Zabeer Ahmed

Design and synthesis of novel N,N′-glycoside derivatives of 3,3′-diindolylmethanes as potential antiproliferative agents

A library of 34 compounds containing the DIM core have been synthesized and tested for their anticancer efficacy by measuring their cytotoxicity to cancer cell lines A549, HeLa and MCF-7. Some of the selected derivatives were N-glycosylated to increase their efficacy. Compound 7d, an N-glycosylated DIM derivative, was found to be effective at 1.3, 0.3 and 0.9 μmol concentrations against A549, HeLa and MCF-7, respectively. Immunochemistry studies revealed that it could induce apoptosis by upregulating a pro-apoptotic protein Par-4 and concomitantly diminishing the expression of pro-survival proteins Bcl-2 and GRP78. Flow cytometry studies showed that the compound arrested cells in the G1 phase of the cell cycle and significantly abrogated the motility of HeLa cells. Computer docking simulations of 7d with GRP78 suggested its involvement in two H-bonds with Asp78, two H-bonds with Arg290, one with Arg367, and one water mediated H-bond interaction. The interaction patterns also demonstrated that the presence of bromide in the vicinity (within 3.5 A) of Lys294 generates the possibility of a halogen bond, which may also contribute in providing some extra stability to the complex. Hence, compounds of this class will be useful for the design of new anticancer agents.

Par-4 dependent modulation of cellular β-catenin by medicinal plant natural product derivative 3-azido Withaferin A

Here, we provide evidences that natural product derivative 3‐azido Withaferin A (3‐AWA) abrogated EMT and invasion by modulating β‐catenin localization and its transcriptional activity in the prostate as well as in breast cancer cells. This study, for the first time, reveals 3‐AWA treatment consistently sequestered nuclear β‐catenin and augmented its cytoplasmic pool as evidenced by reducing β‐catenin transcriptional activity in these cells. Moreover, 3‐AWA treatment triggered robust induction of pro‐apoptotic intracellular Par‐4, attenuated Akt activity and rescued Phospho‐GSK3β (by Akt) to promote β‐catenin destabilization. Further, our in vitro studies demonstrate that 3‐AWA treatment amplified E‐cadherin expression along with sharp downregulation of c‐Myc and cyclin D1 proteins. Strikingly, endogenous Par‐4 knock down by siRNA underscored 3‐AWA mediated inhibition of nuclear β‐catenin was Par‐4 dependent and suppression of Par‐4 activity, either by Bcl‐2 or by Ras transfection, restored the nuclear β‐catenin level suggesting Par‐4 mediated β‐catenin regulation was not promiscuous. In vivo results further demonstrated that 3‐AWA was effective inhibitor of tumor growth and immunohistochemical studies indicated that increased expression of total β‐catenin and decreased expression of phospho‐β‐catenin and Par‐4 in breast cancer tissues as compared to normal breast tissue suggesting Par‐4 and β‐catenin proteins are mutually regulated and inversely co‐related in normal as well as cancer condition. Thus, strategic regulation of intracellular Par‐4 by 3‐AWA in diverse cancers could be an effective tool to control cancer cell metastasis. Conclusively, this report puts forward a novel approach of controlling deregulated β‐catenin signaling by 3‐AWA induced Par‐4 protein.

The anti-angiogenic and cytotoxic effects of the boswellic acid analog BA145 are potentiated by autophagy inhibitors

BackgroundWhile angiogenesis inhibitors represent a viable cancer therapy, there is preclinical and clinical data to suggest that many tumors develop resistance to such treatments. Moreover, previous studies have revealed a complex association between autophagy and angiogenesis, and their collective influence on tumorigenesis. Autophagy has been implicated in cytoprotection and tumor promotion, and as such may represent an alternative way of targeting apoptosis-resistant cancer cells. This study explored the anti-cancer agent and boswellic acid analog BA145 as an inducer of autophagy and angiogenesis-mediated cytoprotection of tumor cells.MethodsFlow cytometry, western blotting, and confocal microscopy were used to investigate the role of BA145 mediated autophagy. ELISA, microvessel sprouting, capillary structure formation, aortic ring and wound healing assays were performed to determine the relationship between BA145 triggered autophagy and angiogenesis. Flow cytometery, western blotting, and microscopy were employed to examine the mechanism of BA145 induced cell death and apoptosis. Live imaging and tumor volume analysis were carried out to evaluate the effect of BA145 triggered autophagy on mouse tumor xenografts.ResultsBA145 induced autophagy in PC-3 cancer cells and HUVECs significantly impeded its negative regulation on cell proliferation, migration, invasion and tube formation. These effects of BA145 induced autophagy were observed under both normoxic and hypoxic conditions. However, inhibition of autophagy using either pharmacological inhibitors or RNA interference enhanced the BA145 mediated death of these cells. Similar observations were noticed with sunitinib, the anti-angiogenic properties of which were significantly enhanced during combination treatments with autophagy inhibitors. In mouse tumor xenografts, co-treatment with chloroquinone and BA145 led to a considerable reduction in tumor burden and angiogenesis compared to BA145 alone.ConclusionThese studies reveal the essential role of BA145 triggered autophagy in the regulation of angiogenesis and cytoprotection. It also suggests that the combination of the autophagy inhibitors with chemotherapy or anti-angiogenic agents may be an effective therapeutic approach against cancer.

In silico identification of viper phospholipaseA2 inhibitors: validation by in vitro, in vivo studies

AbstractSnake venom, particularly of vipers from the Indian subcontinent, contains Phospholipase A2 (PLA2) as one its constituents which is widely implicated in hemorrhagic, cardiac arrest and death. Development of inhibitors of the protein can facilitate the weakening or annihilation of the venom toxicity and save many human lives. In the present communication, our studies relate to the design and development of structure-based ligands as inhibitors of PLA2 of Viper venom. The study involves the computational approach towards evaluating a library of molecules comprising of natural products, and synthetic molecules through docking studies on the venom protein PDB ID: 1OXL (a dimer, available in the literature). In silico experiments have resulted in the identification of several of them as PLA2 inhibitors. The inhibitory effect of PLA2 by these compounds is attributed to a great extent to their interaction with the residues Phe 46 and Val47 of chain B of the target protein and hence these two residues are identified as the key contributor for the said activity. In order to validate the in silico findings, a selected panel of compounds have been tested by in vitro and in vivo experiments against the venom, which has led to the observance of significant corroboration between the wet lab and in silico findings, validating thereby the in silico approach used in the present study. FigureInteraction of the potent inhibitor with PLA2

Synthesis and Structure-Activity Relationships of Vasicine Analogues as Bronchodilatory Agents

The series of vasicine (1) analogues, an alkaloid from Adhatoda vasica Nees., were synthesized with changes in A, B or C rings. Compounds 13-19 were evaluated for in vitro bronchodilatory activity using isolated guinea pig tracheal chain. Compounds 3-8 were also synthesized in good yields using microwave-mediated synthesis under solvent free conditions. Compounds 5 and 8 with seven-member C ring were more active than etofylline and caused 100% relaxation of both the histamine and acetycholine pre-contracted guinea pig tracheal chain. The structure-activity relationship studies showed that the quinazoline and oxo functionalities were essential for activity. The compounds without C ring and instead having aliphatic and phenyl substitutions in B ring showed relaxation against histamine pre-contracted tracheal chain only, 2-methyl substituted analogues, 12 and 13, being most active with 100% relaxation effect.

Protective Effect of a Standardized Fraction from Vitex negundo Linn. AgainstAcetaminophen and Galactosamine Induced Hepatotoxicity in Rodents

The use of Vitex negundo Linn. (Family: verbenaceae) is well documented in ayurveda and traditional Indian system of medicine for variety of diseases and liver ailments. The aim of the study was to investigate liver protective efficacy of a standardized bioactive fraction (SF) from Vitex negundo Linn. against acetaminophen (APAP) and galactosamine (GalN) hepatotoxicity. SF was tested at doses 12.5, 25, 50 and 100mg/kg, p.o. using both prophylactic and curative treatment schedule against APAP and GalN hepatotoxicity in mice and rats respectively. Isolated markers agnuside and negundoside were tested against APAP toxicity. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), bilirubin and albumin were estimated in serum and triglycerides (TG), total protein, glutathione (GSH) and lipid peroxidation (LP) in liver homogenate. Histopathological studies were carried out against APAP induced hepatotoxicity. SF exhibited significant hepatoprotection against APAP and GalN evident by restoration of ALT, AST, LDH, ALP, bilirubin, TG, albumin and total protein. Levels of LP and GSH also exhibited significant dose dependent recovery when treated with SF. Agnuside and negundoside also exhibited dose dependent protection against APAP induced hepatotoxicity. Microscopic examination of histopathological sections of liver confirmed hepatoprotective potential of SF. Results of the study suggest significant value of SF as hepatoprotective with sufficient safety margin as no mortality and change in general gross behavior was observed up to 2000 mg/kg, p.o. Hepatoprotective mechanism of SF may be due to its antioxidant activity exhibited by protection against increased lipid peroxidation and maintained glutathione status. It is apparent from the present study that agnuside and negundoside are the active ingredients in SF and can be responsible for the activity of SF.

Analogues of boswellic acids as inhibitors of pro-inflammatory cytokines TNF-α and IL-6

A library of boswellic acid analogues were synthesized and tested for their anti-inflammatory potential on key inflammatory mediators, TNF-α and IL-6. The study led to the identification of lead compounds showing significant inhibition of the cytokines, TNF-α and IL-6 both in vitro and in vivo.

Anti-inflammatory potential of hentriacontane in LPS stimulated RAW 264.7 cells and mice model

Hentriacontane, has various pharmacological effects including anti-inflammatory, antitumor and antimicrobial activities. Its anti-inflammatory potential has been demonstrated in peritoneal macrophages. However detailed studies on other models elucidating the mechanistic description of the mode of action has not been done. Hence, the aim of the present study is to evaluate the anti-inflammatory potential of hentriacontane both in-vivo (Balb/c mice) and in-vitro (RAW 264.7 cells). Cytokine inhibition of both pro-inflammatory (TNF-α, IL-6, MCP-1 and IL-1β) and anti-inflammatory (IL-10) cytokines was studied in RAW 264.7 cells and Balb/c mice. Suppressive potential of hentriacontane on NO, PGE2, LTB4 and on LPS induced translocation of NF-κB in RAW 264.7 cells was studied. Further investigations on the effect of hentriacontane on phagocytic index, carrageenan induced paw oedema in mice and on organ weight were done. It was found that hentriacontane significantly reduced all the parameters of inflammation in the experiments under study at all the concentrations, 10μM, 5μM and 1μM (in-vitro) and 5mg/kg, 2mg/kg and 1mg/kg (in-vivo). The highest concentration used in the two models presented the most significant results. The results indicate that hentriacontane is a potent suppressor of inflammatory cytokines and other mediators. Moreover it also has regulatory effect on NF-κB. Hence, hentriacontane is a potential candidate for investigations to develop anti-inflammatory drug.

Development and validation of a highly sensitive LC–MS/MS-ESI method for quantification of IIIM-019—A novel nitroimidazole derivative with promising action against Tuberculosis: Application to drug development

The study aims to illustrate an analytical validation of a rapid and sensitive liquid chromatography (LC) coupled to tandem mass spectrometry (MS-MS) and electrospray ionization (ESI) method for quantification of IIIM-019 (a novel nitroimidazole derivative with potential activity against Tuberculosis) in mice plasma. The extraction of the analyte and the internal standard (Tolbutamide) from the plasma samples involves protein precipitation using acetonitrile. The chromatographic separation was accomplished using a gradient mode and the mobile phase comprised of acetonitrile and 0.1% formic acid in water. The flow rate used was 0.7 ml/min on a C18e high performance Chromolith column. IIIM-019 and Tolbutamide (IS) were analyzed by combined reversed-phase LC/MS-MS with positive ion electrospray ionization. The MS-MS ion transitions used were 533>170.1, 533>198 for IIIM-019 and 271>74, 271>155 for internal standard (IS) respectively. The method was linear over a concentration range of 0.5-1000 ng/ml and the lower limit of quantification was 0.50 ng/ml. The entire study was validated for accuracy, precision, linearity, range, selectivity, lower limit of quantification (LLOQ), recovery, and matrix effect in accordance with the FDA guidelines of method validation. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The intra and inter-day precisions were in the range of 0.51-11.18% and 0.51-7.55%. The pharmacokinetics was performed on male Balb/c mice by oral (2.5mg/kg), intraperitoneal (2.5mg/kg) and intravenous (1mg/kg) routes. The oral bioavailability of IIIM-019 was 51.6%. The method was also applied successfully in determining microsomal stability wherein the compound was found to be very slightly metabolized by rat liver microsomes.

TNF-α and IL-6 inhibitory effects of cyclic dipeptides isolated from marine bacteria Streptomyces sp

Inflammation is mediated by a variety of soluble factors, including a group of secreted polypeptides known as cytokines. The anti-inflammatory cytokines are a series of immune regulatory molecules that control the pro inflammatory cytokine response. Cytokines act in concert with specific cytokine inhibitors and soluble cytokine receptors to regulate the human immune response. The aim of the present study is to probe the anti-inflammatory potential of the crude extract and the bioactive metabolite isolated from marine bacteria Streptomyces sp. on key inflammatory mediators like tumor necrosis factor-α and interleukin-6. Here, we isolated ten known pyrazine-1,4-dione substituted cyclic dipeptide by semi-preparative HPLC and studied their anti-inflammatory activities against tumor necrosis factor-α and interleukin-6. Compound 3, 4, 5, 7 and 8 showed good inhibition of the both the cytokines in lipopolysaccharide-stimulated macrophages. The study reveal that compound 7 was to be specific inhibitor for tumor necrosis factor-α which efficiently inhibited tumor necrosis factor-α release in a dose-dependent manner and decreased lipopolysaccharide induced tumor necrosis factor-α production in human peripheral blood mononuclear cells in both the in vitro and in vivo experiments.