Virtual analysis of structurally diverse synthetic analogs as inhibitors of snake venom secretory phospholipase A2.

Due to the toxic pathophysiological role of snake venom phospholipase A2 (PLA2 ), its compelling limitations to anti-venom therapy in humans and the need for alternative therapy foster considerable pharmacological interest towards search of PLA2 specific inhibitors. In this study, an integrated approach involving homology modeling, molecular dynamics and molecular docking studies on VRV-PL-V (Vipera russellii venom phospholipase A2 fraction-V) belonging to Group II-B secretory PLA2 from Daboia russelli pulchella is carried out in order to study the structure-based inhibitor design. The accuracy of the model was validated using multiple computational approaches. The molecular docking study of this protein was undertaken using different classes of experimentally proven, structurally diverse synthetic inhibitors of secretory PLA2 whose selection is based on IC50 value that ranges from 25 µM to 100 µM. Estimation of protein-ligand contacts by docking analysis sheds light on the importance of His 47 and Asp 48 within the VRV-PL-V binding pocket as key residue for hydrogen bond interaction with ligands. Our virtual analysis revealed that compounds with different scaffold binds to the same active site region. ADME analysis was also further performed to filter and identify the best potential specific inhibitor against VRV-PL-V. Additionally, the e-pharmacophore was generated for the best potential specific inhibitor against VRV-PL-V and reported here. The present study should therefore play a guiding role in the experimental design of VRV-PL-V inhibitors that may provide better therapeutic molecular models for PLA2 recognition and anti-ophidian activity.

Identification of potential transmembrane protease serine 4 inhibitors as anti-cancer agents by integrated computational approach.

Transmembrane protease serine 4 is a well known cell surface protease facilitating the extracellular matrix degradation and epithelial mesenchymal transition in hepatocellular carcinoma. Henceforth targeting transmembrane protease serine 4 is strongly believed to provide therapeutic intervention against hepatocellular carcinoma. Owing to lack of crystal structure for human transmembrane protease serine 4, we predicted its three dimensional structure for the first time in this study. Experimentally proven inhibitor-Tyroserleutide (TSL) against hepatocellular carcinoma via transmembrane protease serine 4 was used as a benchmark to identify structurally similar candidates from PubChem database to create the TSL library. Virtual screening of TSL library against modeled transmembrane protease serine 4 revealed the top four potential inhibitors. Further binding free energy (ΔGbind) analysis of the potential inhibitors revealed the best potential lead compound against transmembrane protease serine 4. Drug likeliness nature of the top four potential hits were additionally analyzed in comparison to TSL to confirm on the best potential lead compound with the highest % of human oral absorption. Consequently, e-pharmacophore mapping of the best potential lead compound yielded a six point feature. It was observed to contain four hydrogen bond donor sites (D), one positively ionizable site (P) and one aromatic ring (R). Such e-pharmacophore insight obtained from structural determinants by integrated computational analysis could serve as a framework for further advancement of drug discovery process of new anti-cancer agents with less toxicity and high specificity targeting transmembrane protease serine 4 and hepatocellular carcinoma.

Preclinical Targeting of the PGRMC1-CK2 Axis with Silmitasertib: A Potential Strategy for Lung Adenocarcinoma Therapy.

Progesterone receptor membrane component 1 (PGRMC1) is a pleiotropic protein over-expressed in lung adenocarcinoma (LUAD). The precise molecular mechanisms underlying the signature motif of Casein kinase (CK2) presence in PGRMC1 and their role in LUAD remain unclear. X-ray crystallographic structure for CK2 and PGRMC1 from the PubChem database was obtained and subjected to protein-protein interaction (PPI) analysis to identify their interactions. In addition, the CK2 inhibitor - Silmitasertib was also utilised to understand the interaction between PGRMC1-CK2. The PPI complex (PGRMC1-CK2) and the PPI-ligand interaction analysis and their Molecular Dynamics (MD) studies revealed the stability of their interactions and critical amino acid contacts within the 5Ǻ vicinity of the CK2 signature motif "T/S-x-x-E/D". Moreover, in-vitro colony formation assay, migration assay, and gene expression analysis using quantitative Real-time PCR revealed that Silmitasertib (IC50-2.5 µM) was highly influential in suppressing the PGRMC1-CK2 expression axis. In conclusion, our study infers that PGRMC1-CK-2 axis inhibition could be a potential therapeutic option to limit the promotion and progression of lung cancer.

Artesunate acts as fuel to fire in sensitizing HepG2 cells towards TRAIL mediated apoptosis via STAT3 inhibition and DR4 augmentation.

In the present study, we investigated in vitro, the role of artesunate (ATS) with comparable potency to oxaliplatin (OXP) in sensitizing tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) resistant HepG2 cells towards apoptosis. ATS in consistency with OXP was found to reverse TRAIL resistant HepG2 cells towards TRAIL mediated apoptosis by enhancing caspase-3 and cleavage of PARP. Additionally, ATS also suppressed the nuclear translocation of activated signal transducers and activators of transcription 3 (STAT3) thereby sensitizing the HepG2 cells towards only death receptor 4 (DR4) mediated apoptosis. Furthermore, ATS exposure in TRAIL resistant cells resulted in significant increase of both DR4/DR5 expression and STAT3 inhibition thereby arbitrating TRAIL mediated apoptosis in HepG2 cells. The increase in expression was comparable to that of STAT3 silenced cells. From all the above observations, we conclude that ATS up-regulated DR4 expression by targeting STAT3, which in turn sensitized HepG2 cells to TRAIL mediated apoptosis.

Viper venom hyaluronidase and its potential inhibitor analysis: a multipronged computational investigation.

Viper venom hyaluronidase (VV-HYA) inhibitors have long been used as therapeutic agents for arresting the local and systemic effects caused during its envenomation. Henceforth, to understand its structural features and also to identify the best potential inhibitor against it the present computational study was undertaken. Structure-based homology modeling of VV-HYA followed by its docking and free energy-based ranking analysis of ligand, the MD simulations of the lead complex was also performed. The sequence analysis and homology modeling of VV-HYA revealed a distorted (ß/α)8 folding as in the case of hydrolases family of proteins. Molecular docking of the resultant 3D structure of VV-HYA with known inhibitors (compounds 1-25) revealed the importance of molecular recognition of hotspot residues (Tyr 75, Arg 288, and Trp 321) other than that of the active site residues. It also revealed that Trp 321 of VV-HYA is highly important for mediating π-π interactions with ligands. In addition, the molecular docking and comparative free energy binding analysis was investigated for the VV-HYA inhibitors (compounds 1-25). Both molecular docking and relative free energy binding analysis clearly confirmed the identification of sodium chromoglycate (compound 1) as the best potential inhibitor against VV-HYA. Molecular dynamics simulations additionally confirmed the stability of their binding interactions. Further, the information obtained from this work is believed to serve as an impetus for future rational designing of new novel VV-HYA inhibitors with improved activity and selectivity.

Artesunate as an Anti-Cancer Agent Targets Stat-3 and Favorably Suppresses Hepatocellular Carcinoma.

BACKGROUND: Aberrant signal transducer and activator of transcription 3 (STAT-3) molecular signaling elicit hepatocellular carcinoma (HCC) in humans. Therefore, targeting STAT-3 is considered as an attractive option towards suppression of HCC in humans. OBJECTIVE: Our objective is to identify a potential small molecule inhibitor that can specifically target STAT-3 and suppress HCC. METHODS: In this study, we analyze a group of sesquiterpene lactone (STL) candidates that has been recently reported in preclinical trials against cancer by a unified computational and experimental approach. RESULTS: Our virtual analysis of the STL candidates revealed Artesunate (ATS) as the best potential inhibitor of STAT-3 with comparable potency to specific inhibitor S3I-201. We also observed that ATS inhibited IL-6 driven STAT-3-DNA binding activity with comparable potency to S3I-201 in a cell free system. Furthermore ATS was observed to interfere with STAT-3 dimerization and suppression of both constitutive and IL-6 inducible STAT-3 in vitro. Nevertheless, we also observed that ATS modulated STAT-3 dependent targets (procaspase-3, Bcl-xl and survivin) favoring occurrence of apoptosis in vitro. Overall, the putative inhibition of STAT-3 by ATS suggested its capacity to interfere with STAT-3 dimerization by binding to the SH2 domain of STAT-3 monomer. It resulted in suppression of STAT-3 and also favored promotion of in vitro cells towards apoptosis. Consequently, ATS also exhibited selective cytotoxicity of cancer cells over normal cells in vitro. CONCLUSION: All the above observations substantiated by unified computational and in vitro experimental approaches suggested its potential role as a therapeutic anti-cancer agent against HCC.

Artesunate obliterates experimental hepatocellular carcinoma in rats through suppression of IL-6-JAK-STAT signalling.

Activation of the IL-6 mediated JAK-STAT (Janus associated kinase-signal transducer and activator of transcription) oncogenic signalling plays a major role in hepatocellular carcinoma pathogenesis. The aim of this study is to assess the anti-tumour, anti-proliferative and apoptotic potential of artesunate and its capacity to modulate JAK-STAT pathway in a nitrosodiethylamine mediated experimental hepatocellular carcinoma model. Administration of nitrosodiethylamine (200mg/kg body weight by i.p. Injections) to rats resulted in alterations of liver pathophysiological parameters such as increased relative liver weight, and increased tumour nodule occurrence. It also increased the levels of serum marker enzymes (AST, ALT, ALP, LDH, and γGT) and tumour biomarker (AFP) levels suggestive of its capacity to cause liver tumourigenesis. Additionally, the immunohistochemistry of liver sections pertaining to nitrosodiethylamine administered animals showed increased detection of AgNOR, PCNA, and GST-Pi positive cells suggestive of its capacity to promote liver proliferation associated tumourigenesis. On the contrary, artesunate (25mg/kg bodyweight) supplementation to nitrosodiethylamine administered animals decreased all the above mentioned pathophysiological, biochemical, and immunohistochemistry parameters suggesting its anti-tumour and anti-proliferative potential. Furthermore, immunoblot analysis showed significant up-regulation of IL-6, GP130, JAK-2, STAT-3 (pY705), Bcl-xL, Bcl-2 and simultaneous down-regulation of Caspase-3, PARP and SOCS-3 in nitrosodiethylamine administered animals. Nevertheless, the immunoblot analysis revealed vice-versa on artesunate supplementation to nitrosodiethylamine administered animals, indicating promotion of the feedback loop inhibition mechanism through SOCS3 up-regulation thereby leading to suppression of JAK-STAT signalling. Overall all these findings substantiate that artesunate promotes anti-tumour, anti-proliferation and apoptosis against nitrosodiethylamine mediated hepatocellular carcinoma.

Anticarcinogenic effects of some Indian plant products.

The anticarcinogenic properties of some commonly consumed spices and leafy vegetables were investigated. The effects of feeding the plant products on the induction of squamous cell carcinomas in the stomachs of Swiss mice by feeding benzo[a]pyrene(B[a]P) and on the induction of hepatomas in Wistar rats by feeding 3'-methyl-4-dimethylaminoazobenzene (3'MeDAB) were investigated. Among the nine plant products tested, cumin seeds (Cuminum cyminum Linn) and basil leaves (Ocimum sanctum Linn) significantly decreased the incidence of both B[a]P-induced neoplasia and 3'MeDAB-induced hepatomas. Poppy seeds (Papaver somniferum Linn) significantly inhibited B[a]P-induced neoplasia alone, while the other plant products, asafoetida, kandathipili, turmeric, drumstick leaves, solanum leaves and alternanthera leaves were ineffective. These results suggest that cumin seeds, basil leaves and to a lesser extent poppy seeds, which are all widely used in Indian cooking, may prove to be valuable anticarcinogenic agents.

An evaluation of the tumour affinity of the radioactive cobalt chelate of tallysomycin S10b in lymphoma-bearing mice.

The tumour affinity of the radioactive cobalt chelate of tallysomycin S10b (Tlm S10b), a promising structural analogue of Bleomycin, was assessed using a mouse lymphoma model. The highest concentrations (%dose/gram tissue) were observed in the kidneys, followed by the tumour (4.1%/g 1 h p.i.) at 1 h, 4 h and 48 h. The tumour had the highest concentration among all tissues at 24 h. The biodistribution profile of 60Co-Tlm S10b was distinctly different from that obtained with 60CoCl2, demonstrating the in vivo stability of the chelate. More than half of the chelate (56.8% of the injected dose) was excreted in the urine at 1 h. The highest tumour/non-tumour ratios were obtained for blood (41.3, 4 h), bone (30.5, 4 h) and muscle (29.2, 48 h). Scintigraphy at 24 h, using 57Co-Tlm S10b, showed the tumour, liver, kidney and bladder clearly. The similarities and differences exhibited by Co-Tlm S10b with reference to the literature on cobalt chelates of Bleomycin and naturally occurring tallysomycin (A + B) are discussed.

Genotoxic effects of some foods & food components in Swiss mice.

A number of commonly consumed foods and food components in south India were screened for their genotoxic effects on Swiss mice. Salted, sundried and oil fried vegetables and fishes induced chromosomal aberrations, sperm head abnormalities and micronuclei production, which were comparable to the effect of the positive control viz., 20-methylcholanthrene. Spices like Cissus quadrangularis (an indigenous herb used in certain south Indian dishes) and pyrolysed cumin and aniseeds showed moderate effects. Calamus oil, widely used in pharmaceuticals was highly effective. All the three parameters of genotoxicity gave similar results.

Plant products as protective agents against cancer.

Out of various spices and leafy vegetables screened for their influence on the carcinogen-detoxifying enzyme, glutathione-S-transferase (GST) in Swiss mice, cumin seeds, poppy seeds, asafoetida, turmeric, kandathipili, neem flowers, manathakkali leaves, drumstick leaves, basil leaves and ponnakanni leaves increased GST activity by more than 78% in the stomach, liver and oesophagus, - high enough to be considered as protective agents against carcinogenesis. Glutathione levels were also significantly elevated in the three tissues by these plant products. All of them except neem flowers, significantly suppressed (in vivo) the chromosome aberrations (CA) caused by benzo(a)pyrene in mouse bone marrow cells. Multiple CA and exchanges reflecting the severity of damage within a cell were significantly suppressed by these nine plant products. The results suggest that these nine plant products are likely to suppress carcinogenesis and can act as protective agents against cancer.

Presence of polycyclic aromatic hydrocarbons in some South Indian food components.

South Indian food dishes, comprising several deep fried items have been proved to be mutagenic. Polycyclic aromatic hydrocarbons (PAHs) such as, benzo(a)pyrene, chrysene and dibenzanthracene, which are potent and proven carcinogens have been identified and quantified in several of the commonly consumed South Indian food dishes and food components. Most of the pyrolysed items contained PAHs in appreciable quantities. PAHs were detected in significant levels in salted, sundried and oil fried vegetables and fishes. Some of the raw and uncooked food components also revealed the presence of PAHs.

Mutagenicity of nitrosated food items.

Several food items, commonly consumed in South India, after nitrite treatment under simulated gastric conditions were found to be mutagenic in Salmonella typhimurium tester strain TA 100. Dichloromethane extracts containing the volatile nitroso compounds and ethyl-acetate extracts with the non-volatile nitroso compounds of some of the food items exhibited mutagenicity.

Mutagenic activity of south Indian food items.

Dietary components and food dishes commonly consumed in South India were screened for their mutagenic activity. Kesari powder, calamus oil, palm drink, toddy and Kewra essence were found to be strongly mutagenic; garlic, palm oil, arrack, onion and pyrolysed portions of bread toast, chicory powder were weakly mutagenic, while tamarind and turmeric were not. Certain salted, sundried and oil fried food items were also mutagenic. Cissus quadrangularis was mutagenic, while 'decoctions' of cumin seeds, aniseeds and ginger were not. Several perfumes, essential oils and colouring agents, which are commonly used were also screened and many of them exhibited their mutagenic potential by inducing the 'reverse mutation' in Salmonella typhimurium tester strains.

Formylchromone exhibits salubrious effects against nitrosodiethylamine mediated early hepatocellular carcinogenesis in rats.

The salubrious effects of 3-Formylchromone (3-FC) against nitrosodiethylamine (NDEA) mediated early hepatocellular carcinogenesis was investigated in vivo by this study. Male Wistar rats were administered three interspersed intraperitoneal injections of NDEA (200 mg/kg body weight) until sixth week, followed by, thrice a week oral dose of 3-FC (25 mg/kg body weight) from the seventh week to eleventh week. Oral supplementation of Wistar rats with 3-FC prevented the increase in serum marker enzymes (AST, ALT, LDH) and serum pre-neoplastic marker (γ-GT) induced by NDEA. Biochemical observations were found to be further correlated with histological studies, indicating the potential of 3-FC to mediate suppression of hepatic damage/pre-neoplastic lesions. Argyrophilic nucleolar organizer region (AgNOR) staining was done in histology sections to confirm the anti-proliferative potential of 3-FC against NDEA-induced early hepatocellular carcinogenesis. RT-PCR and immunoblot analysis was done to find the modulations in the gene transcript/protein level expression of pre-neoplastic marker (GST-pi), proliferation marker (PCNA), apoptotic mediators (PPARγ, NFκB-p65 and p53). 3-FC was found to favorably modulate the expressions of GST-pi, PCNA, PPARγ, NFκB-p65, p53 clearly confirming the anti-proliferative and apoptotic potential of 3-FC. Further, the apoptotic effect of 3-FC against NDEA-induced early hepatocellular carcinogenesis was confirmed by caspase-3 activity assay and DNA fragmentation analysis. Based on these findings, it is concluded that 3-FC possesses hepatoprotective, anti-pre-neoplastic, anti-proliferative and apoptosis inducing capability against NDEA-induced early hepatocellular carcinogenesis.

Homology modeling, molecular dynamics and atomic level interaction study of snake venom 5' nucleotidase.

5' Nucleotidase (5' NUC) is a ubiquitously distributed enzyme known to be present in snake venoms (SV) that is responsible primarily for causing dysregulation of physiological homeostasis in humans by inducing anticoagulant effects and by inhibiting platelet aggregation. It is also known to act synergistically with other toxins to exert a more pronounced anti-coagulant effect during envenomation. Its structural and functional role is not yet ascertained clearly. The 3D structure of snake venom 5' nucleotidase (SV-5' NUC) is not yet known and was predicted by us for the first time using a comparative homology modeling approach using Demansia vestigiata protein sequence. The accuracy and stability of the predicted SV-5' NUC structure were validated using several computational approaches. Key interactions of SV-5' NUC were studied using experimental studies/molecular docking analysis of the inhibitors vanillin, vanillic acid and maltol. All these inhibitors were found to dock favorably following pharmacologically relevant absorption, distribution, metabolism and excretion (ADME) profiles. Further, atomic level docking interaction studies using inhibitors of the SV-5' NUC active site revealed amino acid residues Y65 and T72 as important for inhibitor-(SV-5' NUC) interactions. Our in silico analysis is in good agreement with experimental inhibition results of SV-5' NUC with vanillin, vanillic acid and maltol. The present study should therefore play a guiding role in the experimental design of new SV-5' NUC inhibitors for snake bite management. We also identified a few pharmacophoric features essential for SV-5' NUC inhibitory activity that can be utilized further for the discovery of putative anti-venom agents of therapeutic value for snake bite management.

Induction of apoptosis by methanolic extract of Rubia cordifolia Linn in HEp-2 cell line is mediated by reactive oxygen species.

Rubia cordifolia Linn, which belongs to the Rubiaceae family, is a well-known herb used in Ayurvedic medicine. In the present study, we investigated the influence of a methanolic extract (RC) on the induction of apoptosis in HEp-2 (human laryngeal carcinoma) cell line, as evidenced by cytotoxicity, morphological changes and modification in the levels of pro-oxidants. Inhibition of cell proliferation and lactate dehydrogenase (LDH) release increased in a time and dose-dependent manner. Further, reduced glutathione (GSH), glutathione transferase (GST) and protein levels decreased and lipid peroxidation increased significantly on RC treatment in a dose dependent manner when compared to controls. Based on the results we determined the optimal dose as 30 mg/ ml and the apoptotic effect of RC extract (30 mg/ml) on HEp-2 cells was confirmed by fluorescent microscopy and transmission electron microscopy (TEM) based on morphological and ultrastructural changes. RC extract suppressed the proliferation of HEp-2 oral cancer cells inducing apoptotic cell death in vitro. These results point to potential of RC extract as an agent for the treatment of laryngeal squamous cell carcinoma.