Structure-based screening and molecular dynamics simulations offer novel natural compounds as potential inhibitors of <i>Mycobacterium tuberculosis</i> isocitrate lyase

<p><i>Mycobacterium tuberculosis</i> is the etiological agent of tuberculosis in humans and is responsible for more than two million deaths annually. <i>M. tuberculosis</i> isocitrate lyase (MtbICL) catalyzes the first step in the glyoxylate cycle, plays a pivotal role in the persistence of <i>M. tuberculosis</i>, which acts as a potential target for an anti-tubercular drug. To identify the potential anti-tuberculosis compound, we conducted a structure-based virtual screening of natural compounds from the ZINC database (<i>n</i> = 1,67,748) against the MtbICL structure. The ligands were docked against MtbICL in three sequential docking modes that resulted in 340 ligands having better docking score. These compounds were evaluated for Lipinski and ADMET prediction, and 27 compounds were found to fit well with re-docking studies. After refinement by molecular docking and drug-likeness analyses, three potential inhibitors (ZINC1306071, ZINC2111081, and ZINC2134917) were identified. These three ligands and the reference compounds were further subjected to molecular dynamics simulation and binding energy analyses to compare the dynamic structure of protein after ligand binding and the stability of the MtbICL and bound complexes. The binding free energy analyses were calculated to validate and capture the intermolecular interactions. The results suggested that the three compounds had a negative binding energy with −96.462, −143.549, and −122.526 kJ mol⁻¹ for compounds with IDs ZINC1306071, ZINC2111081, and ZINC2134917, respectively. These lead compounds displayed substantial pharmacological and structural properties to be drug candidates. We concluded that ZINC2111081 has a great potential to inhibit MtbICL and would add to the drug discovery process against tuberculosis.</p

Molecular model of sll0067.

<p>The structure of a dimer is shown in this figure. The blue and green colors represent the two monomers. The red color shows motif I while the yellow color shows motif II. The 3D model was made using the Swiss model. The model was visualized with UCSF Chimera.</p

Studies on the effect of pH and temperature of the enzymatic activity of sll0067.

<p>A. Effect of pH on the catalytic activity of sll0067. B. Effect of temperature on the activity of sll0067.</p

Multiple amino acid sequence alignment.

<p>sll0067 (Q55139), TeGST (NP_680998), SeGST (YP_171005) and PmGSTB1-1 (P15214) are aligned using ESpript 3.0 software, which utilizes the Clustal W algorithm. Similar residues are shown in yellow boxes; the red boxes represent identical amino acid residues while the amino acid residues with different properties have no boxes. The arrow represents conserved Pro-53. The motif I (49–73) and motif II (130–147) are shown with arrows.</p

Titration of the intrinsic tryptophan fluorescence of sll0067.

<p>The binding of GSH to the protein was studied by quenching tryptophan fluorescence at various pH values. The GSH binds to the protein at physiological pH while at other pH values, it does not interact with the protein.</p

Overexpression of sll0067 in <i>E</i>. <i>coli</i> and purification of the recombinant protein on Ni-NTA agarose.

<p>A. SDS-PAGE analysis of cell lysate showing overexpression of sll0067 and the purified protein. Lanes 1–4 represent molecular weight markers, supernatant of un-induced culture lysate, supernatant of induced culture lysate, and purified protein, respectively. B. Molecular weight and subunit structure of sll0067. SEC profile of sll0067 on Superdex 200 10/300 GL column at pH 8.0 and 25°C. Inset shows the column calibration curve. The column was calibrated with the gel filtration calibration kit containing-conalbumin (75kDa), ovalbumin (44kDa), carbonic anhydrase (29kDa), ribonuclease A (13.7 kDa), and aprotinin (6.5 kDa).</p

Confirmation of the integrity of the sll0067 structure.

<p>A. Effects of the pH on the CD signal at 222 nm (■) and tryptophan fluorescence emission maxima (□) of sll0067. B. SEC profile of sll0067 on Superdex 200 10/300 GL column at pH 3 and 10 at 25°C. Solid lines represent the protein at pH 3 while the dashed lines represent the protein at pH 10. The curves have been displaced on Y-axis for presentation.</p

Steady state kinetic parameters.

<p>Enzymatic activities are measured at various concentrations of GSH and PITC as described in the Experimental section. Kinetic constants are based on three independent experiments for each measurement. Results are mean ± standard deviation (S.D.) of five independent measurements as described in the Experimental Section.</p><p>Steady state kinetic parameters.</p

Identification of novel natural inhibitors of <i>Opisthorchis felineus</i> cytochrome P450 using structure-based screening and molecular dynamic simulation

<p><i>Opisthorchis felineus</i> is the etiological agent of opisthorchiasis in humans. <i>O. felineus</i> cytochrome P450 (OfCYP450) is an important enzyme in the parasite xenobiotic metabolism. To identify the potential anti-opisthorchid compound, we conducted a structure-based virtual screening of natural compounds from the ZINC database (<i>n</i> = 1,65,869) against the OfCYP450. The ligands were screened against OfCYP450 in four sequential docking modes that resulted in 361 ligands having better docking score. These compounds were evaluated for Lipinski and ADMET prediction, and 10 compounds were found to fit well with re-docking studies. After refinement by docking and drug-likeness analyses, four potential inhibitors (ZINC2358298, ZINC8790946, ZINC70707116, and ZINC85878789) were identified. These ligands with reference compounds (itraconazole and fluconazole) were further subjected to molecular dynamics simulation (MDS) and binding energy analyses to compare the dynamic structure of protein after ligand binding and the stability of the OfCYP450 and bound complexes. The binding energy analyses were also calculated. The results suggested that the compounds had a negative binding energy with −259.41, −110.09, −188.25, −163.30, −202.10, and −158.79 kJ mol⁻¹ for itraconazole, fluconazole, and compounds with IDs ZINC2358298, ZINC8790946, ZINC70707116, and ZINC85878789, respectively. These lead compounds displayed significant pharmacological and structural properties to be drug candidates. On the basis of MDS results and binding energy analyses, we concluded that ZINC8790946, ZINC70707116, and ZINC85878789 have excellent potential to inhibit OfCYP450.</p