Synthesis of a β-turn mimetic suitable for incorporation in the peptide hormone LHRH

LHRH is a decapeptide hormone which plays a central role in neuroendocrinology. Conformational studies have suggested that LHRH may adopt a β-turn involving residues 5-8 when bound to its receptor. A β-turn mimetic with side chains corresponding to those of a Tyr-Gly-Leu-Orn tetrapeptide has therefore been synthesized for incorporation at positions 5-8 in LHRH. In the turn mimetic, residues i and i+1 are connected by a ψ[CH₂O] isostere instead of an amide bond, while a covalent ethylene bridge replaces the hydrogen bond which is often found between residues i and i+3 in β-turns. The turn mimetic was assembled from three types of building blocks: an azido aldehyde, an Fmoc protected amino acid and a protected dipeptide amine.     

Thiazolidinediones with thyroid hormone receptor agonistic activity.

Several thiazolidinedione derivatives (3-7) were designed and synthesized as candidate thyromimetic drugs. Among them, the dihydrogenated compounds, such as 5-2-[[4-(3-tert-butyl-4-hydroxyphenyl)oxy-3,5-diiodophenyl] ethyl]-2,4-thiazolidinedione (6b) and its 3-isopropyl analog (7b), exhibited potent thyroid hormone receptor alpha 1 (TR alpha 1) activation activity.     

Analysis of frankincense from various Boswellia species with inhibitory activity on human drug metabolising cytochrome P450 enzymes using liquid chromatography mass spectrometry after automated on-line extraction

In our search for herbal remedies with inhibitory activity on cytochrome P450 (CYP) enzymes, we identified extracts of the gum-resin of Boswellia carteri, Boswellia frereana, Boswellia sacra and Boswellia serrata as equally potent, non-selective inhibitors of the major drug metabolising CYP enzymes 1A2/2C8/2C9/2C19/2D6 and 3A4. LC/LC/ESI-MS fingerprint analyses of the boswellic acids 11-keto-beta-boswellic acid, alpha-boswellic acid, beta-boswellic acid and their 3-O-acylated derivatives were used for the authentication of the commercially obtained frankincense samples. Although the boswellic acids could be identified as moderate to potent inhibitors of the applied CYP enzymes, they are not the major CYP inhibitory principle of frankincense.     

Application to drug-food interactions of living cells as in vitro model expressing cytochrome P450 activity: enzyme inhibition by lemon juice

Classical inhibitors of human cytochrome P450 3A4 activity, such as ketoconazol and quercetin, are tested to prove the efficiency of a new metabolisation model using living entire cells. Grapefruit juice is a well-known potent inhibitor of cytochrome P450 3A4 activity. With regard to the clinical relevance of grapefruit juice-drug interactions, an investigation of other common juices is undertaken with this in vitro model. The CYP3A4 activity is measured by the formation of the 6beta-hydroxytestosterone, which is quantified by an isocratic high performance liquid chromatography. It is demonstrated for the first time that lemon juice significantly inhibits by 60+/-3% the CYP3A4-mediated oxidation. Grapefruit juice inhibits this activity by 82+/-4%. The mechanism of lemon juice inhibition is competitive, whereas it is mixed for grapefruit juice. These results suggest that our in vitro model combined with our analytical method is applicable for the investigation of the inhibition of CYP3A4 not only by chemical inhibitors but also by natural food products.     

Diaporthichalasin, a novel CYP3A4 inhibitor from an endophytic Diaporthe sp.

A novel CYP3A4 inhibitor, diaporthichalasin, together with pycnidione were isolated from an endophytic fungus, Diaporthe sp. Their structures were elucidated on the basis of spectral data and the structure of diaporthichalasin was confirmed by X-ray crystallographic analysis. Diaporthichalasin exhibited significantly potent inhibition of CYP3A4 with an IC₅₀ value of 0.626 μM, while the IC₅₀ value of pycnidione was 465 μM.     

Rapid determination of six metabolites from multiple cytochrome P450 probe substrates in human liver microsome by liquid chromatography/mass spectrometry: application to high-throughput inhibition screening of terpenoids

A rapid liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the determination of six cytochrome P450 (CYP) probe substrate metabolites including paracetamol (PAR) for CYP1A2, 4-hydroxytolbutamide (OHTOL) for CYP2C9, 5-hydroxyomeprazole (OHOMe) for CYP2C19, dextrorphan (DEXM) for CYP2D6, 6-hydroxychlorzoxazone (OHCHL) for CYP2E1 and dehydronifedipine (DNIF) for CYP3A4. The triple-quadrupole mass spectrometer was operated in both positive and negative modes, and selective reaction monitoring was used for quantification. The method was validated over the concentration ranges (0.075/0.04/0.05/0.02/0.1/0.0625 microM to 4.8/2.56/3.2/1.28/6.4/4.0 microM) for PAR/OHTOL/OHOME/DEXP/OHCHL/DNIF analytes with acceptable accuracy and precision. The inhibitory effect on the six CYP enzymes has been verified with their known specific inhibitors. This high-throughput inhibition screening approach has been successfully applied to study the inhibitory effects of 18 terpenoids on CYP enzymes. Among them, tanshinone IIA and cryptotanshinone are found to be potent inhibitors to CYP1A2, while artemisinin is a marginal inhibitor to CYP1A2 and glycyrrhetic acid is a weak inhibitor to CYP2C9.     

Corydaline inhibits multiple cytochrome P450 and UDP-glucuronosyltransferase enzyme activities in human liver microsomes

Corydaline is a bioactive alkaloid with various antiacetylcholinesterase, antiallergic, and antinociceptive activities found in the medicinal herb Corydalis Tubers. The inhibitory potential of corydaline on the activities of seven major human cytochrome P450 and four UDP-glucuronosyltransferase enzymes in human liver microsomes was investigated using LC-tandem MS. Corydaline was found to inhibit CYP2C19-catalyzed S-mephenytoin-4'-hydroxylatoin and CYP2C9-catalyzed diclofenac 4-hydroxylation, with K(i) values of 1.7 and 7.0 mM, respectively. Corydaline also demonstrated moderate inhibition of UGT1A1-mediated 17b-estradiol 3-glucuronidation and UGT1A9-mediated propofol glucuronidation with K(i) values of 57.6 and 37.3 mM, respectively. In the presence of corydaline, CYP3A-mediated midazolam hydroxylation showed a decrease with increasing preincubation time in a dose-dependent manner with K(i) values of 30.0 mM. These in vitro results suggest that corydaline should be evaluated for potential pharmacokinetic drug interactions in vivo due to potent inhibition of CYP2C19 and CYP2C9.     

Modulating the coupling efficiency of human cytochrome P450 CYP3A4 at electrode surfaces through protein engineering

This study shows that regulating the electron flow to the heme of human cytochrome P450 CYP3A4, using artificial redox chains, can significantly enhance its coupling efficiency and catalytic activity at electrode surfaces. The human CYP3A4 was fused at the genetic level either to the reductase domain of CYP102A1 (BMR) to create the CYP3A4/BMR or to Desulfovibrio vulgaris flavodoxin (FLD) to create the CYP3A4/FLD. Direct electrochemistry of the CYP3A4, CYP3A4/BMR and CYP3A4/FLD on glassy carbon and gold electrodes showed that the BMR and FLD flavo-proteins reduced the electron transfer rate to the CYP3A4 heme. Electrocatalysis resulted in appreciably higher product formation with the immobilized CYP3A4/BMR and CYP3A4/FLD on both surfaces due to an increased coupling efficiency. Rotating disk electrode studies and quantification of hydrogen peroxide were consistent with the proposed mechanism of a longer lived iron-peroxy species in the immobilized CYP3A4/BMR and CYP3A4/FLD. The approaches in this study provide a better understanding of cytochrome P450 uncoupling at electrode surfaces and aids in the construction of improved cytochrome P450 biosensors and bioelectrocatalysts.     

Ureido-modification of the resin-bound LHRH analogue with N,N''-carbonyldiimidazole

The ureido-modification of the resin-bound a luteinizing hormone releasing hormone (LHRH) analogue was investigated by CDI-activating method. The amino group at the side chain of LHRH analogue could be transformed into various substituted urea moieties in high yields. However, its terminal amino group was partially converted to a hydantoin structure due to the attack of the N atom of the adjacent amide bond.     

Engineering of Human CYP3A Enzymes by Combination of Activating Polymorphic Variants

The use of human cytochrome P450 (CYP) enzymes is increasing for the production of drug metabolites used for drug safety testing and doping analysis. Major challenges are high-priced cofactors, poor stability, and comparatively low activities. We have shown previously that production of specific metabolites in milligrams to gram scale is feasible using human CYPs recombinantly expressed in fission yeast. In this study, we sought to improve the activities of human CYP3A enzymes by genetic engineering. Two side chains (Pro293 and Arg409) of known activating human CYP3A polymorphic variants were??separately or together??introduced into the wild-type forms of each of the three enzymes CYP3A4, CYP3A5, and CYP3A7, respectively. Different effects of the two mutations and their combination on enzyme activity were monitored using both polar and nonpolar substrates. Interestingly, the CYP3A7 double mutant displayed a strong increase in activity with respect to testosterone 6??-hydroxylation (300?% of wild-type activity) and luciferin-6??-pentafluoro-benzyl ether turnover (400?% compared to wild type), while the single mutant CYP3A5^Pro293 showed 370 and 400?% of wild-type activity towards 6??-hydroxylation of testosterone and 16??-hydroxylation of dehydroepiandrosterone, respectively. Overall, six out of seven newly created mutants displayed increased activity with at least one of the tested substrates. These results support the notion that pharmacogenetic knowledge can directly contribute to the improvement of biotechnological processes.     

Growth hormone secretagogues

Growth hormone secretagogues (GHSs) are synthetic molecules that stimulate and amplify pulsatile pituitary growth hormone release, via a separate pathway distinct from GH releasing hormone/somatostatin. The activity of GHSs is not fully specific for GH secretion; some GHSs also have slight releasing activity on other pituitary hormones and mediate GH independent biological activities. The first GHSs were discovered in 1977. Since then, an intensive research to synthesize a potent oral GHSs has been undertaken. Although the potential applications of GHSs are numerous, long term trials are needed to evaluate the clinical efficacy and safety of these substances. In the present article we review the historic background of GHSs, their potential clinical uses, the types and the main GHSs that have been synthesized hitherto.     

Hammett analysis of selective thyroid hormone receptor modulators reveals structural and electronic requirements for hormone antagonists

Selective thyroid hormone modulators that function as isoform-selective agonists or antagonists of the thyroid hormone receptors (TRs) might be therapeutically useful in diseases associated with aberrant hormone signaling. The most potent thyroid hormone antagonist reported to date is NH-3. To explore the significance of the 5'-p-nitroaryl moiety of NH-3 and understand what chemical features are important to confer antagonism, we sought to expand the structure-activity relationship data for the class of 5'-phenylethynyl GC-1 derivatives. Herein, we describe an improved synthetic route utilizing palladium-catalyzed chemistry for efficient access to a series of 5'-phenylethynyl compounds with varying size and electronic properties. We prepared and tested sixteen analogues for TR binding and transactivation activity. Substitution at the 5'-position decreased binding affinity, but retained TRbeta-selectivity. In transactivation assays, the analogues displayed a spectrum of agonist, antagonist, and mixed agonist/antagonist activity that correlated with electronic character in a Hammett analysis between sigma substituent value and TR modulation. Analogues NH-5, NH-7, NH-9, NH-11, and NH-23 displayed full antagonist activity with reduced potency compared to NH-3, indicating the nitro group is not required for antagonism. However, para-substitution with strong electron withdrawing properties on the 5'-aryl extension is important for antagonist activity, and antagonist potency-but not ligand receptor binding-was found to correlate linearly with the sigma values for the electron withdrawing substituents.     

Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry of luteinizing hormone releasing hormone-metal ion complexes

Complexes of luteinizing hormone releasing hormone (LHRH) with divalent metal ions (Ni, Zn, Cu) have been studied by matrix-assisted laser desorption ionization (MALDI) and Fourier transform mass spectrometry. LHRH-metal complexes were detected in high abundance for all three metals from synthesized samples, particularly in negative ion mode. The mixture of the apopeptide with the metal salts yielded in most cases a very minor signal of metal-complex ions. As opposed to Ni and Zn, copper complex ions were mostly observed as Cu(I) adducts. This can be partly attributed to plume reactions of Cu(I) with the apopeptide. the Cu(II) complexes appeared only for the synthetic complex. We show how to distinguish between the contribution to the overall signal from desorbed complexes and from Cu(I) complexes formed in the MALDI plume.     

An NH2-terminal truncated cytochrome P450 CYP3A4 showing catalytic activity is present in the cytoplasm of human liver cells

Cytochrome P450 3A4 (CYP3A4), is the dominant human liver hemoprotein enzyme localized in the endoplasmic reticulum (ER), and is responsible for the metabolism of more than 50% of clinically relevant drugs. While we were studying CYP3A4 expression and activity in human liver, we found that anti-CYP3A4 antibody cross-reacted with a lower band in liver cytoplasmic fraction. We assessed the activities of CYP3A4 and its truncated form in the microsomal and cytoplasmic fraction, respectively. In the cytoplasmic fraction, truncated CYP3A4 showed catalytic activity when reconstituted with NADPH-cytochrome P-450 reductase and cytochrome b5. In order to determine which site was deleted in the truncated form in vitro, we transfected cells with N-terminal tagged or C-terminal tagged human CYP3A4 cDNA. The truncated CYP3A4 is the N-terminal deleted form and was present in the soluble cytoplasmic fraction. Our result shows, for the first time, that N-terminal truncated, catalytically active CYP3A4 is present principally in the cytoplasm of human liver cells.     

Synthesis of the major metabolites of Tolvaptan

Tolvaptan is a nonpeptide arginine vasopressin（AVP） V₂-receptor antagonist and used in the treatment of heart failure,cirrhosis, syndrome of inappropriate antidiuretic hormone secretion or other high-volume capacity of hyponatremia.The metabolites of tolvaptan are mainly produced by CYP3A4,including two major compounds named DM-4103 and DM-4107.Herein,the chemical synthesis of those two metabolites is described in this article for further study.     

Design and synthesis of new 7, 8-dimethoxy-α-naphthoflavones as CYP1A1 inhibitors

The flavonoids as inhibitors of CYP1Al exhibit chemopreventive effects against certain procarcinogens and have been considered as the promising cancer preventive agents.A series of novel 7,8-dimethoxy-αnaphthoflavones as the substrate analogs were designed and prepared.The enzyme assay suggested that all of these new flavones were stronger inhibitors of CYP1 Al than the lead compoundα-naphthoflavone. Among the tested ones,3h showed the most potent inhibitory effects.     

Evaluation of the effects of Mitragyna speciosa alkaloid extract on cytochrome P450 enzymes using a high throughput assay

The extract from Mitragyna speciosa has been widely used as an opium substitute, mainly due to its morphine-like pharmacological effects. This study investigated the effects of M. speciosa alkaloid extract (MSE) on human recombinant cytochrome P450 (CYP) enzyme activities using a modified Crespi method. As compared with the liquid chromatography-mass spectrometry method, this method has shown to be a fast and cost-effective way to perform CYP inhibition studies. The results indicated that MSE has the most potent inhibitory effect on CYP3A4 and CYP2D6, with apparent half-maximal inhibitory concentration (IC(50)) values of 0.78 μg/mL and 0.636 μg/mL, respectively. In addition, moderate inhibition was observed for CYP1A2, with an IC(50) of 39 μg/mL, and weak inhibition was detected for CYP2C19. The IC(50) of CYP2C19 could not be determined, however, because inhibition was <50%. Competitive inhibition was found for the MSE-treated CYP2D6 inhibition assay, whereas non-competitive inhibition was shown in inhibition assays using CYP3A4, CYP1A2 and CYP2C19. Quinidine (CYP2D6), ketoconazole (CYP3A4), tranylcypromine (CYP2C19) and furafylline (CYP1A2) were ACCESSused as positive controls throughout the experiments. This study shows that MSE may contribute to an herb-drug interaction if administered concomitantly with drugs that are substrates for CYP3A4, CYP2D6 and CYP1A2.     

Synthetic models related to furanocoumarin-CYP 3A4 interactions. comparison of furanocoumarin, coumarin, and benzofuran dimers as potent inhibitors of CYP3A4 activity

Furanocoumarin derivatives (dimers and monomers) present in commercially available grapefruit juice have the capacity to inhibit the activity of human CYP3A4. Such interactions are believed to result from the mechanism-based inhibition of CYP3A4 activity in the intestine. The aim of this work was to synthesize and test a series of dimers with a view to determining the relationship between structure and inhibitory activity and determining whether they might make suitable probes of CYP3A4 activity. We prepared a series of furanocoumarin, coumarin, and benzofuran derivatives that have inhibitory effects on the activity of human CYP3A4. A synthetic benzofuran dimer, which is more accessible than furanocoumarin dimers, exhibited activity against CYP3A4 comparable to that of furanocoumarin dimers.