Identification of a novel GSK3ß inhibitor involved in abrogating KRas dependent pancreatic tumors in Wnt/beta-catenin and NF-kB dependent manner.

Pancreatic cancer is an aggressive malignancy with a poor survival rate because it is difficult to diagnose the disease during its early stages. The currently available treatments, which include surgery, chemotherapy and radiation therapy, offer only limited survival benefit. Pharmacological interventions to inhibit Glycogen Synthase Kinase-3beta (GSK3ß) activity is an important therapeutic strategy for the treatment of pancreatic cancer because GSK3ß is one of the key factors involved in the onset, progression as well as in the acquisition of chemoresistance in pancreatic cancer. Here, we report the identification of MJ34 as a potent GSK3ß inhibitor that significantly reduced growth and survival of human mutant KRas dependent pancreatic tumors. MJ34 mediated GSK3ß inhibition was seen to induce apoptosis in a ß-catenin dependent manner and downregulate NF-kB activity in MiaPaCa-2 cells thereby impeding cell survival and anti-apoptotic processes in these cells as well as in the xenograft model of pancreatic cancer. In vivo acute toxicity and in vitro cardiotoxicity studies indicate that MJ34 is well tolerated without any adverse effects. Taken together, we report the discovery of MJ34 as a potential drug candidate for the therapeutic treatment of mutant KRas-dependent human cancers through pharmacological inhibition of GSK3ß.

Discovery of a novel and highly selective JAK3 inhibitor as a potent hair growth promoter.

JAK-STAT signalling pathway inhibitors have emerged as promising therapeutic agents for the treatment of hair loss. Among different JAK isoforms, JAK3 has become an ideal target for drug discovery because it only regulates a narrow spectrum of γc cytokines. Here, we report the discovery of MJ04, a novel and highly selective 3-pyrimidinylazaindole based JAK3 inhibitor, as a potential hair growth promoter with an IC50 of 2.03 nM. During in vivo efficacy assays, topical application of MJ04 on DHT-challenged AGA and athymic nude mice resulted in early onset of hair regrowth. Furthermore, MJ04 significantly promoted the growth of human hair follicles under ex-vivo conditions. MJ04 exhibited a reasonably good pharmacokinetic profile and demonstrated a favourable safety profile under in vivo and in vitro conditions. Taken together, we report MJ04 as a highly potent and selective JAK3 inhibitor that exhibits overall properties suitable for topical drug development and advancement to human clinical trials.

Purification, functional characterization and enhanced production of serratiopeptidase from Serratia marcescens MES-4: An endophyte isolated from Morus rubra.

Serratiopeptidase, a proteolytic enzyme serves as an important anti-inflammatory and analgesic medication. Present study reports the production and purification of extracellular serratiopeptidase from an endophyte, Serratia marcescens MES-4, isolated from Morus rubra. Purification of the enzyme by Ion exchange chromatography led to the specific activity of 13,030 U/mg protein of serratiopeptidase, showcasing about 3.1 fold enhanced activity. The catalytic domain of the purified serratiopeptidase, composed of Zn coordinated with three histidine residues (His 209, His 213, and His 219), along with glutamate (Glu 210) and tyrosine (Tyr 249). The molecular mass, as determined by SDS-PAGE was ∼51 kDa. The purified serratiopeptidase displayed optimal activity at pH 9.0, temperature 50°C. Kinetic studies revealed Vmax and Km values of 33,333 U/mL and 1.66 mg/mL, respectively. Further, optimized conditions for the production of serratiopeptidase by Taguchi design led to the productivity of 87 U/mL/h with 87.9 fold enhanced production as compared to the previous conditions.

Orally bioavailable styryl derivative of rohitukine-N-oxide inhibits CDK9/T1 and the growth of pancreatic cancer cells.

The chromone alkaloid is one of the classical pharmacophores for cyclin-dependent kinases (CDKs) and represents the first CDK inhibitor to reach clinical trials. Rohitukine (1), a chromone alkaloid isolated from Dysoxylum binectariferum inspired the discovery of several clinical candidates. The N-oxide derivative of rohitukine occurs naturally, with no reports on its biological activity. Herein, we report isolation, biological evaluation, and synthetic modification of rohitukine N-oxide for CDK9/T1 inhibition and antiproliferative activity in cancer cells. Rohitukine N-oxide (2) inhibits CDK9/T1 (IC50 7.6 µM) and shows antiproliferative activity in the colon and pancreatic cancer cells. The chloro-substituted styryl derivatives, 2b, and 2l, inhibit CDK9/T1 with IC50 values of 0.17 and 0.15 µM, respectively. These derivatives display cellular antiproliferative activity in HCT 116 (colon) and MIA PaCa-2 (pancreatic) cancer cells with GI50 values of 2.5-9.7 µM with excellent selectivity over HEK293 (embryonic kidney) cells. Both analogs induce cell death in MIA PaCa-2 cells via inducing intracellular ROS production, reducing mitochondrial membrane potential, and inducing apoptosis. These analogs are metabolically stable in liver microsomes and have a decent oral pharmacokinetics in BALB/c mice. The molecular modeling studies indicated their strong binding at the ATP-binding site of CDK7/H and CDK9/T1.

Identification of a stretch of four discontinuous amino acids involved in regulating kinase activity of IGF1R.

IGF1R is pursued as a therapeutic target because of its abnormal expression in various cancers. Recently, we reported the presence of a putative allosteric inhibitor binding pocket in IGF1R that could be exploited for developing novel anti-cancer agents. In this study, we examined the role of nine highly conserved residues surrounding this binding pocket, with the aim of screening compound libraries in order to develop small-molecule allosteric inhibitors of IGF1R. We generated GFP fusion constructs of these mutants to analyze their impact on subcellular localization, kinase activity and downstream signaling of IGF1R. K1055H and E1056G were seen to completely abrogate the kinase activity of IGF1R, whereas R1064K and L1065A were seen to significantly reduce IGF1R kinase activity. During molecular dynamics analysis, various structural and conformational changes were observed in different conserved regions of mutant proteins, particularly in the activation loop, compromising the kinase activity of IGF1R. These results show that a stretch of four discontinuous residues within this newly identified binding pocket is critical for the kinase activity and structural integrity of IGF1R. This article has an associated First Person interview with the first author of the paper.

Prevalence, Risk Factors, and Molecular Detection of Campylobacter in Farmed Cattle of Selected Districts in Bangladesh.

A cross-sectional survey was conducted in selected districts of Bangladesh to estimate prevalence, risk factors, and molecular detection of Campylobacter isolates from 540 farmed cattle of 90 herds. As an individual sample, 540 feces, and as a pooled sample, 180 milk samples, 90 feed samples, 90 water samples, 90 manure samples, and 90 animal attendants' hand-rinse water were collected and tested via culture, biochemical, and molecular assays. A pretested semi-structured questionnaire was used to collect herd-level data on risk factors with the herd owners. The herd-level data on risk factors were analyzed through univariate and multivariate analyses, and a p-value <0.05 was considered statistically significant for all analyses. Overall, farm-level prevalence of bovine Campylobacter was enumerated to be 53.3% (95% confidence interval [CI]: 42.5-63.9%). The feces sample was found to be a high level of contamination of 30.9% (95% CI: 27-35%) followed by the manure swab (pooled) at 15.6% (95% CI: 8.8-24.7%). Campylobacter jejuni was documented as an abundant species (12.6%), followed by Campylobacter coli (5.1%), and Campylobacter fetus (0.3%). Older farms (>5 years of age), no/minimum cleaning and disinfection practices, along with animal roaming outside of the farm, were documented as significant risk factors for farm-level Campylobacter occurrence. Evidence-based control measures need to be taken through stringent biosecurity and hygienic measurement to lessen the load of the Campylobacter pathogen in the farm environment and prevent further transmission to animals and humans.

Purification and characterization of thermoactive serratiopeptidase from Serratia marcescens AD-W2.

Serratiopeptidase is a proteolytic enzyme extensively used as an anti-inflammatory and analgesic drug. Present work reports a thermoactive serratiopeptidase from Serratia marcescens AD-W2, a soil isolate from the North-Western Himalayan region of India. The extracellular metalloprotease has been purified by a simple two-step procedure resulting in a specific activity of 20,492 Units/mg protein with 5.28-fold purification. The molecular mass of the metalloprotease, as determined by SDS-PAGE was ~ 51 kDa. The purified serratiopeptidase presented optimum activity at pH 9.0, temperature 50 °C and stability in wide pH and temperature range. Critical temperature of 50 °C confirmed the thermoactivity of the purified serratiopeptidase. The kinetic studies of the purified serratiopeptidase revealed Vmax and Km of 57,256 Units/mL and 1.57 mg/mL, respectively, for casein. The purified serratiopeptidase from S. marcescens AD-W2 was found to be 100% identical to serralysin from Serratia marcescens ATCC 21074/E-15. The catalytic domain comprising of Zn coordinated with three histidine residues (His192, His196, His202), along with glutamate (Glu193) and tyrosine (Tyr232) residues, further confirmed that the purified protein is identical to serralysin.

Identification of a cross-talk between EGFR and Wnt/beta-catenin signaling pathways in HepG2 liver cancer cells.

EGFRis a transmembrane receptor tyrosine kinase involved in regulating cell proliferation, differentiation and survival. EGFR is actively pursued as a therapeutic target because its aberrant expression or activity has been reported in several cancers. Several studies have reported the nuclear localization of the EGFR in various cell types, however, its exact nuclear functions are not clear yet. In this study, we have generated GFP fusion constructs of EGFR and its mutants to analyze their subcellular localizationin normal and cancer cells and impact of its sub-cellular location on its various activities using immunoblotting, confocal microscopy, reporter assays, loss-of-function EGFR mutants, and EGFR specific small molecule inhibitors. We show that EGFR is involved in modulating TCF dependent ß-catenin transcriptional activity in HepG2 cells in a similar fashion as IGF1R tyrosine kinase. Moreover, we show that cytoplasmic and nuclear functions are two independent activities of EGFR.

Analyzing structural differences between insulin receptor (IR) and IGF1R for designing small molecule allosteric inhibitors of IGF1R as novel anti-cancer agents.

IR and insulin-like growth factor-1 receptor (IGF-1R) share high degree of sequence and structural similarity that hinders the development of anticancer drugs targeting IGF1R, which is dysregulated in many cancers. Although IR and IGF1R mediate their activities through similar signalling pathways, yet they show different physiological effects. The exact molecular mechanism(s) how IR and IGF1R exert their distinct functions remain largely unknown. Here, we performed in silico analysis and generated GFP-fusion proteins of wild type IR and its K1079R mutant to analyze their subcellular localization, cytoplasmic and nuclear activities in comparison to IGF1R and its K1055R mutant. We showed that, like K1055R mutation in IGF1R, K1079R mutation does not impede the subcellular localization and nuclear activities of IR. Although K1079R mutation significantly decreases the kinase activity of IR but not as much as K1055R mutation, which was seen to drastically reduce the kinase activity of IGF1R. Moreover, K1079 residue in IR is seen to be sitting in a pocket which is different than the allosteric inhibitor binding pocket present in its homologue (IGF1R). This is for the first time such a study has been conducted to identify structural differences between these receptors that could be exploited for designing small molecule allosteric inhibitor(s) of IGF1R as novel anti-cancer drugs.

Qualitative risk assessment of transmission pathways of highly pathogenic avian influenza (HPAI) virus at live poultry markets in Dhaka city, Bangladesh.

Analysis of environmental samples obtained from the Live Poultry Markets (LPMs) of Dhaka City, Bangladesh, has revealed that the highest degree of prevalence of highly pathogenic avian influenza A (HPAI, H5N1), besides other subtypes of the LPAI virus, poses the plausible risk of transmission of these viruses between human and poultry species. The present study was conducted using the OIE risk analysis framework to assess the risk level of each pathway successively. The estimated risk parameters were integrated towards to obtain the overall risk level for each specific HPAI transmission pathway using the matrix adapted by Cristobel Zepeda accompanying other expert consultations. The relevant data obtained from published and unpublished sources, together with survey data of field observations, were used to formulate and confirm the risk pathways and their associated risks. The results revealed that the risk of the release of the HPAI virus was medium when exposure was high. Additionally, the consequence would be considered very high with a medium degree of uncertainty for all parameters. Ultimately, the overall risk for transmission was estimated as medium with a medium degree of uncertainty. The findings of this study reveal that there is a significant threat that HPAI virus transmission could occur among poultry and humans and effectively sustain within the environment of the LPMs. Our findings are primarily focused on public health considerations, the hygienic slaughter of poultry and the relevant cleaning and sanitation practices conducted in the LPMs to support evidence-based decision-making processes. The findings of the study have the potential to be used to formulate effective risk reduction measures and can be further adapted in low-resource settings without major infrastructural changes required of the LPMs. All of which would reduce the risk of HPAI virus release and further lessen the degree of exposure and transmission in established LPMs.

A further outbreak of classical swine fever in indigenous pigs in Kurigram district, Bangladesh.

Deaths of native scavenging pigs were reported in mid-November 2015 at Nageswari sub-district, Kurigram district of Bangladesh. The investigation for a suspected classical swine fever (CSF) outbreak was accomplished via a joint outbreak investigation team from Department of Livestock Services (DLS) and Food and Agriculture Organization, Emergency Center for Transboundary Animal Disease (FAO-ECTAD), Bangladesh. Out of 592 pigs, 396 were infected and among them 263 died. The attack rate and case fatality rate were 66.9% and 66.4%, respectively. The epidemic curve constructed using the data captured from the CSF outbreak site was nearly bell-shaped, indicating a point source epidemic. The basic reproduction numbers (R0) were estimated to be 1.6 (95% Confidence interval [CI]: 1.5-1.7) and 1.5 (95% CI: 1.3-1.7) based on attack rate and exponential growth rate methods, respectively. Adult pigs showed signs of high fever, staggering gait and depression, whereas piglets either died without any premonitory signs or purulent exudates in the eyes were observed. Post-mortem examination was carried out on a 6-month-old piglet. The necropsy findings included were swollen lymph nodes deep red in colour, and haemorrhages on serous and mucous membranes of the intestinal organs together with button-like ulceration in the intestines. Nasal swabs and tissue samples (spleen, lung and liver) were tested using real-time reverse transcriptase polymerase chain reaction (RT-qPCR) and found to be positive for CSF virus. One-step RT-PCR was used to amplify 1,148 base pair of E2 gene in extracted RNA and was sequenced using standard Sanger's sequencing. Phylogenetic analysis revealed the virus as genotype 2.2 that clustered with CSF virus sequences from Bangladesh and India. This is only the second report of a CSF outbreak in Bangladesh. CSF appears to be an emerging transboundary disease in this country. A special programme for controlling swine diseases is needed since pigs are being reared by marginalized communities of Bangladesh to provide livelihoods and food security.

Identification of dinactin, a macrolide antibiotic, as a natural product-based small molecule targeting Wnt/ß-catenin signaling pathway in cancer cells.

PURPOSE: Despite the fact that hyper-activation of Wnt/ß-catenin signaling pathway has been seen in many cancers, including liver, colorectal and lung carcinoma, no small molecule inhibitors are available that specifically target this pathway. In this study, we analyzed the impact of dinactin (DA), an antibiotic ionophore produced by Streptomyces species, as an effective small molecule targeting Wnt/ß-catenin signaling pathway in cancer cells. METHODS: We performed MTT assays to investigate cell viability and proliferation after exposure to small molecules. Protein expression analysis was carried out by western blotting. Top-Flash reporter assays were used to score for ß-catenin signaling and cell cycle analysis was carried out by flow cytometry. RESULTS: In the first set of experiments, DA was seen to selectively inhibit the proliferation of HCT-116 and HepG2 cancer cells, unlike HEK-293 cells (a low tumorigenic cell line), in apoptosis-independent manner. Further, DA was seen to block the G1/S progression and decrease the expression of cyclin D1 in cancer cells. Since cyclin D1 is the downstream target gene of Wnt/ß-catenin signaling, we examined the impact of DA on TCF-dependent ß-catenin activity using Top-Flash reporter assay. Interestingly, DA significantly decreased Top-Flash activity at lower nano-molar concentrations when compared with salinomycin in HCT-116 and HepG2 cells. CONCLUSION: We report the identification of dinactin as a natural product-based small molecule that effectively blocks the Wnt/ß-catenin signaling pathway in cancer cells at nano-molar concentration. We anticipate that DA could be developed as a novel drug for anti-cancer therapy and for the management of neuropathic pain.

p110α and p110ß isoforms of PI3K are involved in protection against H2O2 induced oxidative stress in cancer cells.

PURPOSE: Phosphatidylinositol-3 kinases (PI3Ks) are involved in regulating cell growth, proliferation, differentiation, apoptosis and survival. p110α and p110ß, two ubiquitously expressed isoforms of PI3K signalling, are involved in growth factor mediated signaling and survival by generating second messengers. Earlier, we have generated GFP-fusion proteins of p110α and p110ß and expressed them in normal and cancer cell-lines to investigate their subcellular localization and their role in various activities. Here, we sought to examine the role of p110α and p110ß isoforms in protecting MCF-7 breast cancer cells against oxidative stress. MATERIAL METHODS: We performed cytotoxicity assays, DNA transfection, Plasmid DNA preparation, western blotting, flourscence microscopy and statistical analysis. RESULTS: To know whether p110α and p110ß are involved in protecting MCF-7 breast cancer cells against oxidative stress, we subjected MCF-7 cells to H2O2 treatment and observed a dose dependent decrease in cell viability and a marked increase in the levels of pro-apoptotic markers which include PARP, Bcl-2, Bax and procaspase-9. We then over-expressed recombinant GFP-fusion p110α and p110ß proteins in MCF-7 cells and observed a significant decrease in apoptosis and a concomitant increase in pAkt levels. CONCLUSION: We report the involvement of p110α and p110ß isoforms of Class 1A PI3K signalling in rescue from oxidative stress-induced apoptosis in MCF-7 cells in Akt dependent manner.