Exploring Leishmania donovani 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) as a potential drug target by biochemical, biophysical and inhibition studies

Abstract

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (HMGR), an NADPH dependant enzyme catalyzes the synthesis of mevalonic acid from HMG-CoA required for isoprenoid biosynthesis. The HMGR gene from Leishmania donovani was cloned and expressed. Genome analysis of L. donovani revealed that HMGR gene having an open reading frame of 1305 bp encodes a putative protein of 434 amino acids. LdHMGR showed optimal activity at pH 7.2 and temperature 37 °C. Kinetic analysis of this enzyme revealed Km values of 35.7 ± 2.5 μM for (R,S)-HMG-CoA and 70 ± 7.9 μM for the cofactor NADPH. On tryptophan fluorescence quenching, the Stern Volmer constant (Ksv), binding constant (Ka) and protein:cofactor stoichiometry for interaction of NADPH cofactor with the enzyme were found to be 6.0 ± 0.7 M(-1), 0.17 μM and 0.72 respectively. Polyclonal anti-rat HMGR antibody detected a band of ∼45 kDa in all phases of promastigote growth. Biophysical analysis of the secondary structure of LdHMGR confirmed the presence of 25.7 ± 0.35% alpha helicity. Thermal denaturation studies showed extreme stability of the enzyme with 60% helical structure retained at 90 °C. Statins (simvastatin and atorvastatin) and non-statin (resveratrol) effectively inhibited the growth of L. donovani promastigotes as well as the catalytic activity of the recombinant LdHMGR. Atorvastatin was found to be most potent antileishmanial inhibitor with an IC50 value of 19.4 ± 3.07 μM and a very lower concentration of 315.5 ± 2.1 nM was enough to cause 50% recombinant LdHMGR enzyme inhibition suggesting direct interaction with the rate limiting enzyme of the ergosterol biosynthetic pathway. Exogenous supplementation of ergosterol in case of atorvastatin and resveratrol treated cells caused complete reversal of growth inhibition whereas simvastatin was found to be ergosterol refractory. Cholesterol supplementation however, failed to overcome growth inhibition in all the cases. Overall our study emphasizes on exploring LdHMGR as a potential drug target for the development of novel antileishmanial agents.

Keywords: Cholesterol; Ergosterol; HMGR; Leishmania donovani; Statins.