A restriction site-free cloning method has been developed for inserting a PCR product into a vector flexibly and precisely at any desired location with high efficiency. The method uses a pair of DNA integration primers with two portions. The 3' portion isolates the inserts by PCR, and the 5' portion integrates the PCR products into the homologous region of the vector. For mutagenesis, a third portion of mutation-generating sequences can be placed in between the 3' and 5' portions. This method has been used to clone the E. coli gene that codes for peptidyl-tRNA hydrolase, expressing it as a native protein and as a glutathione S-transferase fusion protein. It was also applied to convert a construct of the E. coli fatty acid biosynthesis protein with an N-terminal hexa-histidine tag into a construct with a C-terminal hexa-histidine tag.

An understanding of the postnatal development of hepatic UDP‐glucuronosyltransferase (UGT) enzymes is required for accurate prediction of the age‐dependent changes in pharmacokinetics of many drugs used in children. However, the maturation rate of hepatic UGT isoforms remains a major knowledge gap. This study aimed to establish the age‐associated changes in glucuronidation activity of 10 major hepatic UGT isoforms in humans, namely, UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17. Human liver microsomes from pediatric and adult donors were incubated under optimized incubation conditions to assess the activity rates of hepatic UGT isoforms using a panel of 19 in vitro UGT probe substrates and clinically used drugs. Statistically strong correlations of glucuronidation activities allowed the ontogeny of UGT1A1, UGT1A4, UGT2B7, UGT2B10, and UGT2B15 to be established using multiple selective UGT substrates and matched human liver microsome samples. The postnatal development of hepatic UGTs is isoform‐dependent using either individual or cross‐correlated selective isoform substrates. Maximal adult activity was reached at different times ranging from within a month (UGT1A1, UGT2B4, UGT2B7, UGT2B10, and UGT2B15), during infancy (UGT1A3, UGT1A4, and UGT1A9), to adolescence (UGT1A6 and UGT2B17). This study provides an extensive characterization of the postnatal ontogeny profiles of hepatic UGT enzymes that are instrumental for predicting drug disposition via in vitro–in vivo extrapolation algorithms and verifying pharmacokinetic predictions against in vivo observations via pediatric physiologically based pharmacokinetic modeling in pediatric patients.

UDP-glucuronosyltransferase (UGT)-mediated metabolism is possibly the most important conjugation reaction for marketed drugs.

BiP is a member of the Hsp7O heat shock protein family found in the lumen of the endoplasmic reticulum, that binds to a variety of proteins destined to be secreted. Substance P (SP) has been used as a model peptide to study the interaction of BiP with protein substrates. SP stimulates BiP ATPase activity and forms a stable complex with BiP that is dissociated in the presence of levels of ATP > 50 pM. At lower concentrations of ATP, the SP remains bound to BiP, and the results are consistent with the view that a BiP-ATP complex is initially formed that reacts with SP to form a ternary complex, SP-BiP-ATP. Hydrolysis of ATP in this complex yields a SP-BiP-ADP complex. An exchange of ATP with ADP bound to BiP has also been demonstrated, and the results suggest that the interactions of BiP with ATP resemble those seen with GTP-binding proteins and GTP.Heat shock proteins (Hsps) or stress proteins, such as members of the Hsp7O family, interact with proteins to facilitate proper folding and assembly within the cell (1-3). These proteins are known to interact with ATP (4, 5) and possess a week ATPase activity (6-9) that can be stimulated by peptides and proteins that bind to the chaperone (7,10,11). ATP binding and/or hydrolysis is also believed to be involved in the dissociation of the Hsp70-protein complex (1)(2)(3)(10)(11)(12)(13)(14)(15), and recently, several studies have examined the interaction of ATP, ADP, and polypeptides with members of the Hsp7O family (4,(10)(11)(12)(13)(14).One member of this group called BiP (immunoglobulin heavy chain binding protein) or Grp78 (78-kDa glucoseregulated protein) is found in the lumen of the endoplasmic reticulum (ER), where it interacts transiently with some, but not all, nascent polypeptides destined to be secreted (3,(16)(17)(18)(19)(20). However, BiP also forms much tighter complexes with malfolded proteins (e.g., mutated and underglycosylated proteins), unassembled polypeptides, etc. (21)(22)(23)(24). In these cases the BiP-protein complexes are so tight that transport through the ER is prevented. Previously we initiated experiments to investigate the interaction of ATP with BiP, using either BiP isolated from liver (IBiP) or a recombinant form (rBiP) made in Escherichia coli (9, 13). We showed, using gel-filtration chromatography, that ATP could dissociate BiP dimers to monomers and that ATP bound to the monomer species (9, 13). In addition, in cell extracts a BiP-IgE Fc complex that accumulates in the ER was dissociated by ATP to yield a BiP monomer (13 (11) proposed a similar scheme for the interaction of BiP with substrates. In the present study we show that the 11-amino acid peptide substance P (SP), which was reported to bind to BiP (27), acts as a model substrate for BiP interactions on the basis of criteria first described by Flynn et al. (7). A rapid filter-binding assay has been used to examine the effect of SP on nucleotide binding to BiP, and our results are consistent with an initial interaction of SP with a BiP-ATP complex and a subsequent series...

The expression of ten major drug-metabolizing UDP-glucuronosyl transferase (UGT) enzymes in a panel of 130 human hepatic microsomal samples was measured using a LC-MS/MS-based approach. Simultaneously, ten cytochrome P450s and P450 reductase were also measured and activity-expression relationships assessed for comparison. The resulting data sets demonstrated that, with the exception of UGT2B17, 10 th -90 th percentiles of UGT expression spanned 3-to 8fold ranges. These ranges were small relative to ranges of reported mean UGT enzyme expression across different laboratories. We tested correlation of UGT expression with enzymatic activities using selective probe substrates. A high degree of abundance-activity correlation (rs > 0.6) was observed for UGT1As (1A1, 3, 4, 6) and CYPs. In contrast, protein abundance and activity did not correlate strongly for UGT1A9 and UGT2B enzymes (2B4, 7, 10, 15 and 17). Protein abundance was strongly correlated for UGTs 2B7, 2B10 and 2B15. We suggest a number of factors may contribute to these differences including incomplete selectivity of probe substrates, correlated expression of these UGT2B isoforms, and the impact of splice and This article has not been copyedited and formatted. The final version may differ from this version.

Basimglurant, a novel mGlu5-negative allosteric modulator under development for the treatment of major depressive disorder, is cleared via cytochrome P450 (P450)-mediated oxidative metabolism. Initial enzyme phenotyping studies indicated that CYP3A4/5 dominates basimglurant metabolism and highlights a risk for drug-drug interactions when it is comedicated with strong CYP3A4/5 inhibitors or inactivators; however, a clinical drug-drug interaction (DDI) study using the potent and selective CYP3A4/5 inhibitor ketoconazole resulted in an area under the curve (AUC) AUCi/AUC ratio of only 1.24. A further study using the CYP3A4 inducer carbamazepine resulted in an AUCi/AUC ratio of 0.69. More detailed in vitro enzyme phenotyping and kinetics studies showed that, at the low concentrations attained clinically, basimglurant metabolic clearance is catalyzed mainly by CYP1A2. The relative contributions of the enzymes were estimated as 70:30 CYP1A2:CYP3A4/5. Using this information, a clinical study using the CYP1A2 inhibitor fluvoxamine was performed, resulting in an AUCi/AUC ratio of 1.60, confirming the role of CYP1A2 and indicating a balanced DDI risk profile. Basimglurant metabolism kinetics show enzyme dependency: CYP1A2-mediated metabolism follows Michaelis-Menten kinetics, whereas CYP3A4 and CYP3A5 follow sigmoidal kinetics [with similar constant (K) and S values]. The interplay of the different enzyme kinetics leads to changing fractional enzyme contributions to metabolism with substrate concentration, even though none of the metabolic enzymes is saturated. This example demonstrates the relevance of non-Michaelis-Menten P450 enzyme kinetics and highlights the need for a thorough understanding of metabolism enzymology to make accurate predictions for human metabolism in vivo.

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The enantiomeric forms of chiral compounds have identical physical properties but may vary greatly in their metabolism by individual enzymes. Enantioselectivity in UDP-glucuronosyl transferase (UGT) metabolism has been reported for a number of compounds and with different UGT isoforms involved. However, the impact of such individual enzyme results on overall clearance stereoselectivity is often not clear. The enantiomers of medetomidine, RO5263397, and propranolol and the epimers testosterone and epitestosterone exhibit more than 10-fold difference in glucuronidation rates by individual UGT enzymes. In this study we examined the translation of human UGT stereoselectivity to hepatic drug clearance considering the combination of multiple UGTs to overall glucuronidation, the contribution of other metabolic enzymes such as cytochromes P450, and the potential for differences in protein binding and blood/plasma partitioning. For medetomidine and RO5263397, the high individual enzyme (UGT2B10) enantioselectivity translated into ~3-to >10-fold differences in predicted human hepatic in vivo clearance. For propranolol the UGT enantioselectivity was irrelevant in the context of high cytochrome P450 (CYP) metabolism. For testosterone a complex picture emerged, due to differential epimeric selectivity of various contributing enzymes and potential for extrahepatic metabolism. Quite different patterns of CYP and UGT-mediated metabolism were observed across species, as well as differences in stereoselectivity, indicating that extrapolation from human enzyme and tissue data is essential when predicting human clearance enantioselectivity.