Studies on the optimization of expression and purification & Functional characterization of Class C – GPCRs

G-protein coupled receptor (GPCRs) is a multigene family consisting of more than 1000 genes. They are the most abundant membrane proteins found on a cell surface and are involved in several signaling pathways. In a cell, the signal is transduced by diverse activating endogenous ligands binding on the extracellular surface. This results in the uncoupling of G-proteins from the cytoplasmic loops, leading to the activation of the second messengers. GPCRs have enormous therapeutic importance due to their involvement in basic physiological processes including sensory perception, neurotransmission, metabolism, hormonal balance, etc. Structural and biochemical data are the pre-requisites for designing the drugs involving GPCRs. The current study was focused on the optimization of expression, purification and functional characterization of class-C GPCRs involved in neurotransmission. The first part of the study was aimed at optimizing the expression and purification of the ligand binding extracellular domain (ECD) of rat metabotropic GABAB1b receptor (GBR1bNT) in the recombinant baculovirus (RBV) and E.coli expression systems. GBR1bNT was modeled based on the crystal structure coordinates of ECD of metabotropic glutamate receptor (mGluR). Depending on this GBR1bNT model, molecular cloning strategies were developed for the expression of GBR1bNT. In both systems, GBR1bNT was well expressed and purified. The second part of the study was aimed at biochemical characterization of a putative cholesterol binding motif (pCBM) in Drosophila metabotropic glutamate receptor (DmGluRA). This pCBM might be involved in regulating the binding of DmGluRA to glutamate with high affinity. During the study, it was inferred that a 12- amino-acid amphipathic peptide containing the pCBM might be crucial for the activity of the receptor. Upon conducting [3H]-glutamate binding and detergent-resistant-membrane (DRM) association studies on different truncation constructs of DmGluRA (1-910 amino acids), the N-terminal construct (1-624 amino acids) containing the ECD with one single pCBM was found to be capable of binding glutamate in high affinity state as observed with the full length DmGluRA. Together this study shows that 1) Using an interdisciplinary approach (computational and experimental strategies) GBR1bNT was efficiently expressed and purified in both E.coli and RBV expression systems and 2) the role of pCBM was studied in DmGluRA receptor regulation

Propensity based classification: Dehalogenase and non-dehalogenase enzymes

The present work was designed to classify and differentiate between the dehalogenase enzyme to non–dehalogenases (other hydrolases) by taking the amino acid propensity at the core, surface and both the parts. The data sets were made on an individual basis by selecting the 3D structures of protein available in the PDB (Protein Data Bank). The prediction of the core amino acid were predicted by IPFP tool and their structural propensity calculation was performed by an in-house built software, Propensity Calculator which is available online. All datasets were finally grouped into two categories namely, dehalogenase and non-dehalogenase using Naïve Bayes, J-48, Random forest, K-means clustering and SMO classification algorithm. By making the comparison of various classification methods, the proposed tree method (Random forest) performs well with a classification accuracy of 98.88 % (maximum) for the core propensity data set. Therefore we proposed that, the core amino acid propensity could be approved as a novel potential descriptor for the classification of enzymes

A Randomised, Double Blind, Placebo-Controlled Pilot Study of Oral Artesunate Therapy for Colorectal Cancer.

BACKGROUND: Artesunate is an antimalarial agent with broad anti-cancer activity in in vitro and animal experiments and case reports. Artesunate has not been studied in rigorous clinical trials for anticancer effects. AIM: To determine the anticancer effect and tolerability of oral artesunate in colorectal cancer (CRC). METHODS: This was a single centre, randomised, double-blind, placebo-controlled trial. Patients planned for curative resection of biopsy confirmed single primary site CRC were randomised (n = 23) by computer-generated code supplied in opaque envelopes to receive preoperatively either 14 daily doses of oral artesunate (200 mg; n = 12) or placebo (n = 11). The primary outcome measure was the proportion of tumour cells undergoing apoptosis (significant if > 7% showed Tunel staining). Secondary immunohistochemical outcomes assessed these tumour markers: VEGF, EGFR, c-MYC, CD31, Ki67 and p53, and clinical responses. FINDINGS: 20 patients (artesunate = 9, placebo = 11) completed the trial per protocol. Randomization groups were comparable clinically and for tumour characteristics. Apoptosis in > 7% of cells was seen in 67% and 55% of patients in artesunate and placebo groups, respectively. Using Bayesian analysis, the probabilities of an artesunate treatment effect reducing Ki67 and increasing CD31 expression were 0.89 and 0.79, respectively. During a median follow up of 42 months 1 patient in the artesunate and 6 patients in the placebo group developed recurrent CRC. INTERPRETATION: Artesunate has anti-proliferative properties in CRC and is generally well tolerated

Human Lung Stem Cell-Based Alveolospheres Provide Insights into SARS-CoV-2-Mediated Interferon Responses and Pneumocyte Dysfunction

Tata and colleagues report defined conditions for long-term expansion and differentiation of adult human primary alveolar stem cells. Cultured AT2s are conducive to SARS-CoV-2 infection and elicit transcriptome-wide changes that mirror COVID-19 histopathology, including upregulation of inflammatory responses, cell death, and downregulation of surfactant expression, leading to pneumocyte dysfunction. © 2020 Elsevier Inc.Coronavirus infection causes diffuse alveolar damage leading to acute respiratory distress syndrome. The absence of ex vivo models of human alveolar epithelium is hindering an understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here, we report a feeder-free, scalable, chemically defined, and modular alveolosphere culture system for the propagation and differentiation of human alveolar type 2 cells/pneumocytes derived from primary lung tissue. Cultured pneumocytes express the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor angiotensin-converting enzyme receptor type-2 (ACE2) and can be infected with virus. Transcriptome and histological analysis of infected alveolospheres mirror features of COVID-19 lungs, including emergence of interferon (IFN)-mediated inflammatory responses, loss of surfactant proteins, and apoptosis. Treatment of alveolospheres with IFNs recapitulates features of virus infection, including cell death. In contrast, alveolospheres pretreated with low-dose IFNs show a reduction in viral replication, suggesting the prophylactic effectiveness of IFNs against SARS-CoV-2. Human stem cell-based alveolospheres, thus, provide novel insights into COVID-19 pathogenesis and can serve as a model for understanding human respiratory diseases

An Artemisinin-Derived Dimer Has Highly Potent Anti-Cytomegalovirus (CMV) and Anti-Cancer Activities

We recently reported that two artemisinin-derived dimers (dimer primary alcohol 606 and dimer sulfone 4-carbamate 832-4) are significantly more potent in inhibiting human cytomegalovirus (CMV) replication than artemisinin-derived monomers. In our continued evaluation of the activities of artemisinins in CMV inhibition, twelve artemisinin-derived dimers and five artemisinin-derived monomers were used. Dimers as a group were found to be potent inhibitors of CMV replication. Comparison of CMV inhibition and the slope parameter of dimers and monomers suggest that dimers are distinct in their anti-CMV activities. A deoxy dimer (574), lacking the endoperoxide bridge, did not have any effect on CMV replication, suggesting a role for the endoperoxide bridge in CMV inhibition. Differences in anti-CMV activity were observed among three structural analogs of dimer sulfone 4-carbamate 832-4 indicating that the exact placement and oxidation state of the sulfur atom may contribute to its anti-CMV activity. Of all tested dimers, artemisinin-derived diphenyl phosphate dimer 838 proved to be the most potent inhibitor of CMV replication, with a selectivity index of approximately 1500, compared to our previously reported dimer sulfone 4-carbamate 832-4 with a selectivity index of about 900. Diphenyl phosphate dimer 838 was highly active against a Ganciclovir-resistant CMV strain and was also the most active dimer in inhibition of cancer cell growth. Thus, diphenyl phosphate dimer 838 may represent a lead for development of a highly potent and safe anti-CMV compound

Role of Transferrin Receptor and the ABC Transporters ABCB6 and ABCB7 for Resistance and Differentiation of Tumor Cells towards Artesunate

The anti-malarial artesunate also exerts profound anti-cancer activity. The susceptibility of tumor cells to artesunate can be enhanced by ferrous iron. The transferrin receptor (TfR) is involved in iron uptake by internalization of transferrin and is over-expressed in rapidly growing tumors. The ATP-binding cassette (ABC) transporters ABCB6 and ABCB7 are also involved in iron homeostasis. To investigate whether these proteins play a role for sensitivity towards artesunate, Oncotest's 36 cell line panel was treated with artesunate or artesunate plus iron(II) glycine sulfate (Ferrosanol®). The majority of cell lines showed increased inhibition rates, for the combination of artesunate plus iron(II) glycine sulfate compared to artesunate alone. However, in 11 out of the 36 cell lines the combination treatment was not superior. Cell lines with high TfR expression significantly correlated with high degrees of modulation indicating that high TfR expressing tumor cells would be more efficiently inhibited by this combination treatment than low TfR expressing ones. Furthermore, we found a significant relationship between cellular response to artesunate and TfR expression in 55 cell lines of the National Cancer Institute (NCI), USA. A significant correlation was also found for ABCB6, but not for ABCB7 in the NCI panel. Artesunate treatment of human CCRF-CEM leukemia and MCF7 breast cancer cells induced ABCB6 expression but repressed ABCB7 expression. Finally, artesunate inhibited proliferation and differentiation of mouse erythroleukemia (MEL) cells. Down-regulation of ABCB6 by antisense oligonucleotides inhibited differentiation of MEL cells indicating that artesunate and ABCB6 may cooperate. In conclusion, our results indicate that ferrous iron improves the activity of artesunate in some but not all tumor cell lines. Several factors involved in iron homeostasis such as TfR and ABCB6 may contribute to this effect