In fission yeast, calcineurin dephosphorylates and activates the Prz1 transcription factor. Here, we identified the calcineurin-dependent response element (CDRE) in the promoter region of prz1 ؉ gene and monitored the calcineurin activity in living cells using a destabilized luciferase reporter gene fused to three tandem repeats of CDRE. Elevated extracellular CaCl 2 caused an increase in calcineurin activity with an initial peak and then approached a sustained constant level in a concentration-dependent manner. In CaCl 2 -sensitive mutants such as ⌬pmc1, the response was markedly enhanced, reflecting its high intracellular Ca 2؉ . Agents expected to induce Ca 2؉ influx showed distinct patterns of the CDRE-reporter activity, suggesting different mechanisms of calcineurin activation. Knockout of yam8 ؉ or cch1 ؉ encoding putative subunits of a Ca 2؉ channel abolished the activation of calcineurin upon exposure to various stimuli, including high extracellular NaCl and cell wall-damaging agents. However, knockout of yam8 ؉ or cch1 ؉ did not affect the activation of calcineurin upon stimulation by elevated extracellular Ca 2؉ . The Pck2 protein kinase C-Pmk1 mitogen-activate protein kinase pathway was required for the stimulation of calcineurin via Yam8/Cch1-mediated Ca 2؉ influx, but it was not required for the stimulation by elevated extracellular Ca 2؉ , suggesting two distinct pathways for calcineurin activation.

We performed a genomewide screen for altered sensitivity to antifungal drugs, including clotrimazole and terbinafine, that target ergosterol biosynthesis using a Schizosaccharomyces pombe gene deletion library consisting of 3,004 nonessential haploid deletion mutants. We identified 109 mutants that were hypersensitive and 11 mutants that were resistant to these antifungals. Proteins whose absence rendered cells sensitive to these antifungals were classified into various functional categories, including ergosterol biosynthesis, membrane trafficking, histone acetylation and deacetylation, ubiquitination, signal transduction, ribosome biosynthesis and assembly, regulation of transcription and translation, cell wall organization and biogenesis, mitochondrion function, amino acid metabolism, nucleic acid metabolism, lipid metabolism, meiosis, and other functions. Also, proteins whose absence rendered cells resistant to these antifungals were classified into functional categories including mitochondrion function, ubiquitination, membrane trafficking, cell polarity, chromatin remodeling, and some unknown functions. Furthermore, the 109 sensitive mutants were tested for sensitivity to micafungin, another antifungal drug that inhibits (1,3)-β-D-glucan synthase, and 57 hypersensitive mutants were identified, suggesting that these mutants were defective in cell wall integrity. Altogether, our findings in fission yeast have shed light on molecular pathways associated with the cellular response to ergosterol biosynthesis inhibitors and may provide useful information for developing strategies aimed at sensitizing cells to these drugs.

Covalent binding of reactive metabolites to cytochrome P450s (P450s) often causes their mechanism-based inactivation (MBI), resulting in drug-drug interactions or toxicity. The detection and identification of the P450 sites to which reactive metabolites bind would elucidate MBI mechanisms. We describe a proteomic approach using nano-LC/linear ion trap-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to characterize the binding of a reactive metabolite of raloxifene, which is a known P450 3A4 inhibitor, to the P450 3A4 isozyme. LTQ-FT analyses revealed that the metabolic reaction of raloxifene in a reconstituted P450 3A4 system formed a reactive metabolite adduct to P450 3A4 apoprotein, accompanied by a mass shift of 471 Da relative to intact P450 3A4 apoprotein. The reaction mixtures were digested with trypsin, and then the tryptic digests were analyzed by nano-LC-MS/MS. This technique revealed that VWGFYDGQQPVLAITDPDMIK (position 71-91) was a tryptic peptide modified by the reactive metabolite derived from raloxifene. The site of adduction with the reactive metabolite was further postulated to be the nucleophilic OH group of Tyr-75 of P450 3A4. A proteomic approach using LTQ-FT can yield direct information on the P450 3A4 modification site without radiolabeled compounds. In addition, this information can elucidate mechanisms involved in the covalent binding of reactive metabolites and the inactivation of P450 3A4.

A sample treatment procedure and high-sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for quantitative determination of nicardipine in human plasma were developed for a microdose clinical trial with nicardipine, a non-radioisotope labeled drug. The calibration curve was linear in the range of 1-500 pg/mL using 1 mL of plasma. Analytical method validation for the clinical dose, for which the calibration curve was linear in the range of 0.2-100 ng/mL using 20 microL of plasma, was also conducted. Each method was successfully applied to making determinations in plasma using LC/MS/MS after administration of a microdose (100 microg) and clinical dose (20 mg) to each of six healthy volunteers. We tested new approaches in the search for metabolites in plasma after microdosing. In vitro metabolites of nicardipine were characterized using linear ion trap-fourier transform ion cyclotron resonance mass spectrometry (LIT-FTICRMS) and the nine metabolites predicted to be in plasma were analyzed using LC/MS/MS. There is a strong possibility that analysis of metabolites by LC/MS/MS may advance to utilization in microdose clinical trials with non-radioisotope labeled drugs.

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