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Aromatic amino acids and their derivatives are diverse primary and secondary metabolites with critical roles in protein synthesis, cell structure and integrity, defense and signaling. All de novo aromatic amino acid production relies on a set of ancient and highly conserved chemistries. Here we introduce a new enzymatic transformation for l-tyrosine synthesis by demonstrating that the β-subunit of tryptophan synthase—which natively couples indole and l-serine to form l-tryptophan—can act as a latent ‘tyrosine synthase’. A single substitution of a near-universally conserved catalytic residue unlocks activity toward simple phenol analogs and yields exclusive paracarbon–carbon bond formation to furnish l-tyrosines. Structural and mechanistic studies show how a new active-site water molecule orients phenols for a nonnative mechanism of alkylation, with additional directed evolution resulting in a net >30,000-fold rate enhancement. This new biocatalyst can be used to efficiently prepare valuable l-tyrosine analogs at gram scales and provides the missing chemistry for a conceptually different pathway to l-tyrosine.

Cytochrome P450 3A4 (CYP3A4) is the most important drug-metabolizing enzyme in humans and has been associated with harmful drug interactions. The activity of CYP3A4 is known to be modulated by several compounds and by the electron transfer partner, cytochrome P450 reductase (CPR). The underlying mechanism of these effects, however, is poorly understood. We have used hydrogen–deuterium exchange mass spectrometry to investigate the impact of binding of CPR and of three different substrates (7-benzyloxy-4-trifluoromethyl-coumarin, testosterone, and progesterone) on the conformational dynamics of CYP3A4. Here, we report that interaction of CYP3A4 with substrates or with the oxidized or reduced forms of CPR leads to a global rigidification of the CYP3A4 structure. This was evident from the suppression of deuterium exchange in several regions of CYP3A4, including regions known to be involved in protein–protein interactions (helix C) and substrate binding and specificity (helices B′ and E, and loop K/β1). Furthermore, the bimodal isotopic distributions observed for some CYP3A4-derived peptides were drastically impacted upon binding to CPR and/or substrates, suggesting the existence of stable CYP3A4 conformational populations that are perturbed by ligand/CPR binding. The results have implications for understanding the mechanisms of ligand binding, allostery, and catalysis in CYP enzymes.

Because of its exceptional substrate promiscuity, human P450 3A4 (CYP3A4) is arguably the most important drug-metabolizing enzyme. CYP3A4 also has the particularity of binding multiple ligands simultaneously, which is associated with heterotropic or homotropic, positive or negative, cooperativity or allostery. Solving the kinetics of such complex systems remains challenging, and so is identifying the binding pockets involved. Progesterone (PRG) is a known allosteric activator of CYP3A4-catalyzed 7-benzyloxy-4-trifluoromethylcoumarin (BFC) debenzylation. We report herein the use of bioconjugation as a successful strategy to identify this PRG allosteric site. A progesterone analogue (PGM) was covalently attached, separately at several locations, near a peripheral binding pocket previously proposed to be an allosteric site. Studies of BFC debenzylation in the presence of free PRG revealed that two of the bioconjugates successfully positioned the covalently attached PGM moiety in a way that mimics the allosteric activation observed with free PRG. Interestingly, the PGM bioconjugate with the better fit yielded a higher permanent activation of the enzyme.

The use of sub‐2‐μm particle columns for fast high throughput metabolite ID applications was investigated. Three LC‐MS methods based on different sub‐2‐μm particle size columns using the same analytical 3 min gradient were developed (Methods A, B, and C). Method A was comprised of a 1.8 μm particle column coupled to an MS, methods B and C utilized a 1.7 μm particle column (BEH 50×2.1 mm2id) and 1.8 μm particle column coupled to a Q‐TOF MS. The precision and the separation efficiency of the methods was compared with repeated standard injections (N= 10) of reference compounds verapamil (VP), propranolol, and fluoxetine. Separation efficiency and MS/MS spectral quality were also evaluated for separation and detection of VP and its two major metabolites norverapamil (NVP) and O‐demethylverapamil (ODMVP) in human‐liver microsomal incubates. Results show that 1.8 μm particle columns show similar performance for separation of VP and its major metabolites and comparable spectral quality in MSEmode of the Q‐TOF instrument compared to 1.7 μm particle columns. Additionally, the study also confirmed that sub‐2‐μm particle size columns can be operated with standard analytical HPLC but that performance is maximized by integrating column in UPLC method with reduced void volumes. All the methods are suitable for the determination of major metabolites for compounds with high metabolic turnover. The high throughput metabolite profile analysis using 384‐well plate format of up to 48 compounds in incubates of human‐liver microsomes was discussed.

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Many opioids are substrates of the efflux transporter P-glycoprotein (P-gp) in the blood–brain barrier (BBB). In situ brain perfusion in wild-type and mdr 1a(−/−) P-gp-deficient mice was utilized to investigate potential P-gp-mediated transport of novel nonpeptidic δ agonists (AR-M δ compounds). Because radioactive compounds were not available for this series, liquid chromatography–mass spectrometric detection (LC–MS) was the assay methodology of choice. Verapamil in the perfusion buffer (0.5 μM) served as a positive control for P-gp-mediated efflux and as an experimental internal standard for P-gp modulation by AR-M δ compounds. LC–MS provided excellent assay sensitivity with no significant interferences. In P-gp-competent mice, the brain extraction of AR-M δ compounds ranged from 1.1 to 96%. The ratio of initial brain uptake clearances (Clup) in P-gp-deficient and wild-type mice (P-gp effect) ranged from 0.96 to 4.91. Some compounds increased the Clup of verapamil in P-gp-competent mice, consistent with P-gp inhibition. These results demonstrate that LC–MS is an appropriate assay methodology for mouse brain perfusion samples, that AR-M δ compounds may interact with P-gp in the BBB, and that the internal strategy can provide useful information concerning P-gp modulation by compounds of interest. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:244–252, 2002

Many opioids are substrates of the efflux transporter P‐glycoprotein (P‐gp) in the blood–brain barrier (BBB). In situbrain perfusion in wild‐type and mdr 1a(−/−) P‐gp‐deficient mice was utilized to investigate potential P‐gp‐mediated transport of novel nonpeptidic δ agonists (AR‐M δ compounds). Because radioactive compounds were not available for this series, liquid chromatography–mass spectrometric detection (LC–MS) was the assay methodology of choice. Verapamil in the perfusion buffer (0.5 μM) served as a positive control for P‐gp‐mediated efflux and as an experimental internal standard for P‐gp modulation by AR‐M δ compounds. LC–MS provided excellent assay sensitivity with no significant interferences. In P‐gp‐competent mice, the brain extraction of AR‐M δ compounds ranged from 1.1 to 96%. The ratio of initial brain uptake clearances (Clup) in P‐gp‐deficient and wild‐type mice (P‐gp effect) ranged from 0.96 to 4.91. Some compounds increased the Clupof verapamil in P‐gp‐competent mice, consistent with P‐gp inhibition. These results demonstrate that LC–MS is an appropriate assay methodology for mouse brain perfusion samples, that AR‐M δ compounds may interact with P‐gp in the BBB, and that the internal strategy can provide useful information concerning P‐gp modulation by compounds of interest. © 2002 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:244–252, 2002

Purpose. This study assessed the influence of mdr1aP-glycoprotein (P-gp) gene disruption, gender and concentration on initial brain uptake clearance (Clup) of morphine, quinidine and verapamil. Methods. Clupof radiolabeled substrates was determined in P-gp-competent and deficient [mdr1a(−/−)] mice by in situbrain perfusion. Brain:plasma distribution of substrates after i.v. administration was determined in both strains. Results. Genetic disruption of mdr1aP-gp resulted in 1.3-, 6.6- and 14-fold increases in Clupfor morphine, verapamil and quinidine, respectively. With the exception of small differences for verapamil, gender did not affect Clup. Saturable transport of verapamil and quinidine was observed only in P-gp-competent mice, with apparent IC50values for efflux of 8.6 ± 2.3 μM and 36 ± 2 μM, respectively. VerapamilClupwas ∼50% higher in mdr1a(+/−) vs.mdr1a(+/+) mice; no such difference was observed for quinidine. In P-gp-competent mice, uptake of verapamil and quinidine was unaffected by organic vehicles. Plasma decreased VER Clupto a greater extent in the presence of P-gp. The influence of P-gp in situwas lower than, but correlated with, the effect in vivo. Conclusions. P-gp decreases Clupof morphine, verapamil and quinidine in situwith little or no influence of gender, but this effect cannot fully account for the effects of P-gp in vivo. P-gp is the only saturable transport mechanism for verapamil and quinidine at the murine blood-brain barrier. The influence of protein binding on Clupmay be enhanced by P-gp-mediated efflux.

We have identified a novel subtype of galanin receptor (GALR-2) in rat dorsal root ganglia and spinal cord. The open reading frame of GALR-2 is 1116 nucleotides long, encoding a protein of 372 amino acids with a theoretical molecular mass of 40.7 kD. Membranes prepared from stable pools of 293 cells expressing GALR-2, but not wild-type 293 cells, demonstrated high affinity galanin binding sites. Rat galanin and galanin-related peptides M40, C7, M15, and galanin1-16effectively competed for binding; peptide C7 demonstrated a lower affinity for rGALR-2, and all these peptides were agonists at rGALR-2 when assessed on a microphysiometer. Studies on the expression of GALR-2 in various tissues by Northern and in situhybridization analyses suggest a low abundance but wide distribution of GALR-2 mRNA, including several discrete areas in brain and spinal cord and a high abundance in the dorsal root ganglia.

This study examined diverse aspects of social functioning relevant to depressives' interpersonal difficulties, including the realism of perceptions of social competency. Sixteen dysphoric clients, 25 dysphoric students, and 25 nondepressed students evaluated their social distress and avoidance, and self-efficacy expectations prior to an interaction task with confederates. Following the interaction, subjects rated their social competency and were rated by their partners and external observers, who also assessed subjects' specific behavioral skills. Dysphorics relative to nondepressives reported significantly greater social distress and avoidance, as well as lower social performance expectancies, which were negatively correlated. However, neither self-, partner, nor observer ratings discriminated between the groups' social performance or specific verbal and nonverbal skills. Moreover, dysphoric students' self-evaluations were in accord with observers' ratings, whereas clients and nondepressed students overestimated their social competency. All subjects' self-ratings were in agreement with the perceptions of their interactive partners. Results are discussed in relation to previous research, and support the potential role of negative expectancies and situational determinants in accounting for dysphoric/depressive social functioning.

The pharmacokinetics of the Rand Senantiomers of ifosfamide (IFF) and of its 2- and 3-N-dechloroethylated metabolites (2-DCE-IFF and 3-DCE-IFF) were investigated in 14 cancer patients treated with a 3-h infusion of (R,S)-IFF (3 g/m2) with mesna uroprotection. An enantioselective gas chromato-graphic-mass spectrometric (GC-MS) assay was used to determine the concentrations in plasma and urine. The AUCs of (R)-IFF were significantly larger than those of (S)-IFF (2480 ± 200 vs 1960 ± 150 µM. h). The terminal half-lives (7.57 ± 0.99 h) and mean residence times (11.17 ± 1.10 h) of (R)-IFF were significantly longer than those of (S)-IFF, 6.03 ± 0.82 h and 9.37 ± 0.88 h, respectively. The mean volume of distribution at steady state of (R)-IFF (25.68 ± 0.80 1/m2) was slightly smaller than that of (S)-IFF (27.35 ± 0.89 1/m2). While the renal clearances of (R)-IFF and (S)-IFF were similar, the nonrenal clearance was significantly lower for (R)-IFF (30.20 ± 2.70 vs 41.40 ± 3.55 ml/m2per min) as was total clearance (41.52 µ 2.90 vs 52.37 µ 3.75 ml/m2per min). The AUC values for all of the DCE metabolites from (S)-IFF were significantly greater than those from (R)-IFF with 47% of the measured AUC accounted for by DCE from (S)-IFF compared to only 20% for (R)-IFF. Therefore, the enantioselective difference in IFF elimination can be partially explained by differences in N-dechloroethylation.

Abstract:  The cumulative urinary excretions of the enantiomers of ifosfamide [(R)-IFF, (S)-IFF)] and their 2-N-dechloroethylated (2-DCE-IFF) and 3-N-dechloroethylated (3-DCE-IFF) metabolites were determined in 11 adult cancer patients who received a single 3-h infusion of IFF (3 g/m2) with mesna uroprotection. The urine samples were analyzed for the compounds of interest using an enantioselective gas chromatographic- mass spectrometric assay. The results indicated an enantioselective excretion of the parent and N-dechloroethylated metabolites: the urinary recovery of (R)-IFF was significantly greater than that of (S)-IFF (1.73 0.45 vs 1.430.41 mmol, P<0.0001); the excretion of (S)-2-DCE-IFF (0.750.53 mmol) was greater than that of (R)-2-DCE-IFF (0.42 0.22 mmol, P=0.071) while the excretion of (R)-3-DCE-IFF (1.64 0.76 mmol) was greater than that of (S)-3-DCE-IFF (0.770.59 mmol, P=0.012). The study also revealed two distinct metabolic patterns in which the urinary recoveries of (R)-2-DCE-IFF and (R)-3-DCE-IFF were linked as were those of (S)-2-DCE-IFF and (S)-3-DCE-IFF. The results suggest that at least two enzymes are involved in the N-dechloroethylation of IFF. The data also demonstrate the importance of following the metabolic fate of (R)-IFF and (S)-IFF and of determining the relative urinary excretion of all dechloroethylated metabolites.

Aims The study was designed to evaluate tamoxifen metabolic profiles in 25 patients (13 M, 12 F) suffering from recurrent high‐grade cerebral astrocytomas who were treated with high oral doses of tamoxifen (120 mg/m2twice daily).

To determine the influence of sampling site on atracurium pharmacokinetic-pharmacodynamic relationships, blood was drawn simultaneously from the radial artery and a peripheral vein during a 20-minute period after injection of atracurium, 0.2 mg/kg, in eight patients. Atracurium and laúdanosme concentrations were measured by HPLC. Neuromuscular blockade was measured at the adductor pollicis, after stimulation of the ulnar nerve. Venous levels were lower than corresponding arterial values for up to 20 minutes, and this difference was marked for the early samples. Neuromuscular blockade was maximum after 5 to 7 minutes, much later than the peak venous concentration (1 to 3 minutes). Nonparametric analysis yielded (mean ± SEM) a rate constant, concentration for 50% blockade, and slope of the effect-concentration relationship of 0.092 ±0.01 min-1, 379 ± 27 ng/ml, and 7.3 ± 1.67, respectively, when based on arterial samples. The values were statistically different (0.135 ± 0.011 min-1, 235 ± 42 ng/ml, and 3.41 ± 0.37, respectively) when venous levels were used (p < 0.05). It is concluded that forearm venous levels do not correspond to adductor pollicis neuromuscular blockade and the kinetics and kinetic-dynamic relationship for atracurium are heavily dependent on sampling site.

The pharmacokinetics and pharmacodynamics of atracurium, a nondepolarizing neuromuscular blocking agent, were compared between morbidly obese patients and nonobese patients. Atracurium besylate (0.2 mg/kg) was administered intravenously as a bolus to patients who had received anesthesia. The force of contraction of the adductor pollicis was measured and plasma samples were collected for a 2-hour period. The concentrations of atracurium and its major end product, laudanosine, were determined by use of a chromatographic method. The pharmacokinetic-pharmacodynamic relationship was characterized by use of several models. No difference was observed between obese patients and nonobese patients in atracurium elimination half-life (19.8 ± 0.7 versus 19.7 ± 0.7 minutes), volume of distribution at steady state (8.6 ± 0.7 versus 8.5 ± 0.7 L), and total clearance (444 ± 29 versus 404 ± 25 ml/min). However, if values were expressed on a total body weight basis, there was a difference between obese and nonobese patients in the volume of distribution at steady state (0.067 versus 0.141 L/kg) and total clearance (3.5 ± 0.2 versus 6.6 ± 0.5 ml/min/kg). Although atracurium concentrations were consistently higher in obese patients than in nonobese patients, there was no difference in the time of recovery from neuromuscular blockade between the two groups. Consequently, the median effective concentration was higher in obese than in nonobese patients (470 ± 46 versus 312 ± 33 ng/ml).Clinical Pharmacology and Therapeutics (1990) 48, 18–25; doi:10.1038/clpt.1990.112

Aims The study was designed to evaluate tamoxifen metabolic profiles in 25 patients (13 M, 12 F) suffering from recurrent high-grade cerebral astrocytomas who were treated with high oral doses of tamoxifen (120 mg/m2 twice daily).

We report a novel approach to study allostery which combines the use of carefully selected bioconjugates and hydrogen–deuterium exchange mass spectrometry (HDX-MS). This strategy avoids issues related to weak substrate binding and ligand relocalization. The utility of our method is demonstrated using human cytochrome P450 3A4 (CYP3A4), the most important drug-metabolizing enzyme. Allosteric activation and inhibition of CYP3A4 by pharmaceuticals is an important mechanism of drug interactions. We performed HDX-MS analysis on several CYP3A4-effector bioconjugates, some of which mimic the allosteric effect of positive effectors, while others show activity enhancement even though the label does not occupy the allosteric pocket (agonistic) or do not show activation while still blocking the allosteric site (antagonistic). This allowed us to better define the position of the allosteric site, the protein structural dynamics associated with allosteric activation, and the presence of coexisting conformers.

For Abstract see ChemInform Abstract in Full Text.