Discovery and evaluation of novel nitrodihydroimidazooxazoles as promising anti-tuberculosis agents.

New analogues of antitubercular drug Delamanid were prepared, seeking drug candidates with enhanced aqueous solubility and high efficacy. The strategy involved replacement of phenoxy linker proximal to the 2-nitroimidazooxazole of Delamanid by piperidine fused 5 or 6-membered ring heterocycles (ring A). The new compounds were all more hydrophilic than Delamanid, and several class of analogues showed remarkable activities against M. bovis. And among these series, the tetrahydro-naphthyridine-linked nitroimidazoles displayed excellent antimycobacterial activity against both replicating (MABA) and nonreplicating (LORA) M. tb H37Rv and low cytotoxicity. Compared to Delamanid, these new compounds (6, 7, 45) demonstrated dramatically improved physicochemical properties and are suitable for further in vitro and in vivo evaluation.

Dimethyl Labeling Coupled with Mass Spectrometry for Topographical Characterization of Primary Amines on Monoclonal Antibodies.

Site-specific solvent accessibility of the primary amines (mainly lysine or the N-termini) on proteins is of great interest in many research areas because amines are an important functional group for protein conjugation. In this study, we coupled dimethyl labeling via reductive amination with liquid chromatography-mass spectrometry (LC-MS) to fully characterize the solvent accessibility of lysine residues and the N-termini on human immunoglobulin G (IgG). Circular dichroism (CD) and fluorescence spectroscopy revealed that dimethyl labeling did not alter the conformation of the native IgG molecule. Based on intact protein measurements, up to 28 (light chain) and 66 (heavy chain) dimethyl tags, covering all lysine residues and the N-termini, were sequentially incorporated into IgG molecules in 1000 s. All labeled sites were identified and quantified by a bottom-up proteomics approach. Some highly exposed hot-spots (for example, the N-termini of both the heavy and the light chains) and some buried sites (for example, K415 in the heavy chain and K39 in the light chain) were unambiguously revealed. This method was also used to characterize aggregation-induced structural changes in IgGs by increasing the temperature. Substantial changes in the labeling percentage of many lysine sites were observed, indicating a non-native aggregation triggered by thermal stress. Due to high labeling yields and the van der Waals surface of the labeling reagents being comparable to that of water, dimethyl labeling is a highly promising technique for probing the amine's surface topography of proteins. It can also be used as a complementary approach to other methods for resolving the higher-order structure of proteins by LC-MS.

In Vitro and in Vivo Inhibitory Effects of Glycyrrhetinic Acid in Mice and Human Cytochrome P450 3A4.

Glycyrrhetinic acid (GA) has been used clinically in the treatment of patients with chronic hepatitis. This study evaluated the effect of GA on the activity of five P450(CYP450) cytochrome enzymes: CYP2A6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, in human liver microsomes (HLMs) and recombinant cDNA-expressed enzyme systems using a HPLC-MS/MS CYP-specific probe substrate assay. With midazolam as the probe substrate, GA greatly decreased CYP3A4 activity with IC50 values of 8.195 µM in HLMs and 7.498 µM in the recombinant cDNA-expressed CYP3A4 enzyme system, respectively. It significantly decreased CYP3A4 activity in a dose- but not time-dependent manner. Results from Lineweaver-Burk plots showed that GA could inhibit CYP3A4 activity competitively, with a Ki value of 1.57 µM in HLMs. Moreover, CYP2C9 and CYP2C19 could also be inhibited significantly by GA with IC50 of 42.89 and 40.26 µM in HLMs, respectively. Other CYP450 isoforms were not markedly affected by GA. The inhibition was also confirmed by an in vivo study of mice. In addition, it was observed that mRNA expressions of the Cyps2c and 3a family decreased significantly in the livers of mice treated with GA. In conclusion, this study indicates that GA may exert herb-drug interactions by competitively inhibiting CYP3A4.

Enhancement of vascular formation but not improvement of ventricular function of infarcted rat hearts by a high dose of adenovirus-carried VEGF transgene.

The purpose of this study was to examine the influence of adenovirus-carried VEGF165 transgene at 5 x 10(10) pfu (Ad-VEGF) on vascular formation, cardiac geometry and ventricular function in infarcted hearts of the rat and to explore the mechanism of Ad-VEGF-mediated actions on ventricular function by quantitative proteomic analysis. Seven days after coronary occlusion, intramyocardial injection with normal saline (vehicle control), adenovirus-carried beta-galactosidase gene (Ad-LacZ, vector control) or Ad-VEGF to infarcted hearts was conducted. Seven days after intramyocardial injection, ventricular function, cardiac morphology and vascular density were assessed after echocardiographic analysis and immunohistological staining. One dimensional gel electrophoresis coupled with stable isotope dimethyl labeling and LC/MS/MS was used to quantify the abundance ratio of each protein pair in Ad-VEGF- and Ad-LacZ-treated hearts. Our data indicated that both Ad-VEGF and Ad-LacZ increased arteriolar densities. However, the former increased arterial densities but the latter did not. Compared with the vehicle control, Ad-LacZ reversed occlusion-induced wall thinning and functional impairment but Ad-VEGF did not. Quantitative proteomic analysis showed increased ratios of plasma proteins (such as albumin) and oxygen carriers (such as myoglobin) by Ad-VEGF and decreased ratios of proteins involved in glycolysis, calcium homeostasis and lipolysis by Ad-VEGF. Taken together, our functional, morphological and proteomic data suggest that intramuscular delivery of Ad-LacZ at higher doses may improve ventricular function and wall thinning with arteriolar formation. Excessive amounts of VEGF by Ad-VEGF may offset Ad-LacZ-induced improvement in ventricular functions by interfering with calcium homeostasis and lipolysis in infarcted hearts.

Synthesis and evaluation of tripeptidyl alpha-ketoamides as human rhinovirus 3C protease inhibitors.

We describe herein the synthesis and biological evaluation of a series of tripeptidyl alpha-ketoamides as human rhinovirus (HRV) 3C protease inhibitors. The most potent inhibitor discussed in this manuscript, 4I, exhibited impressive enzyme inhibitory activity as well as antiviral activity against HRV-14.