Stereoselective separation of underivatized and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatized amino acids using zwitterionic quinine and quinidine type stationary phases by liquid chromatography-High resolution mass spectrometry.

Amino acids play an important role in cellular processes and are building blocks for peptides and proteins, which take part in regulatory processes within each organism. Hence a large variety of biotechnologically or synthetically produced therapeutic drugs are peptides and proteins. Due to the chiral nature of amino acids and the large variety of common, uncommon and newly synthesized amino acid type compounds, stereoselective separation tools combined with mass spectrometric detection are important in research as well as purity control of therapeutics in industry. Since structural isomers and epimers of common amino acids are isobaric to each other, stereoselective separation is key to their identification. For this purpose zwitterionic quinine and quinidine type chiral stationary phases Chiralpak ZWIX(+) and Chiralpak ZWIX(-) were investigated for their separation performance for underivatized and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ) derivatized proteinogenic amino acids, uncommon amino acids and their isobaric analogs such as allo-threonine, homoserine, allo-isoleucine and homocysteine by HPLC-ESI-QTOF-MS. Cystine and homocystine were reduced with dithiothreitol and S-alkylated with iodoacetic acid and iodoacetamide. In general, derivatization with AQC and thiol alkylation increased the detection sensitivity and resolution of acidic, basic and polar amino acids significantly (e.g. separation factor of Asp increased from 1.00 to 2.29 for Asp-AQC). In addition, throughout this study a u-13C15N-L-amino acid metabolomics mixture was added to the DL-amino acid test solution and used as a co-eluting peak assignment standard to identify the corresponding u-12C14N-L-amino acid peak and hence determine the elution order of the enantiomer pairs for complex mixtures within a single run, employing the same separation conditions for underivatized and AQC-derivatized amino acids and their isobaric analogs.

Two sesquiterpene aminoquinones protect against oxidative injury in HaCaT keratinocytes via activation of AMPKα/ERK-Nrf2/ARE/HO-1 signaling.

AIMS: To investigate the cytoprotective effects of two sesquiterpene aminoquinones isolated from the marine sponge Dysidea fragilis, Dysidaminone H (DA8) and 3'-methylamino-avarone (DA14), we examined their effects against hydrogen peroxide (H2O2)-induced oxidative injury in human keratinocyte cell line and elucidated the underlying mechanisms. MAIN METHODS: Cell viability was detected using a CCK-8 assay kit. Intracellular reactive oxygen species (ROS) production was measured by fluorescence of 2, 7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA). Messenger RNA and protein expression were measured by real-time quantitative PCR and western blotting analysis. Immunocytochemistry was performed to determine the intracellular location of nuclear factorerythroid 2 p45 related factor 2 (Nrf2). The antioxidant response element (ARE)-luciferase reporter gene assay and RNA interference were used to establish the role of ARE and Nrf2. KEY FINDINGS: DA8 and DA14 (DAs) resisted H2O2induced decline of cell viability by inhibiting the accumulation of ROS. Meanwhile, DAs increased HO-1 expression and ARE activity and induced Nrf2 expression, as well as the accumulation of Nrf2 in the cell nucleus. However, silencing of Nrf2 abolished DAs-induced HO-1 expression and ARE luciferase activation. In addition, DAs induced the phosphorylation of both cyclic AMP-activated protein kinase-α (AMPKα) and extracellular signal-regulated kinase (ERK), while specific inhibitors of AMPKα and ERK abrogated HO1 upregulation and Nrf2 activation. SIGNIFICANCE: DAs provided cytoprotective effects against H2O2-induced cytotoxicity by activation of the Nrf2/ARE/HO-1 pathway via phosphorylation of AMPKα and ERK. The findings suggested that DA8 and DA14 might be the candidate therapeutic agents for skin diseases caused by oxidative injury.

Identification of a New Benzimidazole Derivative as an Antiviral against Hepatitis C Virus.

UNLABELLED: Aminoquinolines and piperazines, linked or not, have been used successfully to treat malaria, and some molecules of this family also exhibit antiviral properties. Here we tested several derivatives of 4-aminoquinolines and piperazines for their activity against hepatitis C virus (HCV). We screened 11 molecules from three different families of compounds, and we identified anti-HCV activity in cell culture for six of them. Of these, we selected a compound (B5) that is currently ending clinical phase I evaluation for neurodegenerative diseases. In hepatoma cells, B5 inhibited HCV infection in a pangenotypic and dose-dependent manner, and its antiviral activity was confirmed in primary hepatocytes. B5 also inhibited infection by pseudoparticles expressing HCV envelope glycoproteins E1 and E2, and we demonstrated that it affects a postattachment stage of the entry step. Virus with resistance to B5 was selected by sequential passage in the presence of the drug, and reverse genetics experiments indicated that resistance was conferred mainly by a single mutation in the putative fusion peptide of E1 envelope glycoprotein (F291I). Furthermore, analyses of the effects of other closely related compounds on the B5-resistant mutant suggest that B5 shares a mode of action with other 4-aminoquinoline-based molecules. Finally, mice with humanized liver that were treated with B5 showed a delay in the kinetics of the viral infection. In conclusion, B5 is a novel interesting anti-HCV molecule that could be used to decipher the early steps of the HCV life cycle. IMPORTANCE: In the last 4 years, HCV therapy has been profoundly improved with the approval of direct-acting antivirals in clinical practice. Nevertheless, the high costs of these drugs limit access to therapy in most countries. The present study reports the identification and characterization of a compound (B5) that inhibits HCV propagation in cell culture and is currently ending clinical phase I evaluation for neurodegenerative diseases. This molecule inhibits the HCV life cycle by blocking virus entry. Interestingly, after selection of drug-resistant virus, a resistance mutation in the putative fusion peptide of E1 envelope glycoprotein was identified, indicating that B5 could be used to further investigate the fusion mechanism. Furthermore, mice with humanized liver treated with B5 showed a delay in the kinetics of the viral infection. In conclusion, B5 is a novel interesting anti-HCV molecule that could be used to decipher the early steps of the HCV life cycle.

COTI-2, a novel small molecule that is active against multiple human cancer cell lines in vitro and in vivo.

Identification of novel anti-cancer compounds with high efficacy and low toxicity is critical in drug development. High-throughput screening and other such strategies are generally resource-intensive. Therefore, in silico computer-aided drug design has gained rapid acceptance and popularity. We employed our proprietary computational platform (CHEMSAS®), which uses a unique combination of traditional and modern pharmacology principles, statistical modeling, medicinal chemistry, and machine-learning technologies to discover and optimize novel compounds that could target various cancers. COTI-2 is a small molecule candidate anti-cancer drug identified using CHEMSAS. This study describes the in vitro and in vivo evaluation of COTI-2. Our data demonstrate that COTI-2 is effective against a diverse group of human cancer cell lines regardless of their tissue of origin or genetic makeup. Most treated cancer cell lines were sensitive to COTI-2 at nanomolar concentrations. When compared to traditional chemotherapy or targeted-therapy agents, COTI-2 showed superior activity against tumor cells, in vitro and in vivo. Despite its potent anti-tumor efficacy, COTI-2 was safe and well-tolerated in vivo. Although the mechanism of action of COTI-2 is still under investigation, preliminary results indicate that it is not a traditional kinase or an Hsp90 inhibitor.

Identification of LY83583 as a specific inhibitor of Candida albicans MPS1 protein kinase.

Candida albicans is the most common and virulent fungus causing candidiasis in various parts of the body and can be lethal to immunocompromised patients. All currently known antifungal therapies are drugs which cause serious side effects in the host. An inhibitor specific for fungus survival is an ideal therapeutic. C. albicans MPS1 (monopolar spindle 1) has been reported as a kinase essential to its survival. Because CaMps1p shares limited sequence homology with the human ortholog (hMps1p), we screened for a chemical inhibitor in anticipation of finding one with Candida specific cytotoxicity. In vitro screening using a recombinant catalytic domain of CaMps1p identified LY83583 (6-anilino-5,8-quinolinedione), known as a guanylate cyclase inhibitor, to be blocking CaMps1p kinase activity. In addition to its in vitro kinase inhibition, LY83583 reduced the growth rate of C. albicans. Finally, we compared the inhibitory activity on CaMps1p and hMps1p among inhibitors against those kinases. LY83583 showed specific inhibition for CaMps1p with no effect on hMps1p activity. Conversely, the CaMps1p activity was not affected by known hMps1p inhibitors. These findings suggest that CaMps1p may well be an ideal target molecule for antifungal therapy.

Screening a mushroom extract library for activity against Acinetobacter baumannii and Burkholderia cepacia and the identification of a compound with anti-Burkholderia activity.

BACKGROUND: Acinetobacter baumannii and species within the Burkholderia cepacia complex (BCC) are significant opportunistic bacterial pathogens of humans. These species exhibit a high degree of antibiotic resistance, and some clinical isolates are resistant to all currently available antimicrobial drugs used for treatment. Thus, new drugs are needed to treat infections by these species. Mushrooms could be a potential source for new drugs to treat A. baumannii and BCC infections. METHODS: The aim of this study was to screen a library of crude extracts from 330 wild mushrooms by disk diffusion assays for antibacterial activity against A. baumannii and Burkholderia cepacia in the hope of identifying a novel natural drug that could be used to treat infections caused by these species. Once positive hits were identified, the extracts were subjected to bioassay-guided separations to isolate and identify the active drug molecules. MICs were performed to gauge the in vitro activity of the purified compounds. RESULTS: Only three crude extracts (0.9%) had activity against A. baumannii and B. cepacia. Compounds from two of these extracts had MICs greater than 128 microg/ml, and further analyses were not performed. From the third extract, prepared from Leucopaxillus albissimus, 2-aminoquinoline (2-AQ) was isolated. This compound exhibited a modest MIC in vitro against strains from nine different BCC species, including multi-drug resistant clinical isolates (MIC = 8-64 microg/ml), and a weak MIC (128 microg/ml) against A baumannii. The IC50 against a murine monocyte line was 1.5 mg/ml. CONCLUSION: The small number of positive hits in this study suggests that finding a new drug from mushrooms to treat Gram-negative bacterial infections may be difficult. Although 2-AQ was identified in one mushroom, and it was shown to inhibit the growth of multi-drug resistant BCC isolates, the relatively high MICs (8-128 microg/ml) for both A. baumannii and BCC strains suggests that 2-AQ is not suitable for further drug development in its current form.

Capillary electrophoretic separation and computational modeling of inclusion complexes of beta-cyclodextrin and 18-crown-6 ether with primaquine and quinocide.

The antimalarial drug primaquine (PQ) and its contaminant, the positional isomer quinocide (QC) have been successfully separated using capillary electrophoresis with either beta-cyclodextrin (beta-CD) or 18-crown-6 ether (18C6) as chiral mobile phase additive. The interactions of the drugs with cyclodextrins and 18C6 were studied by the semiempirical method (Parametric Model 3) PM3. Theoretical calculations for the inclusion complexes of PQ and QC with alpha-CD, beta-CD and 18C6 were performed. Data from the theoretical calculations are correlated and discussed with respect to the electrophoretic migration behavior. More stable complexes are predicted for the PQ-beta-CD and PQ-18C6 complexes. The coelution of PQ and QC when alpha-CD was used as buffer additive can be explained by their comparable stabilities of the inclusion complex formed, while significant differences in the complexation stabilities of the drugs with beta-CD is responsible for their separation. The stronger hydrogen bonding in PQ-18C6 system is responsible for the separation between PQ and QC when 18C6 was used as chiral mobile phase additive.

Optimization of separation and detection of 6-aminoquinolyl derivatives of amino acids by using reversed-phase liquid chromatography with on line UV, fluorescence and electrochemical detection.

The combined use of UV-absorbance, fluorescence and electrochemical detection was proposed for the analysis of a set of thirteen amino acids by reversed-phase liquid chromatography (RP-HPLC) using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate as a precolumn derivatization reagent. The utility of using three detectors in series was demonstrated. The separation of all derivatized amino acids was optimized with the aid of a computer optimization program from only four simple linear gradient measurements. The effectiveness of a reliable retention prediction of solutes under any gradient profile using other gradient or isocratic data was also examined.

Evaluation of immobilized metal-ion affinity chromatography for the fractionation of natural Cu complexing ligands.

An immobilized metal-ion affinity chromatography (IMAC) method has been developed and validated for the separation of copper complexing ligands from soil solution. We first investigated the retention behavior of simple model ligands on the IMAC column and found that the ability to form ternary complexes of the structure Cu-IDA-ligand was the dominant factor influencing ligand retention on the IMAC column. The logK value of the Cu-complex was found to have only a minor influence on the retention. Legends containing only carboxylic acid functional groups were not retained on the column. To optimize reproducibility and quantitative recovery of copper ligands from soil solution, different composition and pH values of eluting buffer were tested. Soil solution chromatograms exhibited one non-retained fraction and two retained fractions. The elution times of the retained fractions were characteristic of peptides and proteins (first peak) and for compounds containing aromatic amines (second peak). The results show that IMAC is an effective tool for the fractionation of copper complexing ligands that are capable of forming ternary complexes.

Chemical purity and mutagenicity: case study of a drug in development.

During a routine Ames assay of a potential antipsychotic drug candidate, the compound appeared to be a frameshift mutagen in Salmonella typhimurium strains TA98 and TA1538. Additional testing indicated the mutagenic activity was due to one or more contaminants incurred during synthesis. While the compound was initially shown to be greater than 98% pure by high-performance liquid chromatography, the presence of small amounts (0.01-0.1%) of a highly mutagenic impurity produced positive mutagenicity results. The need to assess for chemical purity before discontinuing development of drug candidates found positive in the Ames assay is discussed.

Separation and identification of N4-(guanosin-7-yl)-4-aminoquinoline 1-oxide, a novel nucleic acid adduct of carcinogen 4-nitroquinoline 1-oxide.

A novel nucleic acid adduct of the carcinogen 4-nitroquinoline 1-oxide, N4-(guanosin-7-yl)-4-aminoquinoline 1-oxide, was separated and identified after RNA was treated with activated 4-hydroxyaminoquinoline 1-oxide. This is the first report of a 7-arylaminated guanine adduct produced by arylaminating carcinogens.

[The fluorimetric determination of dabequin in biological fluids]. / Fluorimetricheskoe opredelenie dabekhina v biologicheskikh zhidkostiakh.

A study of dabequin was performed to define its possible quantification by spectrofluorimetry. By this technique, dabequin was shown to present maximum excitation and emission wave length at 400 and 450 nm, respectively. Drug emission was determined at different concentrations of sulfuric acid and no changes were observed. Two quantification procedures are compared to determine, which one produces better results in quantifying total serum and blood levels in patients. Specificity of a thin-layer chromatography method, as well as the drug stability in 0.05 M sulfuric acid medium are shown.

Adducts from the reaction of O,O'-diacetyl or O-acetyl derivatives of the carcinogen 4-hydroxyaminoquinoline 1-oxide with purine nucleosides.

The diacetyl derivative of 4-hydroxyaminoquinoline 1-oxide (4-HAQO), the proximate carcinogen of 4-nitroquinoline 1-oxide, was reacted in vitro with purine nucleosides to give five adducts (three with guanosine and two with adenosine). The same nucleoside modifications were also obtained with a monoacetyl derivative of 4-HAQO which is probably 4-acetoxyaminoquinoline 1-oxide. The structure of the major adduct (the so-called dG III) was identified as N-(deoxyguanosin-C8-yl)-4-aminoquinoline 1-oxide. The isolation of this adduct from the 4-HAQO-modified DNA in vivo provides strong support for the hypothesis that the acetyl derivatives of 4-HAQO constitute a good model for the ultimate carcinogen.