Identification of New Hsp90 Inhibitors: Structure Based Virtual Screening, Molecular Dynamic Simulation, Synthesis and Biological Evaluation.

BACKGROUND: Heat shock protein90 (Hsp90) is overexpressed in tumor cells, thus the inhibition of the Hsp90 ATPase activity would be a meaningfully effective strategy in cancer therapy. OBJECTIVE: The present work was aimed at four steps: designing new Hsp90 inhibitors as anti-cancer by a virtual screening study; synthesize designed compounds; biological evaluation of them and finally molecular dynamic (MD) simulations of best compounds. METHODS: A virtual screening study was performed on a library (100 compounds) of the ZINC database with benzimidazole scaffold; then an extracted compound and two derivatives were synthesized. The anti-proliferative and ATPase inhibitory activities of these compounds were evaluated by MTT and ATPase inhibition assays, respectively. The western blot analysis was performed to the evaluation of the expression level of Hsp70 and Her2 proteins. Finally, 200 ns molecular dynamic simulation was carried out to confirm the stability of the strongest synthesized compound in Hsp90 active site. RESULTS: ZINC00173501 compound with an aminobenzimidazole scaffold was chosen by the virtual screening study. ZINC00173501 compound and two of its derivatives were synthesized. ATPase inhibitory activity of three synthesized compounds shown that ZINC00173501 compound was the most potent inhibitor (IC50= 8.6 µM) with the anti-proliferative activity 14.41 µM, 19.07 µM and more than 100 µM against MCF-7, HeLa and HUVEC cell lines, respectively. The high level of Hsp70 expression and low level of Her2 expression confirmed ZINC00173501 as an Hsp90 inhibitor. Finally, molecular dynamics simulation showed that ZINC00173501 was stable in Hsp90 active site during 200 ns simulation. CONCLUSION: The biological evaluation results show that 2-aminobenzimidazole scaffold could be suggested as a lead for inhibition of Hsp90.

Cycloarta-23-ene-3beta,25-diol a pentacyclic steroid from Euphorbia spinidens, as COX inhibitor with molecular docking, and in vivo study of its analgesic and anti-inflammatory activities in male swiss mice and wistar rats.

BACKGROUND AND AIMS: Euphorbia is a large genus of flowering plants. In Iran, some plants of this family have been used in the treatment of inflammatory disorders and also to relieve back pain. Euphorbia spinidens is a rich source of Cycloarta-23-ene-3beta,25-diol. Cycloartane structures are the starting material for the synthesis of plant steroids, and the aim of this study is to demonstrate COX inhibitory activity, molecular docking and in vivo approach of anti-inflammatory activity of cycloartane compound isolated from Euphorbia spinidens. MATERIAL AND METHODS: Plant material was extracted with acetone-chloroform and submitted to column chromatography for fractionation. Based on preliminary 1H-NMR spectra, cycloartane fraction was selected and purified by repeated recycle HPLC system. The structure and purity of compound were determined by 1H and 13C-NMR, HPTLC, and mass spectra. Inhibitory activities of the tested compounds on COX-1 and COX-2 were evaluated by a colorimetric COX (ovine) inhibitor screening method. Vero cells were used to assess the toxicity against the normal cells, and calculate the selectivity index. COX inhibitory activity results were evaluated and confirmed by molecular docking experiments. In the in vivo approach, analgesic activity was assessed by acetic acid-induced abdominal writhing and formalin tests. Croton oil-induced ear edema in mice and carrageenan-induced rat paw edema in rats were used to evaluate anti-inflammatory activity. Pain tests were carried out on male Swiss mice (25-35 g). Male Wistar rats (160-200 g) were used for the carrageenan test. RESULTS: Cycloart-23-ene-3ß,25-diol showedin vitro cyclooxygenase 1 and 2 inhibitory activities with more selectivity for COX-2. Molecular docking by predicting binding energies in COX protein receptors confirmed in vitro COX inhibitory results, and determined the best position for ligand in COX receptors along with its residue interactions in receptor pockets, which must be considered for designing of their inhibitors. In the in vivo studies, cycloartane inhibited significantly acetic acid-induced abdominal contractions and formalin-induced licking behavior at a dose of 200 mg/kg. The same dose reduced croton oil ear edema in mice and carrageenan-induced paw edema in rats. CONCLUSION: Therefore, according to these findings, cycloart-23-ene-3beta,25-diol showed promising analgesic and anti-inflammatory effects with low toxicity against normal cells and can be suggested as a template lead for designing anti-inflammatory compounds with good selectivity index, and potency for COX-2 inhibitory activity.

Identification of Essential 2D and 3D Chemical Features for Discovery of the Novel Tubulin Polymerization Inhibitors.

BACKGROUND: Tubulin polymerization inhibitors interfere with microtubule assembly and their functions lead to mitotic arrest, therefore they are attractive target for design and development of novel anticancer compounds. OBJECTIVE: The proposed novel and effective structures following the use of three-dimensionalquantitative structure activity relationship (3D-QSAR) pharmacophore based virtual screening clearly demonstrate the high efficiency of this method in modern drug discovery. METHODS: Combined computational approach was applied to extract the essential 2D and 3D features requirements for higher activity as well as identify new anti-tubulin agents. RESULTS: The best quantitative pharmacophore model, Hypo1, exhibited good correlation of 0.943 (RMSD=1.019) and excellent predictive power in the training set compounds. Generated model AHHHR, was well mapped to colchicine site and three-dimensional spatial arrangement of their features were in good agreement with the vital interactions in the active site. Total prediction accuracy (0.92 for training set and 0.86 for test set), enrichment factor (4.2 for training set and 4.5 for test set) and the area under the ROC curve (0.86 for training set and 0.94 for the test set), the developed model using Extended Class FingerPrints of maximum diameter 4 (ECFP_4) was chosen as the best model. CONCLUSION: Developed computational platform provided a better understanding of requirement features for colchicine site inhibitors and we believe the results of this study might be useful for the rational design and optimization of new inhibitors.

Phytochemical Analysis and Cytotoxicity Evaluation of Kelussia odoratissima Mozaff.

BACKGROUND: Kelussia odoratissima Mozaff. (Apiaceae) is an edible, indigenous, and ethnomedicinal plant that grows only in Iran. Although antioxidant and anti-inflammatory properties of K. odoratissima have been reported, cytotoxic activity of this plant has not been investigated previously. OBJECTIVE: This study aims to evaluate the cytotoxicity of K. odoratissima leaf extract against a panel of human cancer cell lines. A secondary aim was to perform a phytochemical analysis of the plant's leaf oil. METHODS: To extract the plant oil, dried leaves were subjected to hydrodistillation using a Clevenger-type apparatus for up to 3 hours. For the phytochemical analysis, essential oil was subjected to gas chromatography/mass spectrometry. Plant extraction was performed by macerating leaf powder of K. odoratissima (50 g) in 70% methanol (500 mL) at room temperature (25-28°C) for 24 hours. To perform cytotoxicity assays, methanolic extract of K. odoratissima was tested against a panel of cell lines including MDA-MB468 (human breast cancer cell line), K562 (human leukemia cell line), SKOV3 (human ovarian cancer cell line), Y79 (human eye cancer cell line), A549 (lung cancer cell line), and HEK 293 (normal human embryonic kidney cell line). RESULTS: Gas chromatography/mass spectrometry analysis revealed that sesquiterpens are dominant volatile components of the plant, followed by phthalides comprising 3-butyldine phthalide and 3-n-butyl phthalide, the latter compound being the major component of the leaf oil (25.1%). The leaf extract showed selective and dose-dependent cytotoxicity against MDA-MB468, K562, SKOV3, Y79, and A549 cancer cell lines with IC50 values (concentration that inhibits cell growth by 50%) of 85 µg/mL, 70 µg/mL, 120 µg/mL, 82 µg/mL, and145 µg/mL, respectively. CONCLUSIONS: The present results suggest a direct cytotoxic activity of K. odoratissima leaf extract against human cancer cell lines. This activity of K. odoratissima may find application in combination with traditional herbal medicines to develop a new anticancer pharmacopuncture therapy.