Combinatorial Chemistry & High Throughput Screening - Volume 19, Issue 1, 2016

Protein-protein interactions (PPI) play a critical role in regulating many cellular processes. Finding novel PPI inhibitors that interfere with specific binding of two proteins is considered a great challenge, mainly due to the complexity involved in characterizing multi-molecular systems and limited understanding of the physical principles governing PPIs. Here we show that the combination of virtual screening techniques, which are capable of filtering a large library of potential small molecule inhibitors, and a unique secondary screening by isothermal titration calorimetry, a label-free method capable of observing direct interactions, is an efficient tool for finding such an inhibitor. In this study we applied this strategy in a search for a small molecule capable of interfering with the interaction of the tumor-suppressor p53 and the E3-ligase MDM2. We virtually screened a library of 15 million small molecules that were filtered to a final set of 80 virtual hits. Our in vitro experimental assay, designed to validate the activity of mixtures of compounds by isothermal titration calorimetry, was used to identify an active molecule against MDM2. At the end of the process the small molecule (4S,7R)-4-(4-chlorophenyl)-5-hydroxy-2,7-dimethyl-N-(6-methylpyridin-2-yl)-4,6,7,8 tetrahydrIoquinoline-3-carboxamide was found to bind MDM2 with a dissociation constant of ~2 μM. Following the identification of this single bioactive compound, spectroscopic measurements were used to further characterize the interaction of the small molecule with the target protein. 2D NMR spectroscopy was used to map the binding region of the small molecule, and fluorescence polarization measurement confirmed that it indeed competes with p53.

Tuberculosis (TB) is known to mankind as one of the most pervasive and persistent of diseases since the early days of civilization. The growing resistance of the causative pathogen Mycobacterium tuberculosis to the standard drug regimen for TB poses further difficulty in its treatment and control. Screening of novel plant-derived compounds with promising anti-tubercular activity has been cited as a prospective route for new anti-tubercular drug discovery and design. Justicia adhatoda L. is a perennial evergreen shrub which is widely mentioned in scientific literature on account of its potent anti-mycobacterial properties. In the present study, we have employed a series of computational methodologies to reveal the probable molecular interactions of vasicine, the principal alkaloid of Justicia adhatoda L., and two of its close natural derivatives- vasicinone and deoxyvasicine, with certain biological targets in M. tuberculosis. Targets were identified from literature and through a reverse Pharmacophore-based approach. Subsequent comparative molecular docking to identify the best ligand-target interactions revealed Antigen 85C of M. tuberculosis as the most potent biological target of vasicine on the basis of optimum molecular docking values. A chemogenomics approach was also employed to validate the molecular interactions between the same class of chemical compounds as vasicine and Antigen 85C. Further, a library of structural analogs of vasicine was created by bioiosterism-based drug design to identify structural analogs with better inhibitory potential against Antigen 85C.

The chemical space is so vast that only a small portion of it has been examined. As a complementary approach to systematically probe the chemical space, virtual combinatorial library design has extended enormous impacts on generating novel and diverse structures for drug discovery. Despite the favorable contributions, high attrition rates in drug development that mainly resulted from lack of efficacy and side effects make it increasingly challenging to discover good chemical starting points. In most cases, focused libraries, which are restricted to particular regions of the chemical space, are deftly exploited to maximize hit rate and improve efficiency at the beginning of the drug discovery and drug development pipeline. This paper presented a valid methodology for fast target-focused combinatorial library design in both reaction-based and production-based ways with the library creating rates of approximately 70,000 molecules per second. Simple, quick and convenient operating procedures are the specific features of the method. SHAFTS, a hybrid 3D similarity calculation software, was embedded to help refine the size of the libraries and improve hit rates. Two target-focused (p38-focused and COX2-focused) libraries were constructed efficiently in this study. This rapid library enumeration method is portable and applicable to any other targets for good chemical starting points identification collaborated with either structure-based or ligand-based virtual screening.

Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) is an important regulator of focal adhesion, actomyosin contraction and cell motility. In this manuscript, a combination of the multi-complex-based pharmacophore (MCBP), molecular dynamics simulation and a hybrid protocol of a virtual screening method, comprised of multipharmacophore- based virtual screening (PBVS) and ensemble docking-based virtual screening (DBVS) methods were used for retrieving novel ROCK1 inhibitors from the natural products database embedded in the ZINC database. Ten hit compounds were selected from the hit compounds, and five compounds were tested experimentally. Thus, these results may provide valuable information for further discovery of more novel ROCK1 inhibitors.

As a part of our ongoing project on the design and synthesis of new thiazole derivatives with antimicrobial activity, fourteen new ethyl 2-(2-((E)-((Z)-5-(4-benzyliden)- 4-oxothiazolidin-2-yliden)amino-4-yl)acetates, carrying halogens, methoxy and other groups were synthesized. Compounds were tested against eight Gram positive and negative bacteria as well as eight yeasts and mold by microdilution assay. All compounds showed good activity against all bacteria tested with MIC ranging between 2.3-39.8 µmol/ml x 10-2 and MBC of 9.2-79.6 µmol/ml x 10-2. As reference drugs Ampicillin (MIC 24.8-74.4 and MBC 37.2-124.0 µmol/ml x 10-2) and Streptomycin (MIC 4,3-17.2 and MBC 8.6-51.6 µmol/ml x 10-2) were used. The best activity was observed for 4-bromo derivative. All tested compounds showed excellent antifungal activity against all fungi tested with MIC in the range between 0.3-38.6 µmol/ml x 10-2 and MFC range of 0.6-77.2 µmol/ml x 10-2, better than that of reference drugs, Ketoconazole (MIC 38.0-475.0 and MFC 95.0-570 µmol/ml x 10-2) and Bifonazole (MIC 48.0-64.0 and MFC 64.0-80.0 µmol/ml x 10-2). The best activity was observed for 3-nitro derivative. It was found that among the 5-arylidene derivatives the inhibitory effect appears to be dependent on the substitution at the benzene ring. Fourteen new ethyl 2-(2-((E)-((Z)-5-(4-benzyliden)-4-oxothiazolidin-2-yliden)amino-4-yl)acetates were synthesized and evaluated for antibacterial and antifungal activity.

Antioxidant (AO) capacity of various medicinal plants extracts and phenolic compounds was assessed by the most widely used spectrophotometric assays such as ferric reducing antioxidant power (FRAP) and scavenging of 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH). In addition, two direct current (DC) polarographic assays, one based on a decrease of anodic current of [Hg(O2H)(OH)] - HydroxoPerhydroxoMercury(II) Complex (HPMC) formation in alkaline solution of H2O2, at the potential of mercury dissolution and another recently developed Mercury Reduction Antioxidant Power (MRAP), based on a a decrease of cathodic current of Hg(II) reduction were employed. Percentage of both currents decrease was plotted versus the volume of gradually added complex samples or the amount of individual ones and the slopes of these plots were used to express AO capacity. Total phenolic content (TPC) of extracts was determined by Folin- Ciocalteu (FC) assay. Correlations between applied assays were calculated by regression analysis. Relative Antioxidant Capacity Index (RACI), calculated by assigning equal weight to all applied assays and Phenolic Antioxidant Coefficients (PAC), calculated as a ratio between particular AO capacity and TPC, were used to achieve more comprehensive comparison between analyzed samples, as well as applied assays.

Centaurea solstitialis L. ssp. solstitialis (CSS) has been used as medicine for various diseases. In this work, root, stem and flower parts of the plant were separately extracted with methanol to execute bioassay-guided isolation. Antiproliferative activities of each extracts on C6 cells (Rat Brain tumor cells) and HeLa cells (human uterus carcinoma) were investigated in vitro. The methanol extract of stem exhibited the most antiproliferative activity therefore isolation of active compounds was carried out for stem of the plant. Methanol extract of stem was boiled at 97 °C for 2 hours in water and then hexane and ethyl acetate were extracted sequentially. Solstitialin A 1 and 15-dechloro-15-hydroxychlorojanerin 2 were isolated from ethyl acetate extract by column chromatography and identified by spectroscopic techniques. Solstitialin A 1 was isolated from CSS and 15-dechloro-15-hydroxychlorojanerin 2 was isolated from Saussurea lipschitz and Rhaponticum pulchrum previously. These two compounds exhibited very high antiproliferative activity on C6 and HeLa cells. IC50 and IC75 values of compound 1 were obtained as 10.78 and 53.65 against C6 cell and as 48.78 and 68.52 against HeLa, respectively. IC50 and IC75 values of compound 2 were determined as 432.43 and 109.79 against C6 cell.

MERTK plays an important role in cell biology and is correlated with many cancers, such as mantle cell lymphomas, pituitary adenomas, and T-cell acute lympholoblastic leukemia. So identification of new MERTK inhibitors is of extreme importance. In this study, 107 MERTK inhibitors with known activities were gathered to generate a ligand-based pharmacophore model (ADDHH.4), followed by building a 3D-QSAR model, which had high value of coefficient of determination (R2=0.9061) and high value of coefficient of determination (Q2=0.7442). For the pharmacophore model, two hydrogen bond donors (D), one hydrogen bond receptor (A), and two hydrophobic groups (H) were considered as the key elements contributing to ligand activity. The model then served to search a drug-like database with 1.5 million molecules, and 47832 hits were obtained. Subsequently, docking procedure was applied on these hits, and 840 compounds were obtained through high-throughput virtual screening (HTVS). Standard precision (SP), extra precision (XP) and rule of five were also used in virtual screening protocol. Finally, six candidates were identified as potential MERTK inhibitors, with the docking mode in MERTK analyzed.