DNA-mediated coupling of ATPase, translocase and nuclease activities of a Type ISP restriction-modification enzyme.

Enzymes involved in nucleic acid transactions often have a helicase-like ATPase coordinating and driving their functional activities, but our understanding of the mechanistic details of their coordination is limited. For example, DNA cleavage by the antiphage defense system Type ISP restriction-modification enzyme requires convergence of two such enzymes that are actively translocating on DNA powered by Superfamily 2 ATPases. The ATPase is activated when the enzyme recognizes a DNA target sequence. Here, we show that the activation is a two-stage process of partial ATPase stimulation upon recognition of the target sequence by the methyltransferase and the target recognition domains, and complete stimulation that additionally requires the DNA to interact with the ATPase domain. Mutagenesis revealed that a ß-hairpin loop and motif V of the ATPase couples DNA translocation to ATP hydrolysis. Deletion of the loop inhibited translocation, while mutation of motif V slowed the rate of translocation. Both the mutations inhibited the double-strand (ds) DNA cleavage activity of the enzyme. However, a translocating motif V mutant cleaved dsDNA on encountering a translocating wild-type enzyme. Based on these results, we conclude that the ATPase-driven translocation not only brings two nucleases spatially close to catalyze dsDNA break, but that the rate of translocation influences dsDNA cleavage.

Enhanced Biopharmaceutical Performance of Rivaroxaban through Polymeric Amorphous Solid Dispersion.

Rivaroxaban (RXB) is an orally active direct inhibitor of the activated serine protease Factor Xa, given as monotherapy in the treatment of venous thromboembolism (VTE). It has been characterized in vitro as a substrate for the active, nonsaturable efflux via P-gp transporter, limiting its high permeability. Therefore, the role of P-gp inhibiting polymers in enhancing the biopharmaceutical performance of RXB by preparing polymeric amorphous solid dispersion and subsequent improvement in solubility and permeability was investigated. Initially, solubility parameter and Flory-Huggins interaction parameter were determined for miscibility studies between drug and polymers. Binary dispersions were prepared by dissolving drug with polymers eudragit S100, eudragit L100, and soluplus in common solvent (5% v/v water in tetrahydrofuran) using spray dryer. Prepared binary dispersions were analyzed by differential scanning calorimetry (DSC), microscopy, powder X-ray diffractometry (PXRD), Fourier transform infrared spectroscopy (FTIR), dynamic vapor sorption (DVS), and solution nuclear magnetic resonance (NMR) spectroscopy. Superior performance of binary dispersions was observed upon dissolution and solubility studies over micronized active pharmaceutical ingredient. Amorphous solid dispersion (ASD) prepared with soluplus showed 10-fold increase in apparent solubility and maintenance of supersaturation for 24 h compared to the crystalline RXB. Further, pharmacokinetic study performed in animals was in good correlation with the solubility data. Increases of 5.7- and 6.7-fold were observed in AUC and Cmax, respectively, for ASDs prepared with soluplus compared to those with crystalline RXB. FTIR and NMR spectroscopy unveiled the involvement of N-H group of RXB with C═O group of polymers in intermolecular interactions. The decreased drug efflux ratio was observed for ASDs prepared with eudragit S100 and soluplus in Caco-2 transport study suggesting improvement in the absorption of RXB. Hence, the present study demonstrates ASD using soluplus as a promising formulation strategy for enhancing the biopharmaceutical performance of RXB by increasing the solubility and circumventing the P-gp activity.

Potential of amphiphilic graft copolymer α-tocopherol succinate-g-carboxymethyl chitosan in modulating the permeability and anticancer efficacy of tamoxifen.

Recent studies showed an enhanced oral bioavailability of tamoxifen (TMX) by hydrophobically modified α-tocopherol succinate-g-carboxymethyl chitosan (Cmc-TS) micelles. As a continued effort, here we evaluated TMX-loaded polymeric micelles (TMX-PMs) for its enhanced permeability with increased anticancer efficacy and decreased hepatotoxicity. We employed co-solvent evaporation technique to encapsulate TMX into Cmc-TS. Apparent permeability assay of TMX-PMs was performed on Caco-2 cell line. The absorptive transport of TMX increased significantly about 3.8-fold when incorporated into Cmc-TS PMs. Cytotoxicity of Cmc-TS PMs was studied on MCF-7 cell line by MTT and; confocal microscopy was used for cellular uptake. Confocal microscopy revealed that Cmc-TS PMs could effectively accumulate in the cytosol of MCF-7 cell lines. In vitro data was further validated using N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis model in Sprague-Dawley rats. Hepatotoxicity profiles of TMX-PMs at three different doses were also evaluated against the free drug TMX. TMX-PMs were more effective in suppressing breast tumor in MNU-induced mammary carcinoma model than free TMX with better safety profile. In addition, histological data shows that tumors are "benign" in TMX-PMs treated group compared with "malignant" tumors in free TMX treated and control groups. Overall, the results implicate that our Cmc-TS PMs may serve as a promising carrier for the intracellular delivery of anticancer drug molecules via oral route.

Alpha-lipoic acid-stearylamine conjugate-based solid lipid nanoparticles for tamoxifen delivery: formulation, optimization, in-vivo pharmacokinetic and hepatotoxicity study.

OBJECTIVES: This study was designed to demonstrate the potential of novel α-lipoic acid-stearylamine (ALA-SA) conjugate-based solid lipid nanoparticles in modulating the pharmacokinetics and hepatotoxicity of tamoxifen (TMX). METHODS: α-lipoic acid-stearylamine bioconjugate was synthesized via carbodiimide chemistry and used as a lipid moiety for the generation of TMX-loaded solid lipid nanoparticles (TMX-SLNs). TMX-SLNs were prepared by solvent emulsification-diffusion method and optimized for maximum drug loading using rotatable central composite design. The optimized TMX-SLNs were stabilized using 10% w/w trehalose as cryoprotectant. In addition, pharmacokinetics and hepatotoxicity of freeze-dried TMX-SLNs were also evaluated in Sprague Dawley rats. KEY FINDINGS: Initial characterization with transmission electron microscopy revealed spherical morphology with smooth surface having an average particle size of 261.08 ± 2.13 nm. The observed entrapment efficiency was 40.73 ± 2.83%. In-vitro release study showed TMX release was slow and pH dependent. Pharmacokinetic study revealed a 1.59-fold increase in relative bioavailability as compared to TMX suspension. A decrease in hepatotoxicity of TMX is evidenced by the histopathological evaluation of liver tissues. CONCLUSIONS: α-lipoic acid-stearylamine conjugate-based SLNs have a great potential in enhancing the oral bioavailability of poorly soluble drugs like TMX. Moreover, this ALA-SA nanoparticulate system could be of significant value in long-term anticancer therapy with least side effects.

Synthesis and evaluation of antibacterial and antioxidant activity of novel 2-phenyl-quinoline analogs derivatized at position 4 with aromatically substituted 4H-1,2,4-triazoles.

A set of novel quinolone-triazole conjugates (12-31) were synthesized in three steps in good yields starting from 2-phenylquinoline-4-carboxylic acid. All the intermediates, as well as the final 1,2,4-triazolyl quinolines were fully characterized by their detailed spectral analysis utilizing different techniques such as IR, 1H NMR, 13C NMR, and finally mass spectrometry. All the synthesized compounds were evaluated in vitro for their potential antibacterial activity and their preliminary safety profile was assessed through cytotoxicity assay. Additionally, six selected conjugates were evaluated for their antioxidative properties on the basis of density functional theory calculations, using radical scavenging assay (DPPH) and cellular antioxidant assay. The reported results encourage further investigation of selected compounds and are shading light on their potential pharmacological use.

Novel aminoalkylated azaphenothiazines as potential inhibitors of T98G, H460 and SNU80 cancer cell lines in vitro.

A set of twenty-one novel aminoalkylated azaphenothiazines is synthesized using a two-step methodology starting from azaphenothiazines. The key step was the selective monoalkylation at position 10 of azaphenothiazines. In all, twenty-five molecules, including intermediates, were investigated for their in vitro anticancer activity, of which fourteen azaphenothiazines (2b, 3a, 3c, 3d, 3e-h, 3j, 3n, 3o, 3p, 3s, and 3u) were found to decrease the metabolic viability and growth of the T98G, H460 and SNU80 cancer cells effectively in a dose-dependent manner. In silico, pharmacokinetic studies suggest that these molecules have good bioavailability, water solubility and other drug-like parameters. Compounds 3a, 3c and 3g were identified as the leading molecules for future investigation due to their (a) high activity against T98G brain, H460 lung and SNU80 thyroid cancer cells; (b) low cytotoxicity with regard to non-malignant HEK293 and MRC5 cells; (c) low toxic risk levels based on in vitro and in silico evaluations; (d) good theoretical oral bioavailability according to Lipinski 'rule of five' pharmacokinetic parameters; and (e) better drug-likeness and drug-score values.

Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes.

Production of endonucleolytic double-strand DNA breaks requires separate strand cleavage events. Although catalytic mechanisms for simple, dimeric endonucleases are known, there are many complex nuclease machines that are poorly understood. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide after convergent ATP-driven translocation. We report the 2.7-Å resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are located upstream of the direction of translocation, an observation inconsistent with simple nuclease-domain dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex in which the nuclease domains are distal. Sequencing of the products of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand-nicking events combine to produce DNA scission.

An efficient and convenient microwave-assisted chemical synthesis of (thio)xanthones with additional in vitro and in silico characterization.

Xanthones and their thio-derivatives are a class of pleiotropic compounds with various reported pharmacological and biological activities. Although these activities are mainly determined in laboratory conditions, the class itself has a great potential to be utilized as promising chemical scaffold for the synthesis of new drug candidates. One of the main obstacles in utilization of these compounds was related to the difficulties in their chemical synthesis. Most of the known methods require two steps, and are limited to specific reagents not applicable to a large number of starting materials. In this paper a new and improved method for chemical synthesis of xanthones is presented. By applying a new procedure, we have successfully obtained these compounds with the desired regioselectivity in a shorter reaction time (50s) and with better yield (>80%). Finally, the preliminary in vitro screenings on different bacterial species and cytotoxicity assessment, as well as in silico activity evaluation were performed. The obtained results confirm potential pharmacological use of this class of molecules.

Development of quercetin nanoformulation and in vivo evaluation using streptozotocin induced diabetic rat model.

Quercetin-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (Qu-NP) were prepared by emulsion-diffusion-evaporation method and characterized as 179.9 ± 11.2 nm in size with 0.128 as polydispersity index, more than 86% drug entrapment efficiency, and zeta potential was -6.06 ± 1.51 mV. D-Trehalose (5% w/v) was found to be a suitable cryoprotectant for lyophilization of Qu-NP, and antioxidant assays indicated that Qu-NP were able to retain the antioxidant property similar to that of free drug at equivalent concentration after formulation development. In vitro release study of Qu-NP showed a controlled release pattern of quercetin. An enhanced oral bioavailability (523% relative increase) was observed in pharmacokinetic study with a 6-day sustained release from Qu-NP as compared to quercetin suspension, which indicated the reduced dosing frequency. Efficacy in diabetic rats suggested that same dose of Qu-NP on every fifth day was sufficient to bring effect similar to daily dose of oral quercetin suspension, and the same effect was also observed for catalase and superoxide dismutase levels in pancreas and kidneys. Thus, the system offers an efficacious oral therapy with reduced dose and dosing frequency for treatment of diabetes and is hence patient compliant.