Molecular rationale delineating the role of lycopene as a potent HMG-CoA reductase inhibitor: in vitro and in silico study

Abstract This study initially aimed to depict the molecular rationale evolving the role of lycopene in inhibiting the enzymatic activity of β-hydroxy-β-methylglutaryl-CoA (HMG-CoA) reductase via in vitro and in silico analysis. Our results illustrated that lycopene exhibited strong HMG-CoA reductase inhibitory activity (IC50 value of 36 ng/ml) quite better than pravastatin (IC50 = 42 ng/ml) and strong DPPH free radical scavenging activity (IC50 value = 4.57 ± 0.23 μg/ml) as compared to ascorbic acid (IC50 value = 9.82 ± 0.42 μg/ml). Moreover, the Ki value of lycopene (36 ng/ml) depicted via Dixon plot was well concurred with an IC50 value of 36 ± 1.8 ng/ml. Moreover, molecular informatics study showed that lycopene exhibited binding energy of −5.62 kcal/mol indicating high affinity for HMG-CoA reductase than HMG-CoA (ΔG: −5.34 kcal/mol). Thus, in silico data clearly demonstrate and support the in vitro results that lycopene competitively inhibit HMG-CoA reductase activity by binding at the hydrophobic portion of HMG-CoA reductase.


Introduction
The prevalence of hyperlipidemia, a disorder of lipid metabolism and one of the major risk factors responsible for cardiovascular diseases (CvD), is currently increasing at a striking rate throughout the world (WHO 2015). Therefore, regulating the lipid metabolism and decreasing the higher levels of serum total cholesterol, triglycerides and lDl cholesterol are considered to be quite advantageous for the therapeutic approaches of CvD (Derosa et al. 2006). Cholesterol ameliorating drugs, commonly statins (pravastatin simvastatin, fluvastatin and lovastatin) work specifically by inhibiting the activity of HMG-CoA reductase, rate limiting enzyme in the cholesterol biosynthetic pathway (Carbonell & Freire 2005). Nonetheless, these oral medications have certain limitation and side effects (Golomb & evan 2008). Therefore, investigation for other possible natural therapeutic agents, which are non-toxic, free radical quencher and are capable to inhibit HMG-CoA reductase activity, could be an important therapeutic approach in treatment and management of hypercholesterolaemia. lycopene, a deep red colour pigment, is the most abundant carotenoid present in human plasma and synthesised by plants and microorganisms. The available literature illustrated various possible mechanisms supporting the inhibitory effect of lycopene on pathogenesis and the progression of CvD (Fuhrman et al. 1997;Silaste et al. 2007), but none of them neither specifically target HMG-CoA reductase enzyme nor illustrate their mode of inhibition at molecule level. Thus, the present manuscript specifically aims to depict the molecular rationale evolving the role of lycopene via in vitro and in silico analysis as well as permediated to justify the consensus between in vitro and in silico work. In addition, the study also provides an ease to use the tool for screening of plant products as a hypolipidemic agent before moving further for preclinical and clinical study.

Results and discussion
The inhibitory effect of lycopene on pathogenesis and the progression of CvD has been illustrated previously (Fuhrman et al. 1997;Navarro-Gonza'lez et al. 2014), but their specific mode of inhibition against HMG-CoA reductase enzyme and its consensus with in silico study is still need to be elucidated. In order to validate the bioactivity of lycopene used in this study, DPPH radical scavenging assay was measured. Our result showed that the lycopene exhibited strong DPPH free radical scavenging activity (IC 50 value = 4.57 ± 0.23 μg/ml) as compared to standard, ascorbic acid (IC 50 value = 9.82 ± 0.42 μg/ml) (Figure S.1), which is in consensus with previously published report (Kaur et al. 2012;Tommonaro et al. 2014), indicating this lycopene is suitable for further studies. HMG-CoA reductase inhibitory activity of lycopene was done following a previously standardised protocol Iqbal, Khan, Khan, Khan, et al. 2014b). The results demonstrated that the lycopene exhibited potent HMG-CoA reductase inhibitory activity with an IC 50 value of 36 ± 1.8 ng/ ml which is quite better than pravastatin (IC 50 = 42 ng/ml) ( Figure S.2A). This is the first time we are demonstrating the in vitro inhibition of HMG-CoA reductase directly by lycopene.
The spectrophotometric time scans demonstrated the ability of pravastatin (Figure S.2B) and lycopene (Figure S.2C) at different concentrations (10-200 ng/ml) to increasingly mimic the inhibitory activity which may suggest direct interaction of lycopene with this enzyme. Furthermore, this compound demonstrated a competitive mode of inhibition against HMG-CoA reductase, which revealed that lycopene inhibits HMG-CoA reductase activity by binding only at the hydrophobic portion of HMG-CoA reductase. While, HMG-CoA and statin bind at hydrophobic as well as hydrophilic portion of catalytic site of HMG-CoA reductase ( Figure  S.3A). Therefore, lycopene may exert steric hindrance between HMG-CoA and HMG-CoA reductase through hydrophobic interactions. By occupying the active site, the inhibitor prevents normal substrates from binding and being catalysed. From the lineweaver-Burk plot analysis, it has been observed that k m value was increased as the concentration of inhibitor was increased, whereas there is no significant effect on V max value, which in turn indicates and justifies the competitive mode of enzyme inhibition. Moreover, the K i value of lycopene, depicted via Dixon plot was 36 ng/ml, which was well concurred with an IC 50 value of 36 ± 1.8 ng/ml observed in Figure S.2A (Figure S.3B).
To further corroborate our in vitro findings, molecular simulation studies were carried out and lycopene showed binding energy of −5.62 kcal/mol, indicating high affinity for the binding site (Table 1), though HMG-CoA has lower affinity with the ligand (ΔG: −5.34 kcal/ mol). Our results illustrate that lycopene fitted tightly with hydrophobic interaction into the conformation of HMG-CoA reductase active site with higher affinity than HMG-CoA. All of the stabilising interactions for lycopene were seen to be van der Waals and hydrophobic in nature.
An important conclusion emerging after the visualisation of the docked complexes of lycopene with HMG-CoA reductase was the similarity in the binding pocket used by these compounds as well as the standard drugs against HMG-CoA reductase. The docked structures of lycopene-HMG-CoA reductase and pravastatin-HMG-CoA reductase were found to be surrounded by Ser865, lys722, Ala564, Arg568, val720 and Arg571 amino acid residues, whereas docked structures of lycopene-HMG-CoA reductase and HMG-CoA-HMG-CoA reductase were found to be surrounded by lys722, Arg568, Gly807 and Glu559 which play important role in stabilising the complex ( Figure S.4A, B and C). Superimpose image shows that lycopene was competitively anchored at the catalytic centre ( Figure S.4D), which might explain why the enzymatic activity of HMG-CoA reductase was successfully blocked. Thus, in Table 1. Molecular interaction studies of lycopene, hMG-coa and pravastatin with β-hydroxy-β-methylglutaryl-coa reductase. silico data clearly demonstrate and support the in vitro results that lycopene inhibits HMG-CoA reductase activity by binding only at the hydrophobic portion of HMG-CoA reductase, whereas HMG-CoA binds both at hydrophilic and hydrophobic residues. Based on our in vitro and in silico results, it has been concluded that lycopene competitively ameliorate the enzymatic activity of HMG-CoA reductase, which revealed that lycopene-HMG-CoA reductase and HMG-CoA-HMG-CoA reductase were found to be surrounded by same hydrophobic residues which play important role in stabilising the complex.

Disclosure statement
No potential conflict of interest was reported by the authors.