In vitro and in silico studies of GST-inhibitory and larvicidal activities of alkaloids against Rhipicephalus microplus

The resistance of Rhipicephalus microplus to the main commercial acaricides has stimulated research for new antiparasitic agents, including natural products as an alternative to eco-friendly control. Among the main classes of secondary metabolites of plants stand out the alkaloids, which are characterized by a variety of biological activities such as enzymatic inhibition. The enzyme Glutathione-S-transferase (GST) is an important catalyst of xenobiotic detox reactions in arthropods and represents a promising target in the acaricide development. This work aimed to associate in silico and in vitro assays for the identification of alkaloids with inhibitory action against GST from R. microplus (RmGST). The model of RmGST was designed using the SWISS-MODEL online server and validated by molecular dynamics. The molecular affinity of alkaloids with RmGST was assessed using the DOCK 6.9 program and the best molecules were employed in in vitro assays. The in vitro assays performed were enzymatic inhibition and larval immersion tests. Molecular dynamics was run with GROMACS for alkaloid selected from in silico and in vitro analyses. The RmGST model shown to be sterically and energetically acceptable. In molecular dynamics simulations, the 3D structure remains stable with Root Mean Square Deviation = 4.35 Å and Root Mean Square Fluctuation = 2.21 Å. All the 20 alkaloids tested in molecular docking analyses show molecular affinity to the RmGST. Colchicine and papaverine show the best GridScocre: −38.09 and −36.09 kcal/mol, respectively. In in vitro enzymatic evaluation, colchicine exhibited more activity (81.73 %, 0.5 mg/mL) in comparison to papaverine (27.8 %, 0.5 mg/mL). However, these alkaloids did not interfere in vitro with the viability of R. microplus larvae. The colchicine contributed to the stability of the RmGST model in molecular dynamics simulations and presented hydrophobic and hydrogen interactions with residues from the orthosteric site. The association of in silico and in vitro techniques allowed the identification of two potential RmGST inhibitors (colchinine and papaverine) capable of interacting in the active site of the enzyme. RmGST inhibitors can be used in association with acaricides for a more effective control of R. microplus reducing parasite resistance.