Flavio Nguyen posted an update 3 months ago
More recently, Zhou and co-workers proposed that PtpB promotes mycobacterial survival in vitro by inhibiting extracellular signal-regulated kinase 1/2 and p38 pathways and increasing the phosphorylation of Akt, resulting in reduced production of interleukin-6 and decreased apoptotic activity, respectively. Alber and coworkers have synthesized a strong, competitive and selective PtpB inhibitor, namely OMTS showing an IC50 of 0.44 μM, and solved the three-dimensional structure of the PtpB-inhibitor complex by means of X-ray crystallography. Other groups also have successfully identified inhibitors of PtpB: indole derivatives with selectivity indexes up to 100, cyclic hexapeptides from cyanobacterium Tychonema sp. with IC50 around 8.0 μM, an isoxazole with Ki value of 0.22 μM, selective indolin-2- on-3-spirothiazolidinones with IC50 values of 35.5 to 1.2 μM and, recently, benzofurans with sub-micromolar inhibitory activity. Based on these evidences, PtpB has emerged as an important target for anti-TB pharmacological intervention and new inhibitors are in high demand. The screening of natural compounds libraries is a consolidated strategy in drug discovery, which employs the criteria of biological prevalidation and relevance to nature. Natural products have long been recognized as an important source of therapeutically effective agents, also because they embody rigid, non-flat three dimensional structures which may positively influence the probability of clinical success of a drug. Indeed, natural products can offer unprecedented opportunities for finding novel hits or leads against a wide range of biological targets. In previous works, we were pioneers in testing libraries of chalcones to find PtpA and PtpB inhibitors, underlining that screening natural products libraries may fuel the discovery of bioactive molecules. Moreover, in silico screening is a widely appreciated and reliable tool for prioritizing small molecules for biological testing. Accordingly, in this work we screened in silico an in house library of natural compounds by means of a structure-based approach composed of molecular docking, rescoring and visual inspection to prioritize few natural compounds as possible PtpB inhibitors that were Wortmannin 19545-26-7 subsequently assayed in vitro. Results of inhibition studies, kinetic measurements and mass spectrometry assays allowed elucidate their mechanism of action. Negative controls were performed in the absence of enzyme or compounds, and positive controls in the presence of enzyme and 4% DMSO. The fraction of residual activity was calculated as the difference in absorbance observed at 2 and 7 minutes of enzyme reaction, obtained by the average of two experiments carried out in triplicate. The IC50 values were determined with increasing concentrations of inhibitor versus percentage of residual activity, which was calculated as the difference between the observed absorbance at 2 and 7 min of enzyme reaction, obtained by the average of three independent experiments carried out in triplicate. The experimental data were analyzed with GraphPad Prism 5.0 and the IC50 values determined by linear regression. It is important to stress the fact that all compounds are soluble in the assay mixtures at the described experimental conditions. The ability of some docking programs to reproduce the X-ray determined binding conformation of OMTS within the active site of PtpB was preliminary checked. The ligand OMTS was self-docked into the binding site of PtpB whose coordinates were retrieved from the X-ray structure and the docked conformation corresponding to the lowest free energy provided by each program was selected as the most probable binding pose. While most programs and functions failed to dock correctly OMTS, the GoldScore function implemented in GOLD provided satisfactory results. In particular, the best agreement between computational and X-ray structural data was obtained by self-docking OMTS toward the receptor structure which was previously energy minimized with Amber11, and including conserved water molecules. The position and number of water molecules retained within the PtpB catalytic site was established by a GRID analysis performed using the OH2 probe atom. The parameter set used for self-docking OMTS was further used to dock the in house unique library within the catalytic site of PtpB. After docking and visual inspection, top 10% ranking compounds were selected for rescoring. It is widely appreciated in computer-aided drug design that rescoring docking poses with a scoring function different to that used in generating docking poses could better describe the ligand binding energy toward a receptor. In previous studies we evaluated the capability of the Molecular Mechanics Generalized Born Surface Area method in rescoring docking poses generated with GOLD. Similar results were discussed also by other research groups.