Discovery of a highly potent,selective and novel CDK9 inhibitor as an anticancer drug candidate

A series of novel hybrid structure derivatives, containing both LEE011 and Cabozantinib pharmacophore, were designed, synthesized and evaluated. Surprisingly, a compound 4d was discovered that highly exhibited effective and selective activity of CDK9 inhibition with IC₅₀ = 12 nM. It effectively induced apoptosis in breast and lung cancer cell lines at nanomolar level. Molecular docking of 4d to ATP binding site of CDK9 kinase demonstrated a new hydrogen bonding between F atom of 4-(3-fluorobenzyloxy) group and ASN116 residue, compared with the positive control, LEE011. The compound 4d could block the cell cycle both in G0/G1 and G2/M phase to prevent the proliferation and differentiation of cancer cells. Mice bared-breast cancer treated with compound 4d showed significant suppression of cancer with low toxicity. Taken together, this novel compound 4d could be a promising drug candidate for clinical application.     

Suppressive effect of Withania somnifera root powder on experimental gouty arthritis: An in vivo and in vitro study

The effect of Withania somnifera L. Dunal root powder on paw volume and serum lysosomal enzyme activities was investigated in monosodium urate crystal-induced rats. The levels of beta-glucuronidase and lactate dehydrogenase were also measured in monosodium urate crystal incubated polymorphonuclear leucocytes (PMNL). A significant increase in the level of paw volume and serum lysosomal enzymes was observed in monosodium urate crystal-induced rats. The increased beta-glucuronidase and lactate dehydrogenase level were observed in untreated monosodium urate crystal incubated polymorphonuclear leucocytes. On treatment with the W. somnifera root powder (500/1000 mg/kg body weight), the above changes were reverted back to near normal levels. W. somnifera also showed potent analgesic and antipyretic effect with the absence of gastric damage at different dose levels in experimental rats. For comparison purpose, non-steroidal anti-inflammatory drug (NSAID) indomethacin was used as a standard. These results provide evidence for the suppressive effect of W. somnifera root powder by retarding amplification and propagation of the inflammatory response without causing any gastric damage.     

Purification and characterization of a glycoprotein inhibitor of toxic phospholipase from Withania somnifera

A phospholipase inhibitor (WSG) has been purified from Withania somnifera using gel-filtration and ion-exchange chromatographies. The WSG is an acidic glycoprotein. Its molecular mass as determined by SDS-PAGE was 27kDa. It neutralized the enzyme activity and pharmacological properties such as cytotoxicity, edema, and myotoxicity of a multi-toxic Indian cobra venom phospholipase (NNXIa-PLA) but failed to neutralize the neurotoxicity. The glycan part of the molecule does not appear to be involved in any of the pharmacological properties studied. The results suggest that the neutralization of the pharmacological effects of the toxic phospholipase is brought about by inhibition of the enzyme activity by formation of a complex between the WSG and the toxic phospholipase. We report the purification and characterization of a glycoprotein phospholipase A inhibitor from Withania somnifera, medicinal plant.     

Germination behaviour of seeds of Withania somnifera (L.) Dunal: a high value medicinal plant

In order to evolve a quick method for smooth and optimum germination for Withania somnifera- a medicinally efficacious multipurpose plant, present investigation was carried to study the effect of physico-chemical treatments, storage, temperature, photoperiod and growth regulators (GA₃, IAA, IBA, 2–4 D and BA) on germinability. The most effective treatment is GA₃ at 150 μg/ml concentration at 25 °C. The optimal temperature for germination is 25 °C and continuous light favored germination showing that photoperiod has a significant role. The seedlings derived from seeds performed well when grown in a glasshouse. The data have implications for conservation and cultivation of the species studied.     

Expression and functional analysis of an inhibitor of apoptosis protein from Trichoplusia ni

An inhibitor of the apoptosis protein (IAP) family gene from Trichoplusia ni, Tn-IAP1v, a variant of lepidopteran Tn-IAP1, was cloned by RT-PCR. There are six single nucleotide polymorphisms between the two Tn-IAP1 variants, resulting in three predicted single amino acid polymorphisms. With the GST fusion expression system, soluble recombinant Tn-IAP1v was highly expressed in Escherichia coli and then purified by affinity chromatography. Caspase inhibition assays indicated that recombinant Tn-IAP1v could specifically inhibit human caspase-9 in vitro instead of caspase-3, -7, and -8, which was further confirmed by the observation that recombinant Tn-IAP1v can directly bind caspase-9 in the protein pull-down assay. These results suggested that Tn-IAP1v might serve as an initiator caspase inhibitor in vivo in the conserved mitochondria apoptotic pathway.     

Enhancement of antitumor effect of paclitaxel in combination with immunomodulatory Withania somnifera on benzo(a)pyrene induced experimental lung cancer

The current experimental work deals with the immunomodulatory studies on the extract of Withania somnifera (L.) Dunal root powder against benzo(a)pyrene induced lung cancer in male Swiss albino mice. In our previous study, we reported the antioxidant and anticarcinogenic effect of W. somnifera (L.) Dunal along with paclitaxel. Immune dysfunction has been found to be associated with cancer and chemotherapy. Benzo(a)pyrene induced cancer animals were treated with 400mg/kg bodyweight of W. somnifera (L.) Dunal extract for 30 days significantly alters the levels of immunocompetent cells, immune complexes and immunoglobulins. Based on the data, the carcinogen as well as the paclitaxel affects the immune system, the toxic side effects on the immune system is more reversible and more controllable by W. somnifera (L.) Dunal. These results concluded the immunomodulatory activity of W. somnifera (L.) Dunal extract, which is a known immunomodulator in indigenous medicine.     

Comprehensive metabolic fingerprinting of Withania somnifera leaf and root extracts

Profiling of metabolites is a rapidly expanding area of research for resolving metabolic pathways. Metabolic fingerprinting in medicinally important plants is critical to establishing the quality of herbal medicines. In the present study, metabolic profiling of crude extracts of leaf and root of Withania somnifera (Ashwagandha), an important medicinal plant of Indian system of medicine (ISM) was carried out using NMR and chromatographic (HPLC and GC-MS) techniques. A total of 62 major and minor primary and secondary metabolites from leaves and 48 from roots were unambiguously identified. Twenty-nine of these were common to the two tissues. These included fatty acids, organic acids, amino acids, sugars and sterol based compounds. Eleven bioactive sterol-lactone molecules were also identified. Twenty-seven of the identified metabolites were quantified. Highly significant qualitative and quantitative differences were noticed between the leaf and root tissues, particularly with respect to the secondary metabolites.     

A standardized root extract of Withania somnifera and its major constituent withanolide-A elicit humoral and cell-mediated immune responses by up regulation of Th1-dominant polarization in BALB/c mice

The effects of graded doses of a chemically standardized aqueous alcoholic (1:1) root extract (AGB) of Withania somnifera on the immune system of SRBC immunized BALB/c mice were investigated. Mice were administrated AGB orally for 15 days. AGB stimulated cell mediated immunity, IgM and IgG titers reaching peak value with 30 mg/kg b.wt. Flow cytometric analysis of lymphocyte surface markers of T cells (CD3(+), CD4(+) and CD8(+)) and B cells (CD19(+)) indicated prominent enhancement in proliferation and differentiation of lymphocytes. The extract selectively, induced type 1 immunity because it guided enhanced expression of T helper cells (Th)1 cytokines interferon (IFN)-gamma and interleukin (IL)-2 while Th2 cytokine IL-4 observed a moderate decline. Confirmation of Th1 polarization was obtained from augmented levels of IgG2a over IgG1 in the blood sera of AGB treated groups. Withanolide-A, a major constituent of AGB appeared responsible for Th1 skewing effect of the extract as it significantly increased the levels of Th1 cytokines, decreased moderately IL-4 and significantly restored the selective dexamethasone inhibition of Th1 cytokines in mouse splenocytes cultures in vitro. In addition, AGB also strongly activated macrophage functions ex vivo and in vitro indicated by enhanced secretion of nitrite, IL-12 and TNF-alpha. In contrast IL-10 remained unchanged again suggesting that AGB critically influenced Th1 profile of the cytokines. The studies suggested that AGB supports predominantly Th1 immunity with increase in macrophage functions. The standardized root extract of no toxicological consequences might therefore, find useful applications against the intracellular pathogens and in the management of immune suppressed diseases.     

Protease inhibitor from Moringa oleifera with potential for use as therapeutic drug and as seafood preservative

Protease inhibitors are well known to have several applications in medicine and biotechnology. Several plant sources are known to return potential protease inhibitors. In this study plants belonging to different families of Leguminosae, Malvaceae, Rutaceae, Graminae and Moringaceae were screened for the protease inhibitor. Among them Moringa oleifera, belonging to the family Moringaceae, recorded high level of protease inhibitor activity after ammonium sulfate fractionation. M. oleifera, which grows throughout most of the tropics and having several industrial and medicinal uses, was selected as a source of protease inhibitor since so far no reports were made on isolation of the protease inhibitor. Among the different parts of M. oleifera tested, the crude extract isolated from the mature leaves and seeds showed the highest level of inhibition against trypsin. Among the various extraction media evaluated, the crude extract prepared in phosphate buffer showed maximum recovery of the protease inhibitor. The protease inhibitor recorded high inhibitory activity toward the serine proteases thrombin, elastase, chymotrypsin and the cysteine proteases cathepsin B and papain which have more importance in pharmaceutical industry. The protease inhibitor also showed complete inhibition of activities of the commercially available proteases of Bacillus licheniformis and Aspergillus oryzae. However, inhibitory activities toward subtilisin, esperase, pronase E and proteinase K were negligible. Further, it was found that the protease inhibitor could prevent proteolysis in a commercially valuable shrimp Penaeus monodon during storage indicating the scope for its application as a seafood preservative. This is the first report on isolation of a protease inhibitor from M. oleifera.     

Modulation of TCA cycle enzymes and electron transport chain systems in experimental lung cancer

The modulatory effect of Withania somnifera along with paclitaxel on tricarboxylic acid (TCA) cycle key enzymes and electron transport chain complexes were investigated against lung cancer induced by benzo(a)pyrene in Swiss albino mice. Decreased activities of TCA cycle key enzymes such as isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH) in lung cancer bearing animals were observed. Upon W. somnifera along with paclitaxel administration the above biochemical changes were inclined towards normal control animal values. Activities of mitochondrial enzymes and electron transport complexes were analyzed in the experimental groups to determine the efficiency of energy production. This study further confirms the chemotherapeutic effect of W. somnifera along with paclitaxel which is found to be more effective in the treatment of lung cancer. Thus these results are consistent with our hypothesis that the combination chemotherapy of W. somnifera along with paclitaxel as a promising chemotherapeutic agent.     

A novel yeast cell-based screen identifies flavone as a tankyrase inhibitor

The telomere-associated protein tankyrase 1 is a poly(ADP-ribose) polymerase and is considered to be a promising target for cancer therapy, especially for BRCA-associated cancers. However, an efficient assay system for inhibitor screening has not been established, mainly due to the difficulty of efficient preparation of the enzyme and its substrate. Here, we report a cell-based assay system for detecting inhibitory activity against tankyrase 1. We found that overexpression of the human tankyrase 1 gene causes a growth defect in the fission yeast Schizosaccharomyces pombe. Chemicals that restore the growth defect phenotype can be identified as potential tankyrase 1 inhibitors. We performed a high-throughput screen using this system, and identified flavone as a compound that restores the growth of yeast cells overexpressing tankyrase 1. Indeed, flavone inhibited poly(ADP-ribosyl)ation of proteins caused by overexpression of tankyrase 1 in yeast cells. This system allows rapid identification of inhibitory activity against tankyrase 1 and is amenable to high-throughput screening using robotics.     

Chondroprotective potential of root extracts of <Emphasis Type="Italic">Withania somnifera</Emphasis> in osteoarthritis

This is the first report describing two novel chondroprotective activities of aqueous extracts of Withania somnifera root powder. First, these extracts had a statistically significant, short-term chondroprotective effect on damaged human osteoarthritic cartilage matrix in 50% of the patients tested. Second, these extracts caused a significant and reproducible inhibition of the gelatinase activity of collagenase type 2 enzyme in vitro.     

Structural characterization of a flavonoid glycosyltransferase from Withania somnifera

Medicinal plants are extensively utilized in traditional and herbal medicines, both in India and around the world due to the presence of diverse low molecular weight natural products such as flavonoids, alkaloids, terpenoids and sterols. Flavonoids which have health benefits for humans are the large class of phenylpropanoid-derived secondary metabolites and are mostly glycosylated by UDP-glycosyltransferases (UGTs). Although large numbers of different UGTs are known from higher plants, very few protein structures have been reported till now. In the present study, the three-dimensional model of flavonoid specific glycosyltransferases (WsFGT) from Withania somnifera was constructed based on the crystal structure of plant UGTs. The resulted model was assessed by various tools and the final refined model revealed GT-B type fold. Further, to understand the sugar donors and acceptors interactions with the active site of WsFGT, docking studies were performed. The amino acids from conserved PSPG box were interacted with sugar donor while His18, Asp110, Trp352 and Asn353 were important for catalytic function. This structural and docking information will be useful to understand the glycosylation mechanism of flavonoid glucosides.

Abbreviations

DOPE - Discrete Optimized Potential Energy, PDB - Protein Data Bank, PSPG - Plant Secondary Product Glycosyltransferase, RMSD - Root Mean Squared Deviation, UDP - Uridine diphosphate, UGT - UDP-glycosyltransferases.     

Purification of a post-synaptic neurotoxic phospholipase A2 from Naja naja venom and its inhibition by a glycoprotein from Withania somnifera

A post-synaptic neurotoxic phospholipase A(2) (PLA(2)) has been purified from Indian cobra Naja naja venom. It was associated with a peptide in the venom. The association was disrupted using 8 M urea. It is denoted to be a basic protein by its behavior on both ion exchange chromatography and electrophoresis. It is toxic to mice, LD(50) 1.9 mg/kg body weight (ip). It is proved to be post-synaptic PLA(2) by chymographic experiment using frog nerve-muscle preparation. A glycoprotein, (WSG) was isolated from a folk medicinal plant Withania somnifera. The WSG inhibited the phospholipase A(2) activity of NN-XIa-PLA(2,) isolated from the cobra venom, completely at a mole-to-mole ratio of 1:2 (NN-XIa-PLA(2): WSG) but failed to neutralize the toxicity of the molecule. However, it reduced the toxicity as well as prolonged the death time of the experimental mice approximately 10 times when compared to venom alone. The WSG also inhibited several other PLA(2) isoforms from the venom to varying extent. The interaction of the WSG with the PLA(2) is confirmed by fluorescence quenching and gel-permeation chromatography. Chemical modification of the active histidine residue of PLA(2) using p-brophenacyl bromide resulted in the loss of both catalytic activity as well as neurotoxicity of the molecule. These findings suggest that the venom PLA(2) has multiple sites on it; perhaps some of them are overlapping. Application of the plant extract on snakebite wound confirms the medicinal value associated with the plant.     

Immunotherapy of neuroblastoma by an Interleukin-21-secreting cell vaccine involves survivin as antigen

AIM: IL-21 is the most recently identified member of the IL-2 cytokine family. Here we studied the therapeutic efficacy of IL-21-gene-modified cells (Neuro2a/IL-21) in a syngeneic metastatic neuroblastoma (NB) model. MATERIALS AND METHODS: Neuro2a/IL-21 cells were tested as subcutaneous (sc) vaccine both in prophylactic and therapeutic settings. Depletion studies, cytotoxicity assay and immunohistochemical analyses were carried out to evaluate the mechanisms involved in tumor rejection. RESULTS: When injected sc in syngeneic A/J mice viable Neuro2a/IL-21 cells were rejected and induced resistance to a subsequent iv challenge with Neuro2a parental cells (Neuro2a/pc), suggesting the involvement of an immune response. More importantly, in mice bearing Neuro2a/pc micrometastases, a single sc injection of Neuro2a/IL-21 cells significantly increased the mean tumor-free survival of treated animals (43 vs. 22 days) and cured 14% of them. The administration of two or three doses of Neuro2a/IL-21 cell vaccine further increased the mean survival time to 54 and 75 days, and the cure rate to 27 and 33%, respectively, whereas the use of unmodified Neuro2a or mock-transfected cells had no effect. In vivo cell subset depletion and a Winn-assay indicated the involvement of CD8 + CTLs. Immunohistochemical analysis indicated a reduction of CD31+ and VEGFR2+ microvessels in late metastases from therapeutically vaccinated mice. A role of survivin as antigen was suggested by in vitro assays using survivin-synthetic CTL-epitopes. CONCLUSIONS: Our present data indicate that IL-21-secreting NB cells are effective as therapeutic vaccine in mice bearing metastatic NB, through a specific CTL response involving survivin as antigen, and suggest a potential interest for IL-21 in NB immuno-gene therapy.     

Pyrrhocoricin as a potential drug delivery vehicle for Cryptosporidium parvum

This study analysed the intracellular delivery capacity of insect derived pyrrhocoricin with a peptide cargo in Cryptosporidium parvum in vitro using fluorescence microscopy. Results revealed that pyrrhocoricin was capable of acting as a delivery vehicle in transducing peptides across the parasite cell membrane for multiple life-cycle stages. The successful transduction may aid in target validation and the delivery of future peptide-based drugs against this important human pathogen.     

Nucleases in homologous recombination as targets for cancer therapy

Genomic DNA is constantly challenged from endogenous as well as exogenous sources. The DNA damage response (DDR) mechanism has evolved to combat these challenges and ensure genomic integrity. In this review, we will focus on repair of DNA double-strand breaks (DSB) by homologous recombination and the role of several nucleases and other recombination factors as suitable targets for cancer therapy. Their inactivation as well as overexpression have been shown to sensitize cancer cells by increasing toxicity to DNA-damaging agents and radiation or to be responsible for resistance of cancer cells. These factors can also be used in targeted cancer therapy by taking advantage of specific genetic abnormalities of cancer cells that are not present in normal cells and that result in cancer cell lethality.     

Phytochemical and genetic analysis in selected chemotypes of Withania somnifera

The main active components and genetic profile of 15 selected accessions of Withania somnifera Dunal. were analysed. Ethanolic extract of the dried roots/leaves of the plant was concentrated under pressure at 50+/-5 degrees C and was analysed for main compounds (withanolides and withaferin A) by HPLC. All the main components were found to be present in accessions (AGB 002, AGB 009, RSS 009, RSS 033). Correlation of these main components with their genetic factors, was undertaken using AFLP (amplified fragment length polymorphism) markers. Among 64 primers 7 yielded optimum polymorphism. A total of 913 polymorphic peaks were generated using these primers. Jaccard's similarity coefficient indicated that accessions having almost the same active compounds clustered together. The present study demonstrates that AFLP can be successfully used to resolve the correlation of AFLP data with the presence of secondary metabolites.