyl acetate, crotocascarin ω, (4-hydroxy-3-methoxyphenyl)-propyl benzoate, β-oplopanone). Among these compounds, six of them (ermiasolide A, ermiasolide B, ermiasolide C, ermiasoid, 11-acetoxy vi

crotocascarin L and crotocascarin ω) were previously undescribed new compounds with anti-HIV activity. The new compounds displayed the highest anti-HIV activity by inhibiting more than 70% of viral replication with an IC50 value of less than 0.04 μg/mL. The in vitro assay showed that the bioactive compounds are more active in inhibiting more than 60 % of HIV-1 protease enzyme activity. In addition, molecular docking study showed that these compounds had strong binding with the HIV-1 PR at free binding energy of less than -7.00 kcal/mol and form hydrophobic and hydrogen bonding with several amino acids in the active site of the HIV protease enzyme and inhibit the binding of viral polypeptide to the catalytic domain of the enzyme. Conclusion The isolated pure compounds have displayed potent inhibition of HIV-1 protease enzyme activity due to the strong binding of the compounds in the enzyme's active site and the formation of several hydrogen bonds with key residues.