Multiple e-Pharmacophore modeling to identify a single molecule that could target both streptomycin and paromomycin binding sites for 30S ribosomal subunit inhibition

The bacterial ribosome is an established target for anti-bacterial therapy since decades. Several inhibitors have already been developed targeting both defined subunits (50S and 30S) of the ribosome. Aminoglycosides and tetracyclines are two classes of antibiotics that bind to the 30S ribosomal subunit. These inhibitors can target multiple active sites on ribosome that have a complex structure. To screen putative inhibitors against 30S subunit of the ribosome, the crystal structures in complex with various known inhibitors were analyzed using pharmacophore modeling approach. Multiple active sites were considered for building energy-based three-dimensional (3D) pharmacophore models. The generated models were validated using enrichment factor on decoy data-set. Virtual screening was performed using the developed 3D pharmacophore models and molecular interaction towards the 30S ribosomal unit was analyzed using the hits obtained for each pharmacophore model. The hits that were common to both streptomycin and paromomycin binding sites were identified. Further, to predict the activity of these hits a robust 2D-QSAR model with good predictive ability was developed using 16 streptomycin analogs. Hence, the developed models were able to identify novel inhibitors that are capable of binding to multiple active sites present on 30S ribosomal subunit.     

Refolding and Purification of Zymomonas mobilis Levansucrase Produced as Inclusion Bodies in Fed-Batch Culture of Recombinant Escherichia coli

Zymomonas mobilis levansucrase was overproduced by the fed-batch culture of recombinant Escherichia coli harboring a novel expression system that is constitutively expressed by the promoter from the Rahnella aquatilis levansucrase gene. Most of the levansucrase was produced as inclusion bodies in the bacterial cytoplasm, accounting for approximately 20% of the total cellular protein. Refolding after complete denaturation by high concentrations of urea or guanidine hydrochloride was not successful, resulting in large amounts of insoluble aggregates. During the development of the refolding method, it was found that direct solubilization of the inclusion bodies with Triton X-100 reactivated the enzyme, with a considerable refolding efficiency. About 65% of inclusion body levansucrase was refolded into active levansucrase in the renaturation buffer containing 4% (v/v) Triton X-100. The in vitro refolded enzyme was purified to 95% purity by single-step DEAE–Sepharose ion exchange chromatography. Triton X-100 was removed by this ion exchange chromatography.     

Circulating tumor cell-derived organoids: Current challenges and promises in medical research and precision medicine

Traditional 2D cell cultures do not accurately recapitulate tumor heterogeneity, and insufficient human cell lines are available. Patient-derived xenograft (PDX) models more closely mimic clinical tumor heterogeneity, but are not useful for high-throughput drug screening. Recently, patient-derived organoid cultures have emerged as a novel technique to fill this critical need. Organoids maintain tumor tissue heterogeneity and drug-resistance responses, and thus are useful for high-throughput drug screening. Among various biological tissues used to produce organoid cultures, circulating tumor cells (CTCs) are promising, due to relative ease of ascertainment. CTC-derived organoids could help to acquire relevant genetic and epigenetic information about tumors in real time, and screen and test promising drugs. This could reduce the need for tissue biopsies, which are painful and may be difficult depending on the tumor location. In this review, we have focused on advances in CTC isolation and organoid culture methods, and their potential applications in disease modeling and precision medicine.     

Inhibition of poly(A)-binding protein synthesis in Friend erythroleukemia cells subsequent to heat shock

When Friend erythroleukemia cells (FEC) are incubated at 43 degrees C there is a rapid and nearly complete inhibition of protein synthesis which can be reversed when cells are returned to their normal growing temperature of 37 degrees C. Examination of the recovery of FEC from heat shock indicates that most cellular mRNAs behave as a cohort and return to translation at approximately the same rate. We found a notable exception to this rule in the case of a 78 kDa basic protein (named protein A) whose rate of return to a normal synthetic rate is markedly inhibited subsequent to heat shock. We show that protein A corresponds to the 78 kDa polypeptide commonly found to be associated with the poly(A) tails of mammalian mRNA.     

Five Xanthomonas type III effectors suppress cell death induced by components of immunity-associated MAP kinase cascades

Mitogen-activated protein kinase (MAPK) cascades play a fundamental role in signaling of plant immunity and mediate elicitation of cell death. Xanthomonas spp. manipulate plant signaling by using a type III secretion system to deliver effector proteins into host cells. We examined the ability of 33 Xanthomonas effectors to inhibit cell death induced by overexpression of components of MAPK cascades in Nicotiana benthamiana plants. Five effectors inhibited cell death induced by overexpression of MAPKKKα and MEK2, but not of MAP3Kϵ. In addition, expression of AvrBs1 in yeast suppressed activation of the high osmolarity glycerol MAPK pathway, suggesting that the target of this effector is conserved in eukaryotic organisms. These results indicate that Xanthomonas employs several type III effectors to suppress immunity-associated cell death mediated by MAPK cascades.     

Molecular docking,QSAR properties and DNA/BSA binding,anti-proliferative studies of 6-methoxy benzothiozole imine base and its metal complexes

Abstract

The molecular and QSAR (Quantitative Structure–Activity Relationship) properties of title compound 2-((6-Methoxybenzo[d]thiazol-2-ylimino)methyl)-6-ethoxyphenol (HL) were evaluated employing HyperChem 7.5 tools. The interaction of the 1a–1e complexes of HL with calf thymus DNA (CT-DNA) was investigated by absorption titrations, Fluorescence quenching and viscosity measurements. The experimental data suggest that these complexes bind to CT-DNA through an intercalative mode, wherein DNA-binding affinity of 1e is found to be greater compared to other complexes. The tryptophan emission-quenching with bovine serum albumin (BSA) experiment revealed stronger binding of 1e than other complexes in the hydrophobic region of protein. The photocleavage of plasmid pBR322 DNA investigated in the presence of the title complexes inferred conversion of supercoiled form of DNA plasmid to circular nicked form. Free-radical scavenging activity studies of HL and its metal complexes determined by their interaction with the stable free-radical DPPH have shown promising antioxidant property. Further cytotoxicity studies with HeLa and MCF-7 cell lines indicated that the compounds can efficiently inhibit the cell proliferation in a dose dependent manner. The DAPI staining assay studies revealed the higher potency of 1e to induce apoptosis. Abbreviations BSA Bovine serum albumin protein

CT-DNA Calf thymus DNA

DMSO Dimethyl sulfoxide

DAPI 4′,-6-Diamidino-2-phenylindole dihydrochloride

ESI–MS Electrospray ionization mass spectrometry

IC₅₀ Half-maximal inhibitory concentration

MBTYE 2-((6-methoxybenzo[d]thiazol-2-ylimino) methyl)-6-ethoxyphenol

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

PBS Phosphate-buffered saline

Tris Tris(hydroxymethyl)aminomethane

Communicated by Ramaswamy H. Sarma     

A Vaccine That Co-Targets Tumor Cells and Cancer Associated Fibroblasts Results in Enhanced Antitumor Activity by Inducing Antigen Spreading

Dendritic cell (DC) vaccines targeting only cancer cells have produced limited antitumor activity in most clinical studies. Targeting cancer-associated fibroblasts (CAFs) in addition to cancer cells may enhance antitumor effects, since CAFs, the central component of the tumor stroma, directly support tumor growth and contribute to the immunosuppressive tumor microenvironment. To co-target CAFs and tumor cells we developed a new compound DC vaccine that encodes an A20-specific shRNA to enhance DC function, and targets fibroblast activation protein (FAP) expressed in CAFs and the tumor antigen tyrosine-related protein (TRP)2 (DC-shA20-FAP-TRP2). DC-shA20-FAP-TRP2 vaccination induced robust FAP- and TRP2-specific T-cell responses, resulting in greater antitumor activity in the B16 melanoma model in comparison to monovalent vaccines or a vaccine encoding antigens and a control shRNA. DC-shA20-FAP-TRP2 vaccination enhanced tumor infiltration of CD8-positive T cells, and induced antigen-spreading resulting in potent antitumor activity. Thus, co-targeting of tumor cells and CAFs results in the induction of broad-based tumor-specific T-cell responses and has the potential to improve current vaccine approaches for cancer.