A mechanism-based study on inhibitory effect of nicotine and cotinine on human lactate dehydrogenase C

Abstract

Lactate dehydrogenase C (LDHC) as the most abundant isoform of lactate dehydrogenase in testis and spermatozoa, has a critical role in energy metabolism of germ cells. Also, LDHC gene is expressed aberrantly with a high prevalence in various kinds of malignancies. So, LDHC is a promising target protein for development of contraceptive and anti-cancer drugs. In the current study, inhibitory effects of nicotine and cotinine on human LDHC were investigated. Partially purified LDHC was extracted from healthy human spermatozoa and treated with various concentrations of nicotine and cotinine at a range of 0.13-0.66 μM. The results showed that at a concentration of 0.6 μM, cotinine and nicotine could inhibit LDHC activity by 47% and 45%, respectively. Mechanisms of these inhibitory effects were further investigated by docking and molecular dynamic (MD) simulation studies. Nicotine and cotinine were docked against available crystal structure of mice LDHC (PDB ID: 2LDX). LDHC alone and in complex with docked ligands was minimized, solvated in explicit water cage, neutralized by addition of required ions, and finally simulated for 50 ns of MD simulation. Analysis of simulated structures revealed that nicotine and cotinine could affect LDHC structure near His192 which play a key role in the interaction of LDHC with pyruvate/lactate. Thus, it seems that nicotine and cotinine could induce their effects via an inhibitory competition mechanism. Enzymatic assays with various concentration of pyruvate in the presence of mean plasma concentration of nicotine and cotinine in active smokers confirmed MD simulation results.