Constitutive activity of the metabotropic glutamate receptor 2 explored with a whole-cell label-free biosensor.

Label-free cellular assays using a biosensor provide new opportunities for studying G protein-coupled receptor (GPCR) signaling. As opposed to conventional in vitro assays, integrated receptor-mediated cellular responses are determined in real-time rather than a single downstream signaling pathway. In this study, we examined the potential of a label-free whole cell impedance-based biosensor system (i.e. xCELLigence) to study the pharmacology of one GPCR in particular, the mGlu2 receptor. This receptor is a target for the treatment of several psychiatric diseases such as schizophrenia and depression. After optimization of assay conditions to prevent interference of endogenous glutamate in the culture medium, detailed pharmacological assessments were performed. Concentration-response curves showed a concentration-dependent increase in impedance for agonists and positive allosteric modulators, whereas receptor inhibition by an antagonist or negative allosteric modulator resulted in a concentration-dependent decrease in cellular impedance. Interestingly, constitutive receptor activity was observed that was decreased by LY341495, which therefore behaved as an inverse agonist here, a property that was heretofore unappreciated. This was confirmed by concentration-dependent modulation of LY341495 potency and efficacy by a allosteric modulators. In summary, the use of the xCELLigence system to study mGlu2 receptor pharmacology was validated. This is the first class C GPCR to be characterized extensively by such method, opening new avenues to study receptor pharmacology including inverse agonism and demonstrating its value for future drug discovery efforts of mGlu receptors as well as other GPCRs.

High sensitivity and selectivity in quantitative analysis of drugs in biological samples using 4-column multidimensional micro-UHPLC-MS enabling enhanced sample loading capacity.

Sensitivity is often a critical parameter in quantitative bioanalyses in drug development. For liquid-chromatography-based methods, sensitivity can be improved by reducing the column diameter, but practical sensitivity gains are limited by the reduced sample loading capacity on small internal diameter (I.D.) columns. We developed a set-up that has overcome these limitations in sample loading capacity. The set-up uses 4 columns with gradually decreasing column diameters along the flow-path (2.1 â†’ 1 → 0.5 â†’ 0.15 mm). Samples are pre-concentrated on-line on a 2.1 mm I.D. trapping column and back flushed to a 1 mm I.D. UHPLC analytical column and separated. The peak(s) of interest are transferred using heartcutting to a second trapping column (0.5 mm I.D.), which is back-flushed to a 0.15 mm I.D. micro-UHPLC analytical column for orthogonal separation. The proof of concept of the set-up was demonstrated by the simultaneous analysis of midazolam and 1'-hydroxy midazolam in plasma by injection of 80 µL of protein precipitated plasma. The 4-column funnel set-up proved to be robust and resulted in a 10-50 times better sensitivity compared to a trap-elute approach and 250-500 fold better compared to direct micro-UHPLC analysis. A lower limit of quantification of 100 fg/mL in plasma was obtained for both probe compounds.

Comparison of triple quadrupole and high-resolution TOF-MS for quantification of peptides.

BACKGROUND: With an increased interest in peptides and proteins as potential new drug candidates, new approaches for sensitive and selective quantitative analysis are required. LC-MS/MS analysis provides a good alternative to immunoassays with reduced method development times and increased specificity. RESULTS: We have evaluated two state-of-the-art triple quadrupole and high-resolution TOF mass spectrometers with respect to their performance for quantification of six peptides (glufibrinopeptide B, somatostatin, enfuvirtide, TRI1144, C34 and exenatide). The peptides were spiked into protein-precipitated plasma supernatant. Triple quadrupole quantification was performed in SRM mode, and in high-resolution, MS narrow-width extracted chromatograms were generated for quantification. Specificity, accuracy, reproducibility and robustness were found to be comparable between the two instruments. The triple quadrupole instrument is still the most sensitive instrument for quantification of peptides with a median factor of about four-times higher sensitivity (based on LLOQ evaluation). CONCLUSION: Based on sensitivity, the newest generation triple quadrupole MS systems are still the preferred technology for quantification of peptides. Since the sensitivity difference between triple quadrupole instruments and the new-generation high-resolution TOF-MS instruments is minor, the latter offer a useful alternative whenever additional selectivity is preferred or the use of a generic approach not requiring method optimization is advantageous.

Identifying metabolite ions of peptide drugs in the presence of an in vivo matrix background.

BACKGROUND: Peptides represent a growing class of potential drugs. Information on metabolic clearance can be valuable for peptide drug development but different challenges are encountered with the identification of peptide metabolites in comparison to the process used for small-molecule therapeutics. RESULTS: Enfuvirtide was selected as a test compound and dosed intravenously at 2 mg/kg to rats. Plasma samples were collected and analyzed on two different quadrupole-TOF instruments in positive and negative ion modes. Different post-acquisition processing tools were evaluated to identify the metabolites of a peptide drug in the presence of an in vivo matrix. Charge state filtering and ion mobility extraction were applied to reduce the matrix background and combined with more comprehensive software tools generally used for large molecule analyses as well as tools designed for small-molecule metabolite identification work. CONCLUSION: Both ion mobility spectrometry and charge state filtration proved to be successful in extracting peptide ions and significantly reducing background signals. Both small- and large-molecule software tools contain specific capabilities that could be usefully combined in a single package for peptide metabolite identification.

A screening UHPLC-MS/MS method for the analysis of amyloid peptides in cerebrospinal fluid of preclinical species.

BACKGROUND: Amyloid peptides are established biomarkers for Alzheimer's disease but their analysis presents major challenges. RESULTS: In this article, we describe the use of ultra high-performance LC and API4000 triple quadrupole instrumentation for the quantification of amyloid peptides (Aß(1-38) and Aß(1-42)) in cerebrospinal fluid (CSF), using electrospray ionization with negative ion multiple reaction monitoring transitions. Sample preparation was simplified by the addition of acetonitrile and ammonium hydroxide. Although excellent sensitivity and precision was demonstrated, we observed differences in matrix suppression effects when using artificial CSF versus canine CSF for calibration curves and quality control samples. CONCLUSION: A case study shows that the method can be used to determine the relative levels of two key peptides (Aß(1-38) and Aß(1-42)) compared with their predose values (a screening assay) in support of preclinical studies.