Design,synthesis and biological evaluation of α-substituted isonipecotic acid benzothiazole analogues as potent bacterial type II topoisomerase inhibitors

The discovery and optimisation of a new class of benzothiazole small molecules that inhibit bacterial DNA gyrase and topoisomerase IV are described. Antibacterial properties have been demonstrated by activity against DNA gyrase ATPase and potent activity against Staphylococcus aureus, Enterococcus faecalis, Streptococcus pyogenes and Haemophilus influenzae. Further refinements to the scaffold designed to enhance drug-likeness included analogues bearing an α-substituent to the carboxylic acid group, resulting in excellent solubility and favourable pharmacokinetic properties.     

NAGbinder: An approach for identifying N‐acetylglucosamine interacting residues of a protein from its primary sequence

N‐acetylglucosamine (NAG) belongs to the eight essential saccharides that are required to maintain the optimal health and precise functioning of systems ranging from bacteria to human. In the present study, we have developed a method, NAGbinder, which predicts the NAG‐interacting residues in a protein from its primary sequence information. We extracted 231 NAG‐interacting nonredundant protein chains from Protein Data Bank, where no two sequences share more than 40% sequence identity. All prediction models were trained, validated, and evaluated on these 231 protein chains. At first, prediction models were developed on balanced data consisting of 1,335 NAG‐interacting and noninteracting residues, using various window size. The model developed by implementing Random Forest using binary profiles as the main principle for identifying NAG‐interacting residue with window size 9, performed best among other models. It achieved highest Matthews Correlation Coefficient (MCC) of 0.31 and 0.25, and Area Under Receiver Operating Curve (AUROC) of 0.73 and 0.70 on training and validation data set, respectively. We also developed prediction models on realistic data set (1,335 NAG‐interacting and 47,198 noninteracting residues) using the same principle, where the model achieved MCC of 0.26 and 0.27, and AUROC of 0.70 and 0.71, on training and validation data set, respectively. The success of our method can be appraised by the fact that, if a sequence of 1,000 amino acids is analyzed with our approach, 10 residues will be predicted as NAG‐interacting, out of which five are correct. Best models were incorporated in the standalone version and in the webserver available at https://webs.iiitd.edu.in/raghava/nagbinder/     

Expression pattern of an evolutionarily conserved splice variant in the rat Tacc2 gene

The transforming acidic coiled‐coil containing protein 2 (Tacc2) gene and its paralogs, Tacc1 and Tacc3 encode proteins that are associated with the centrosome and involved in microtubule assembly during the cell cycle. Tacc2 produces several splice variants, which are poorly characterized, especially in the rat. Characterization of the temporal/spatial expression patterns of these isoforms would be useful in understanding their distinct and overlapping functions. By comparative sequence analyses of Tacc2 in multiple species, we identified a third splice variant in rat, which is much shorter in size (1,021 aa) than the longest isoform (2,834 aa). This newly identified Tacc2 splice variant (isoform 3) uses a distinct first exon and generates a different open reading frame. Although Isoform 3 is expressed predominantly during developmental stages, the long Tacc2 isoform (isoform 1) is distributed mainly in adult tissues. Multiple protein sequence analyses revealed that Tacc2 Isoform 3 could be the ancient form, as it is conserved in mammals, birds, and amphibians; whereas the long Tacc2 isoforms may have evolved in the mammalian lineage by adding exons toward the 5′ region of the ancient isoform. genesis 52:378–386, 2014. © 2014 Wiley Periodicals, Inc.     

Investigation of Critical Core Formulation and Process Parameters for Osmotic Pump Oral Drug Delivery

Push–pull osmotic pump (PPOP) tablets of a practically insoluble model drug were developed and the effect of various formulation and process parameters on tablet performance was evaluated in order to identify critical factors. The formulation factors such as the viscosity grade of polyethylene oxide as the primary polymer as well as the level and location of osmogen within the bilayer tablets led to a difference in performance of osmotic tablets and hence should be critically evaluated in the design of such dosage forms. Modification of granulation process, i.e., the granulating liquid composition or drying method of granules, did not impact the drug release from the osmotic tablets at the evaluated scale of this study. The influence of varying dose and aqueous solubility of other model drugs (i.e., theophylline, acetaminophen, and verapamil HCl) on the developed PPOP template was also investigated. Results showed that irrespective of the perceived complexity of development and manufacturing of osmotic pumps, the osmotic tablets in this study demonstrated a robust and yet flexible platform in accommodating different types of drug candidates, regardless of solubility, for the dose levels below 25% w/w of the pull layer formulation.     

MUC1 extracellular domain confers resistance of epithelial cancer cells to anoikis

Anoikis, a special apoptotic process occurring in response to loss of cell adhesion to the extracellular matrix, is a fundamental surveillance process for maintaining tissue homeostasis. Resistance to anoikis characterises cancer cells and is a pre-requisite for metastasis. This study shows that overexpression of the transmembrane mucin protein MUC1 prevents initiation of anoikis in epithelial cancer cells in response to loss of adhesion. We show that this effect is largely attributed to the elongated and heavily glycosylated extracellular domain of MUC1 that protrudes high above the cell membrane and hence prevents activation of the cell surface anoikis-initiating molecules such as integrins and death receptors by providing them a mechanically ‘homing'' microenvironment. As overexpression of MUC1 is a common feature of epithelial cancers and as resistance to anoikis is a hallmark of both oncogenic epithelial–mesenchymal transition and metastasis, MUC1-mediated cell resistance to anoikis may represent one of the fundamental regulatory mechanisms in tumourigenesis and metastasis.Anoikis, the apoptotic process that occurs in cells that have lost adhesion to the extracellular matrix (ECM),^1,2 is a fundamental process for maintaining tissue homeostasis. It removes displaced epithelial/endothelial cells and thus prevents them from seeding to inappropriate sites. Resistance to anoikis contributes prominently to tumourigenesis and, in particular, to metastasis by allowing survival of cancer cells that have invaded into the blood or lymphatic circulation and thus facilitating their metastatic spread to remote sites.³Initiation of anoikis starts from the cell surface through activation of the cell surface anoikis-initiating molecules, for example, integrins, cadherins and death receptors, in response to loss of cell adhesion. Loss of the integrin-mediated cell basement matrix contact,⁴ loss of the E-cadherin-mediated cell–cell contact^5,6 or ligation of the cell surface death receptors with their ligands^4,7 all induce conformational changes or oligomerization of these cell surface anoikis-initiating molecules. This triggers a series of events leading to activation of either the caspase-8-mediated extrinsic apoptotic signalling pathway or the mitochondrion-mediated intrinsic apoptotic signalling pathway.MUC1 is a large transmembrane mucin protein that is expressed exclusively on the apical side of normal epithelial and some other cell types. MUC1 consists of a large extracellular domain, a transmembrane region and a short cytoplasmic tail. The MUC1 extracellular domain contains a variable number of tandem repeats that are heavily glycosylated (up to 50% of the MUC1 molecular weight) with complex O-linked mucin-type glycans⁸ and flanked by a unique N-terminal domain and an SEA domain. In the SEA domain, autocleavage takes place resulting in a heterodimer but both moieties remain firmly attached. The cytoplasmic tail of MUC1 contains 72 amino acids and harbours several phosphorylation sites and is able to interact with various growth factor receptors and intracellular signalling proteins.^{9, 10, 11}MUC1 is overexpressed up to at least 10-fold in epithelial cancers¹² and overexpression of MUC1 is closely associated with high metastatic potential and poor prognosis in many cancer types.¹³ In epithelial cancer cells, MUC1 loses its apical membrane polarization and becomes expressed over the entire cell surface.^14,15 In epithelial cancer cells, MUC1 also shows reduced expression of complex O-glycans and increased expression of short oncofetal oligosaccharides such as GalNAc-α (Tn antigen), sialylated GalNAc-α (sialyl-Tn antigen) and Galβ1,3GalNAc-α (Thomsen–Friedenreich, TF antigen).¹⁶ Immunological targeting of cancer-associated MUC1 has been under intensive investigation as a strategy for cancer treatment.^17,18 Our recent studies have shown that interaction of TF antigen on cancer-associated MUC1 with the galactoside-binding galectins promotes metastasis by enhancing tumour cell heterotypic adhesion to the vascular endothelium and also by increasing tumour cell homotypic aggregation for the potential formation of tumour emboli.^19–21In this report, we describe a new role of MUC1 in anoikis. We show that overexpression of MUC1 in epithelial cells prevents initiation of anoikis in response to loss of cell adhesion, an effect that is found to be attributed substantially to the MUC1 extracellular domain.     

Protein Array Patterning by Diffusive Gel Stamping

Proteins and small molecules are the effectors of physiological action in biological systems and comprehensive methods are needed to analyze their modifications, expression levels and interactions. Systems-scale characterization of the proteome requires thousands of components in high-complexity samples to be isolated and simultaneously probed. While protein microarrays offer a promising approach to probe systems-scale changes in a high-throughput format, they are limited by the need to individually synthesize tens of thousands of proteins. We present an alternative technique, which we call diffusive gel (DiG) stamping, for patterning a microarray using a cellular lysate enabling rapid visualization of dynamic changes in the proteome as well protein interactions. A major advantage of the method described is that it requires no specialized equipment or in-vitro protein synthesis, making it widely accessible to researchers. The method can be integrated with mass spectrometry, allowing for the discovery of novel protein interactions. Here, we describe and characterize the sensitivity and physical features of DiG-Stamping. We demonstrate the biologic utility of DiG-Stamping by (1) identifying the binding partners of a target protein within a cellular lysate and by (2) visualizing the dynamics of proteins with multiple post-translational modifications.