Covalent Binding Studies Research Articles

Accelerating covalent binding studies: Direct mass shift measurement with acoustic ejection and TOF-MS

Tracking chemical reactions by measuring incurred mass shifts upon successful binding is a direct and attractive alternative to existing assays based on chemical tags. Traditional methods use liquid chromatography-mass spectrometry (LC-MS), and because the required buffers are not amenable to direct MS injection, sample pre-treatment is needed to desalt. This leads to analysis times from ten seconds to minutes per sample, limiting throughput and preventing widespread application.Combining an acoustic ejection (AE) interface with a time-of-flight mass spectrometer (MS) removes this bottleneck, as samples can be directly introduced at rates of up to one second per sample. This article describes a complete workflow for measuring the covalent binding of compounds to proteins in real-time, from assay to data evaluation. It is noteworthy that this is the first instance of using SCIEX Echo® MS+ system with ZenoTOF 7600 system to study the kinetic regimes of covalent binding.

Identification of human liver microsomal proteins adducted by a reactive metabolite using shotgun proteomics.

Covalent modification of cellular proteins by chemically reactive compounds/metabolites has the potential to disrupt biological function and elicit serious adverse drug reactions. Information on the nature and binding patterns of protein targets are critical toward understanding the mechanism of drug induced toxicity. Protein covalent binding studies established in liver microsomes can quantitively estimate the extent of protein modification, but they provide little information on the nature of the modified proteins. In this article, we describe a label-free shotgun proteomic workflow for the identification of target proteins modified in situ by reactive metabolites in human liver microsome incubations. First, we developed a shotgun proteomic workflow for the characterization of the human liver microsomal subproteome, which consists of predominately membrane-bound proteins. Human liver microsomes were solubilized with a combination of MS-compatible organic solvents followed by protein reduction, alkylation, and tryptic digestion. The unmodified samples were analyzed by UHPLC-MS/MS, and the proteins were identified by database searching. This workflow led to the successful identification of 329 human liver microsomal subproteome proteins with 1% FDR (false discovery rate). The same method was then applied to identify the modifications of human liver microsomal proteins by a known reactive metabolite 2-(methylsulfonyl)benzo[d]thiazole (2), either after incubation directly with 2 or with its parent compound 2-(methylthio)benzo[d]thiazole (1). A total of 19 modified constituent peptides which could be mapped to 18 proteins were identified in human liver microsomes incubated directly with 2. Among these, 5 modified constituent peptides which could be mapped to 4 proteins were identified in incubation with 1, which is known to generate 2 in human liver microsomal incubations. This label-free workflow is generally applicable to the identification and characterization of proteins adducted with reactive metabolites in complex matrices and may serve as a valuable tool to understand the link between protein targets and clinically relevant toxicities.

The metabolic fate of [14C]-fenclozic acid in the hepatic reductase null (HRN) mouse

1. The distribution, metabolism, excretion and hepatic effects of fenclozic acid were investigated following a single oral dose of 10 mg/kg to hepatic reductase null (HRN) mice.2. The majority of the [14C]-fenclozic acid was eliminated via the urine/aqueous cage wash, (55 %) with a smaller portion excreted in the faeces, (5 %). The total recovery of radioactivity in the excreta over the 72 h period studied was ca. 60%.3. Metabolism of fenclozic acid in the HRN mice was entirely to the carboxylic acid function and was dominated by amino acid conjugation to glycine and taurine, with lesser amounts of an acyl glucuronide.4. Whole body autoradiography of mice showed general distribution into all tissues except the brain. Radioactivity was still detectable in the kidney and liver of the HRN mice at 72 h post-dose. Covalent binding studies showed evidence of binding to kidney, liver and plasma proteins however, the degree of binding was less than 50 pmol equiv/mg protein for all tissues.5. The HRN mouse appears to be a useful in vivo model for the study of the Phase II conjugation metabolism of fenclozic acid in the absence of hepatic cytochrome P450-related oxidative metabolism.

In vitro exploration of potential mechanisms of toxicity of the human hepatotoxic drug fenclozic acid

The carboxylic acid NSAID fenclozic acid exhibited an excellent preclinical safety profile and promising clinical efficacy, yet was withdrawn from clinical development in 1971 due to hepatotoxicity observed in clinical trials. A variety of modern in vitro approaches have been used to explore potential underlying mechanisms. Covalent binding studies were undertaken with [(14)C]-fenclozic acid to investigate the possible role of reactive metabolites. Time-dependent covalent binding to protein was observed in NADPH-supplemented liver microsomes, although no metabolites were detected in these incubations or in reactive metabolite trapping experiments. In human hepatocytes, covalent binding was observed at lower levels than in microsomes and a minor uncharacterizable metabolite was also observed. In addition, covalent binding was observed in incubations undertaken with dog and rat hepatocytes, where a taurine conjugate of the drug was detected. Although an acyl glucuronide metabolite was detected when liver microsomes from human, rat and dog were supplemented with UDPGA, there was no detectable UDPGA-dependent covalent binding. No effects were observed when fenclozic acid was assessed for P450-dependent and P450-independent cytotoxicity to THLE cell lines, time-dependent inhibition of five major human cytochrome P450 enzymes, inhibition of the biliary efflux transporters BSEP and MRP2 or mitochondrial toxicity to THLE or HepG2 cells. These data suggest that Phase 1 bioactivation plays a role in the hepatotoxicity of fenclozic acid and highlight the unique insight into mechanisms of human drug toxicity that can be provided by investigations of biotransformation and covalent binding to proteins.

Optimized proteomic analysis of rat liver microsomes using dual enzyme digestion with 2D-LC–MS/MS

A systematic approach was developed to optimize the analysis of rat liver microsomes combining ion exchange fractionation with reverse-phase chromatography coupled to high resolution quadrupole-time-of-flight mass spectrometry. A comparison was performed with several conditions to select the most efficient solubilization and proteolysis protocol to achieve highest proteome coverage. Optimal trypsin digestion conditions were achieved with SDS and heat to increase solubilization of microsomal samples, with an increase from 621 to 686 identified proteins when SDS and heat were applied. Pepsin digestion yielded complementary results, especially in terms of hydrophobic environments, thus allowing sequence coverage to be increased substantially. Several dual digestion strategies were tested, with trypsin and pepsin combined in series or in parallel. A parallel tryptic-peptic dual digestion, combining mass spectral data of single enzyme digestions, yielded the best results in terms of number of identified proteins, increasing by 29% from the best single enzyme procedure, and sequence coverage improved by 5% on average for all proteins identified. Using our complete set of data, a total of 1095 proteins were identified with less than 1% FDR, out of which 213 proteins (19.5%) were integral membrane proteins. Proteomics data have been deposited to the ProteomeXchange Consortium with dataset identifier PXD000128. A systematic approach to optimize the proteomic analysis of rat liver microsomes by 2D-LC-MS/MS using several different digestion conditions was performed in order to increase our knowledge of the rat liver proteome, especially important to drug metabolism and toxicology. A parallel (combined) tryptic-peptic digestion yielded best overall performance, when mass spectral data were acquired separately and combined prior to database searching. This large-scale data set will be available publicly on the ProteomeXchange server and will therefore be accessible to a large scientific community interested in using this data for their own studies. One of the main goals of this study is to identify a comprehensive list of proteins for follow-up protein covalent binding studies related to drug toxicity.

Identification and quantification of drug–albumin adducts in serum samples from a drug exposure study in mice

The formation of drug-protein adducts following the bioactivation of drugs to reactive metabolites has been linked to adverse drug reactions (ADRs) and is a major complication in drug discovery and development. Identification and quantification of drug-protein adducts in vivo may lead to a better understanding of drug toxicity, but is challenging due to their low abundance in the complex biological samples. Human serum albumin (HSA) is a well-known target of reactive drug metabolites due to the free cysteine on position 34 and is often the first target to be investigated in covalent drug binding studies. Presented here is an optimized strategy for targeted analysis of low-level drug-albumin adducts in serum. This strategy is based on selective extraction of albumin from serum through affinity chromatography, efficient sample treatment and clean-up using gel filtration chromatography followed by tryptic digestion and LC-MS analysis. Quantification of the level of albumin modification was performed through a comparison of non-modified and drug-modified protein based on the relative peak area of the tryptic peptide containing the free cysteine residue. The analysis strategy was applied to serum samples resulting from a drug exposure experiment in mice, which was designed to study the effects of different acetaminophen (APAP) treatments on drug toxicity. APAP is bioactivated to N-acetyl-p-benzoquinoneimine (NAPQI) in both humans and mice and is known to bind to cysteine 34 (cys34) of HSA. Analysis of the mouse serum samples revealed the presence of extremely low-level NAPQI-albumin adducts of approximately 0.2% of the total mouse serum albumin (MSA), regardless of the length of drug exposure. Due to the targeted nature of the strategy, the NAPQI-adduct formation on cys34 could be confirmed while adducts to the second free cysteine on position 579 of MSA were not detected.

The hepatic reductase null mouse as a model for exploring hepatic conjugation of xenobiotics: Application to the metabolism of diclofenac

The distribution, metabolism, excretion and hepatic effects of diclofenac were investigated following a single oral dose of 10 mg/kg to wild type and hepatic reductase null (HRN) mice.For the HRN strain the bulk of the [14C]-diclofenac-related material was excreted in the urine/aqueous cagewash within 12 h of administration (~82%) with only small amounts eliminated via the faeces (~2% in 24 h). Wild type mice excreted the radiolabel more slowly with ca. 52 and 15% of the dose recovered excreted in urine and faeces, respectively, by 24 h post dose.The metabolic profiles of the HRN mice were dominated by acyl conjugation to either taurine or glucuronic acid. Wild type mice produced relatively small amounts of the acyl glucuronide.Whole Body Autoradiography (WBA) of mice sacrificed at 24 h post dose indicated increased retention of radioactivity in the livers of HRN mice compared to wild type mice. Covalent binding studies showed no differences between the two strains.Metabolism of diclofenac in HRN mice involved mainly acyl glucuronide formation and taurine amide conjugation. This mouse model may find utility in understanding the impact of reactive metabolite formation via routes that involve the production of acyl-CoA or acyl glucuronides of acidic drugs.

Structure–Activity Relationships of GHRP-6 Azapeptide Ligands of the CD36 Scavenger Receptor by Solid-Phase Submonomer Azapeptide Synthesis

The cluster of differentiation 36 (CD36) class B scavenger receptor binds a variety of biologically endogenous ligands in addition to synthetic peptides (i.e., growth hormone-releasing peptides, GHRPs), which modulate biological function related to anti-angiogenic and anti-atherosclerotic activities. Affinity labeling had previously shown that GHRP-6 analogues such as hexarelin, [2-Me-W(2)]GHRP-6 (1), bind to the lysine-rich domain of the CD36 receptor. Moreover, the azapeptide analogue [aza-F(4)]GHRP-6, 2, exhibited a characteristic β-turn conformation as described by CD and NMR spectroscopy and a slightly higher CD36 binding affinity relative to hexarelin (1.34 and 2.37 μM, respectively), suggesting receptor binding was mediated by the conformation and the aromatic residues of these peptide sequences. Ligand-receptor binding interactions were thus explored using azapeptides to examine influences of side-chain diversity and backbone conformation. In particular, considering that aromatic cation interactions may contribute to binding affinity, we have explored the potential of introducing salt bridges to furnish GHRP-6 azapeptide ligands of the CD36 receptor. Fifteen aza-glutamic acid analogues related to 2 were prepared by submonomer solid-phase synthesis. The azapeptide side chains were installed by novel approaches featuring alkylation of resin-bound semicarbazone with Michael acceptors and activated allylic acetates in the presence of phosphazene base (BTPP). Moreover, certain Michael adducts underwent intramolecular cyclization during semicarbazone deprotection, leading to novel pyrrazoline and aza-pyroglutamate N-terminal residues. Structural studies indicated that contingent on sequence the [aza-Glu]GHRP-6 analogues exhibited CD spectra characteristic of random coil, polyproline type II and β-turn secondary structures in aqueous media. In covalent competition binding studies with the GHRP-6 prototype hexarelin bearing a radiotracer, certain [aza-Glu]GHRP-6 azapeptides retained relatively high (2-27 μM) affinity for the CD36 scavenger receptor.

Can In Vitro Metabolism-Dependent Covalent Binding Data Distinguish Hepatotoxic from Nonhepatotoxic Drugs? An Analysis Using Human Hepatocytes and Liver S-9 Fraction

In vitro covalent binding studies in which xenobiotics are shown to undergo metabolism-dependent covalent binding to macromolecules have been commonly used to shed light on the biochemical mechanisms of xenobiotic-induced toxicity. In this paper, 18 drugs (nine hepatotoxins and nine nonhepatotoxins) were tested for their proclivity to demonstrate metabolism-dependent covalent binding to macromolecules in human liver S-9 fraction (9000 g supernatant) or human hepatocytes, as an extension to previous work that used human liver microsomes published in this journal [ Obach et al. ( 2008 ) Chem. Res. Toxicol. 21 , 1814 -1822 ]. In the S-9 fraction, seven out of the nine drugs in each category demonstrated some level of metabolism-dependent covalent binding. Inclusion of reduced glutathione, cofactors needed by conjugating enzymes, and other parameters (total daily dose and fraction of total intrinsic clearance comprised by covalent binding) improved the ability of the system to separate hepatotoxins from nonhepatotoxins to a limited extent. Covalent binding in human hepatocytes showed that six out of the nine hepatotoxins and four out of eight nonhepatotoxins demonstrated covalent binding. Taking into account estimates of total daily body burden of covalent binding from the hepatocyte data showed an improvement over other in vitro systems for distinguishing hepatotoxins from nonhepatotoxins; however, this metabolism system still displayed some false positives. Combined with the previous study using liver microsomes, these findings identify the limitations of in vitro covalent binding data for prospective prediction of hepatotoxicity for new drug candidates and highlight the need for a better understanding of the link between drug bioactivation, covalent adduct formation, and toxicity outcomes. Directly relating covalent binding to hepatotoxicity is likely an oversimplification of the process whereby adduct formation ultimately leads to toxicity. Understanding underlying complexities (e.g., which macromolecules are important covalent binding targets, interindividual differences in susceptibility, etc.) will be essential to any understanding of the problem of metabolism-dependent hepatotoxicity and predicting toxicity from in vitro experiments.

Prediction of in Vivo Potential for Metabolic Activation of Drugs into Chemically Reactive Intermediate: Correlation of in Vitro and in Vivo Generation of Reactive Intermediates and in Vitro Glutathione Conjugate Formation in Rats and Humans

The covalent binding of reactive intermediates to macromolecules might have potential involvement in severe adverse drug reactions. Thus, quantification of reactive metabolites is necessary during the early stage of drug discovery to avoid serious toxicity. In this study, the relationship between covalent binding and glutathione (GSH) conjugate formation in rat and human liver microsomes were investigated using 10 representative radioactive compounds that have been reported as hepatotoxic or having other toxicity derived from their reactive intermediates: acetaminophen, amodiaquine, carbamazepine, clozapine, diclofenac, furosemide, imipramine, indomethacin, isoniazid, and tienilic acid, all at a concentration of 10 microM. The GSH conjugate formation rate correlates well with the covalent binding of radioactivity (both rat and human, r2 = 0.93), which suggests that quantification of the GSH conjugate can be used to estimate covalent binding. To quantify the GSH-conjugation rate with non-radiolabeled compounds in vitro, the validation study for the determination of GSH conjugate formation using 35S-GSH by radio-HPLC was useful to predict metabolic activation. Following oral administration of 20 mg/kg of the radiolabeled compounds to rats, radioactivity that covalently bound to plasma and liver proteins was determined. The in vivo maximum covalent binding level in liver based on the free fraction of plasma area under the concentration curve (AUC) and in vitro covalent binding rate was found to correlate well (r2 = 0.79). Therefore, this model for in vitro covalent binding studies in human and rat and in vivo rat studies might be useful in predicting human metabolic activation of compounds.

Radioiodinated Azide and Isothiocyanate Derivatives of Cocaine for Irreversible Labeling of Dopamine Transporters: Synthesis and Covalent Binding Studies

Two novel N-substituted-3beta-phenyltropane alkaloids have been labeled with iodine-125 for use as irreversible probes of dopamine transporter (DAT) binding sites. One contains an iodoaryl azide moiety for photolabeling, while the other bears an iodoaryl isothiocyanate for direct conjugation. Both radioligands were prepared in a one-flask procedure by electrophilic radioiodination of the corresponding aniline under no-carrier-added conditions, followed either by diazotization and treatment with sodium azide, or by addition of thiophosgene under basic conditions. Specifically, (-)-N-[4-(3-[(125)I]iodo-4-azidophenyl)butyl]-2beta-carbomethoxy-3beta-(4-chlorophenyl)tropane ([(125)I]MFZ-2-24) and (-)-N-[4-(3-[(125)I]iodo-4-isothiocyanophenyl)butyl]-2beta-carbomethoxy-3beta-(4-chlorophenyl)tropane ([(125)I]MFZ 3-37) were synthesized. Isolation by reversed-phase HPLC and solid-phase extraction gave good average yields of [(125)I]MFZ-2-24 (67%, n = 5) and [(125)I]MFZ-3-37 (45%, n = 3) with high radiochemical purities (96-99%) and specific radioactivities (>2000 mCi/micromol). The utility of the radioligands was demonstrated by their covalent linkage to rat striatal membranes, and immunoprecipitation of a single radiolabeled band at 80 kDa corresponding to the full-length DAT.

Mechanism-based inactivation of cytochrome P450 3A4 by 4-ipomeanol.

Earlier phase I and II clinical studies showed that 4-ipomeanol produced selective hepatotoxicity. To investigate the mechanism of bioactivation of 4-ipomeanol, we thoroughly studied the interaction of 4-ipomeanol with human cytochrome P450 3A4 (EC 1.14.14.1). 4-Ipomeanol produced a time- and concentration-dependent inactivation of P450 3A4. More than 80% of the P450 3A4 activity was lost after its incubation with 4-ipomeanol at the concentration of 75 microM in 12 min. The inactivation was characterized by a rate of inactivation (kinact) of 0.15 min(-1) and by an inactivation potency (KI) of 20 microM. In addition, the inhibition of P450 3A4 by 4-ipomeanol was NADPH-dependent and irreversible. Glutathione, catalase, and superoxide dismutase failed to protect P450 3A4 from inactivation by 4-ipomeanol. The presence of testosterone, a substrate of P450 3A4, protected the enzyme from inactivation. The estimated partition ratio of the inactivation was approximately 257. Covalent binding studies demonstrated that reactive metabolites of 4-ipomeanol modified P450 3A4 but not P450 reductase (EC 1.6.2.4). The stoichiometry of binding between reactive metabolites of radiolabeled 4-ipomeanol and P450 3A4 was approximately 1.5:1. In addition to P450 3A4, reactive metabolites of 4-ipomeanol were found to covalently bind to other proteins. 4-Ipomeanol failed to inactivate P450 1A2 in human liver microsomes. In conclusion, 4-ipomeanol irreversibly inhibited P450 3A4, and it was characterized as a mechanism-based inactivator of P450 3A4. This finding facilitates the understanding of the mechanism of bioactivation of 4-ipomeanol by human hepatic enzymes.

Kinetic Studies of Covalent Binding between N-acetyl-L-cysteine and Human Serum Albumin Through a Mixed-disulfide Using an N-methylpyridinium Polymer-based Column

The binding properties of the disulfide covalent bond between N-acetyl-L-cysteine (NAC) and human serum albumin (HSA) were investigated. HSA, purified from either healthy subjects or renal failure patients, was incubated with NAC in buffer and analyzed by 4VP-EG-Me column chromatography, which can distinguish between the redox states of the only free thiol of HSA. Although intact HSA was found to consist of mainly three sub-types, marcaptoalbumin (HMA), cysteine-bound nonmercaptoalbumin (HNA(Cys)) and a further oxidized form (HNA(oxy)), the formation of a new type of nonmercaptoalbumin (HNA(NAC)) was confirmed after incubation with NAC. Interestingly, NAC rapidly dissociated Cys from HNA(Cys) and NAC itself bound very slowly to HSA. These findings suggest that the interaction between NAC and HSA proceeds in a 2-step processes. The first-order binding and dissociation rate constants of NAC to healthy HSA (k(on,NAC)) and Cys from healthy HNA(Cys) (k(off,Cys)) were approximately 0.0032 and 1.3 (h(-1)), respectively. On the other hand, HSA from renal failure patients showed decreased HMA and increased HNA(Cys). The k(on,NAC) and k(off,Cys) were 0.0094 and 0.45 (h(-1)), respectively, suggesting that the pathological state may affect the binding properties of HSA and NAC.

Drug−Protein Adducts: An Industry Perspective on Minimizing the Potential for Drug Bioactivation in Drug Discovery and Development

It is generally accepted that there is neither a well-defined nor a consistent link between the formation of drug-protein adducts and organ toxicity. Because the potential does exist, however, for these processes to be causally related, the general strategy at Merck Research Laboratories has been to minimize reactive metabolite formation to the extent possible by appropriate structural modification during the lead optimization stage. This requires a flexible approach to defining bioactivation issues in a variety of metabolism vectors and typically involves the initial use of small molecule trapping agents to define the potential for bioactivation. At some point, however, there is a requirement to synthesize a radiolabeled tracer and to undertake covalent binding studies in vitro, usually in liver microsomal (and sometimes hepatocyte) preparations from preclinical species and human, and also in vivo, typically in the rat. This paper serves to provide one pragmatic approach to addressing the issue of bioactivation from an industry viewpoint based on protocols adopted by Merck Research Laboratories. The availability of a dedicated Labeled Compound Synthesis group, coupled to a close working relationship between Drug Metabolism and Medicinal Chemistry, represents a framework within which this perspective becomes viable; the overall aim is to bring safer drugs to patients.

559 Mechanistic studies of covalent heme binding to CYP4B1

559 Mechanistic studies of covalent heme binding to CYP4B1

Hydroxyproline reaction with free radicals generated during benzoyl peroxide catalytic decomposition of carbon tetrachloride Structure of reaction products formed

Benzoyl peroxide catalytic decomposition of carbon tetrachloride in a model system produces trichloromethyl and trichloromethylperoxyl free radicals. These radicals are also produced by CCl4 bioactivation in liver and are considered to be responsible for the deleterious effects of this hepatotoxin. In this study, it is attempted to learn about how the .CCl3 and CCl3O2. tend to react with hydroxyproline in a model system. Hydroxyproline was selected because of its role in collagen metabolism. During the interaction of both radicals with hydroxyproline a total of 16 reaction products were isolated and identified by gas chromatographic-mass spectrometric analysis. All of them were hydroxyproline analogs, no single one contained C from CCl4 and only three contained chlorine. Consequently, most adducts would be missed in experiments where formation of reaction products are studied by formation of(14)C or(36)Cl labeled adducts (e.g. covalent binding studies used by toxicologists). If similar hydroxyproline analog reaction products were observed during CCl4 intoxication it might be reasonably expected that they interfered with collagen metabolism and participate in cirrhogenic effects of CCl4 on the liver.

Development of polyclonal antibodies for detection of protein modification by 1,2-naphthoquinone.

Naphthoquinones have been reported to be toxic to liver cells in vitro. Protein modification is associated with naphthoquinone-induced cytotoxicity. In addition, 1,2-naphthoquinone was found to bind covalently to cysteine residues of proteins of lung Clara cells incubated with naphthalene. To further identify the target proteins of the naphthoquinone, we raised polyclonal antibodies by immunizing rabbits with 1,2-naphthoquinone protein adducts. A high titer of polyclonal antibodies was obtained by antiserum dilution tests. Competitive ELISA showed that the antibodies specifically recognize the 1,2-naphthoquinone N-acetylcysteine adduct. Very weak cross reactivity toward N-acetylcysteine and its 1,4-naphthoquinone as well as naphthalene oxide adducts was observed. For covalent binding studies, we incubated mouse liver homogenates with 1,2-naphthoquinone at concentrations of 1.0 and 10 microM at 37 degrees C for 1 h. The resulting protein samples were developed by SDS-PAGE, followed by Western blotting and immunostaining using the polyclonal antibodies. Chemiluminescent bands developed with ECL chemiluminescence kit were observed on the poly(vinylidene difluoride) microporous membrane blotted with the mouse liver homogenates exposed to 1.0 and 10 microM 1,2-naphthoquinone. One chemiluminescent band at a molecular weight of 22 kDa was observed in the lane loaded with the protein sample incubated with 1.0 microM 1,2-naphthoquinone, and many chemiluminescent bands at a wide range of molecular weights were observed in the lane loaded with the protein sample incubated with 10 microM quinone. As expected, no chemiluminescent bands were detected on the membrane blotted with the proteins exposed to vehicle. We have successfully raised polyclonal antibodies to recognize 1,2-naphthoquinone cysteine adducts and developed immunostaining to detect protein modification by 1,2-naphthoquinone.

Escherichia coli Expression of Site-Directed Mutants of Cytochrome P450 2B1 from Six Substrate Recognition Sites: Substrate Specificity and Inhibitor Selectivity Studies

Cytochrome P450 2B1 wild-type and eight site-directed mutations at positions 114, 206, 236, 302, 363, 367, and 478 have been expressed in an Escherichia coli system. Solubilized membrane preparations yielded 100-180 nmol of P450/L of culture. The metabolism of a number of substrates including androstenedione, progesterone, (benzyloxy)resorufin, pentoxyresorufin, and benzphetamine was analyzed. The E. coli-expressed enzymes displayed the same androstenedione metabolite profiles previously observed with a COS cell expression system. Several of the mutants exhibited an increased rate of progesterone hydroxylation, possibly as the result of an enlarged substrate binding pocket and increased D-ring alpha-face binding. (Benzyloxy)resorufin and pentoxyresorufin O-dealkylation by the P450 2B1 mutants exhibited activities ranging from 10% to 99% and 3% to 71% of wild-type, respectively. Interestingly, the Val-363-->Leu mutant showed markedly suppressed pentoxyresorufin but unaltered (benzyloxy)resorufin dealkylase activity. Benzphetamine N-demethylase activities ranged from 28% to 110% of wild-type. Mechanism-based inactivation of the P450 2B1 mutants showed that susceptibility to inactivation by chloramphenicol and D-erythro- and L-threo-chloramphenicol was abolished in the Val-367-->Ala mutant. The Val-363-->Leu mutant was refractory to L-threo-chloramphenicol. Studies of chloramphenicol covalent binding and metabolism by the Val-367-->Ala mutant showed that its resistance to inactivation is largely attributable to an inability to bioactivate the inhibitor. The expression of P450 2B1 wild-type and mutants in E. coli provides an excellent opportunity to study structure/function relationships by site-directed mutagenesis.

Preferential effects of the chemotherapeutic DNA crosslinking agent mitomycin C on inducible gene expression in vivo

The immediate effects of a single dose of the chemotherapeutic DNA crosslinking agent, mitomycin C (MMC), on the expression of several constitutive and drug-inducible genes were examined in a simple in vivo system, the 14 day chick embryo. We observed no effect of MMC on the steady-state mRNA expression of the constitutively expressed beta-actin, transferrin, or albumin genes. In contrast, MMC treatment significantly altered both the basal and drug-inducible mRNA expression of two glutethimide-inducible genes, 5-aminolevulinic acid (ALA) synthase and cytochrome P450 CYP2H1. The basal expression of these genes was transiently but significantly increased over a 24 hr period following a single dose of MMC. Conversely, MMC significantly suppressed the glutethimide-inducible expression of these genes when administered 1 to 24 hr prior to the inducing drug. The effects of MMC on both basal and drug-inducible ALA synthase and CYP2H1 mRNA expression were principally a result of changes in the transcription rates of these genes. In contrast, MMC treatment had little or no effect on glutethimide-induced expression of ALA synthase or CYP2H1 when administered 1 hr after the inducing drug, suggesting that a very early event in the induction process represents the target for these MMC effects. Covalent binding studies demonstrated that the effects of MMC on gene expression were closely correlated temporally with formation of [3H]-porfiromycin-DNA adducts. These results support the hypothesis that genotoxic chemicals specifically target their effects to inducible genes in vivo.

A new series of photoactivatable and iodinatable linear vasopressin antagonists.

A series of new linear photoactivatable and iodinatable antagonists of the neuropeptidic hormone vasopressin was designed and synthesized by a combination of PyBOP-mediated Boc/solid-phase peptide synthesis and solution synthesis approaches. These were based on modifications of a previously reported potent and selective antagonist of the vasopressor response (V1a receptor) to [arginine]vasopressin, phenylacetyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2. (Azidophenyl)alkyl substitutions, of the general structure N3-C6H4(CH2)nCO (n = 0, 1, 2, or 3), were employed in position 1. The seven new analogues are 4-N3-C6H4CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (3), 3-N3-C6H4CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (12), 4-N3-C6H4CH2-CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (13), 3-N3-C6H4CH2CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (14), 4-N3-C6H4(CH2)2CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (15), 3-N3-C6H4(CH2)2CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (16), 4-N3-C6H4-(CH2)3CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (17). All analogues were tested for their affinity of the rat hepatic V1a receptor. Analogues 3 and 12 have a low affinity (Ki approximately 20 nM) and analogues 13-17 show a high affinity (Ki between 0.04 and 0.3 nM). The affinity values appear to be mainly a function of the alkyl chain length and to a lesser extent of the meta or para position of the azido group on the aromatic ring. Analogues 13-17 were iodinated on the Tyr-9 residue, giving compounds 18-22. All these five iodinated derivatives exhibited Ki values of 0.2-1 nM for rat liver membranes. Their affinities for oxytocin and renal V2 vasopressin receptors were much lower. Moreover, all analogues completely antagonized the vasopressin-stimulated inositol phosphates production in WRK1 cells and were devoided of any agonistic potency. Preliminary covalent binding studies showed improved covalent yields as compared to any previously reported results. They are very promising candidates as potential high-affinity, highly selective, photosensitive ligands for the V1a receptor. They could serve as a useful pharmacological tools for studies on the vasopressin binding site.