Antibiotic Effects of Loperamide:Homology of human targets of Loperamide with targets in Acanthamoeba spp

Background: Loperamide is an anti-diarrheal drug prescribed for non-infectious diarrhea. The drug is an opioid receptor agonist, a blocker of voltage-dependent calcium channel (Cav) and a calmodulin (CaM) inhibitor on human cells. Loperamide has been reported to exert anti-amoebic effects against pathogenic strains of Acanthamoeba castellanii. Objectives: The precise mode of antibiotic action, cellular target homology with human counterparts and the pattern of cell death induced by loperamide in Acanthamoeba castellanii remain to be established. Methods: Bioinformatics, 3D structural modelling, ligand binding predictions and apoptotic/amoebicidal assays were used in this study to answer the above queries. Amino acid sequences and structural models were compared between human and A. castellanii proteins that are involved in regulation of calcium homeostasis. Results: Our results show that A. castellanii expresses similar, to near identical types of primitive calcium channel CavAc and CaM that are well known targets of loperamide in humans. The growth assays showed anti-amoebic effects of loperamide at different doses, both alone and in combinations with other Ca+2- CaM inhibitors. The synergistic actions of loperamide with haloperidol showed to be more amoebicidal than when either of them used alone. Imaging with Annexin V, Acridine orange and Propidium iodide showed apoptosis in A. castellanii at a dose of 100μg/ml and necrosis at higher doses of 250μg/ml. Conclusion: Though, Acanthamoeba does not express a homolog of the human mu-opioid receptor, but does shows evidence of the homologs for other known human targets of Loperamide that are involved in Ca+2 uptake and Ca+2 signal transduction pathways. This suggests optimization of similar drug interactions with these targets may be useful in developing new approaches to control the growth of this parasite and possibly the diseases caused by it.