Allosteric Activation of Cytochrome P450 3A4 via Progesterone Bioconjugation (original) (raw)
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Archives of biochemistry and biophysics, 2018
Human cytochrome P450 3A4 (CYP3A4) is an important drug metabolizing enzyme involved in a number of drug-drug and food-drug interactions. As such, much effort has been devoted into investigating its mechanism of interaction with ligands. CYP3A4 has one of the highest levels of substrate promiscuity for an enzyme, and can even bind multiple ligands simultaneously. The location and orientation of these ligands depend on the chemical structure and stoichiometry, and are generally poorly understood. In the case of the steroid testosterone, up to three copies of the molecule can associate with the enzyme at once, likely two in the active site and one at a postulated allosteric site. Recently, we demonstrated that steroid bioconjugation at the allosteric site results in an increase in activity of CYP3A4 toward testosterone and 7-benzyloxy-4-trifluoromethylcoumarin oxidation. Here, using the established bioconjugation methodology, we show how steroid bioconjugation at the allosteric site a...
Proceedings of the National Academy of Sciences, 1998
Cytochrome P450 3A4 is generally considered to be the most important human drug-metabolizing enzyme and is known to catalyze the oxidation of a number of substrates in a cooperative manner. An allosteric mechanism is usually invoked to explain the cooperativity. Based on a structure–activity study from another laboratory using various effector–substrate combinations and on our own studies using site-directed mutagenesis and computer modeling of P450 3A4, the most likely location of effector binding is in the active site along with the substrate. Our study was designed to test this hypothesis by replacing residues Leu-211 and Asp-214 with the larger Phe and Glu, respectively. These residues were predicted to constitute a portion of the effector binding site, and the substitutions were designed to mimic the action of the effector by reducing the size of the active site. The L211F/D214E double mutant displayed an increased rate of testosterone and progesterone 6β-hydroxylation at low s...
Drug Metabolism and Disposition, 2002
Pharmaceutical industry investigators routinely evaluate the potential for a new drug to modify cytochrome P450 (P450) activities by determining the effect of the drug on in vitro probe reactions that represent activity of specific P450 enzymes. The in vitro findings obtained with one probe substrate are usually extrapolated to the compound's potential to affect all substrates of the same enzyme. Due to this practice, it is important to use the right probe substrate and to conduct the experiment under optimal conditions. Surveys conducted by reviewers in CDER indicated that the most common in vitro probe reactions used by industry investigators include the following: phenacetin O-deethylation for CYP1A2, coumarin 7-hydroxylation for CYP2A6, 7-ethoxy-4-trifluoromethyl coumarin O-dealkylation for CYP2B6, tolbutamide 4-hydroxylation for CYP2C9, S-mephenytoin 4-hydroxylation for CYP2C19, bufuralol 1-hydroxylation for CYP2D6, chlorzoxazone 6-hydroxylation for CYP2E1, and testosterone 6-hydroxylation for CYP3A4. We reviewed the validation information in the literature on these reactions and other frequently used reactions, including caffeine N3demethylation for CYP1A2, S-mephenytoin N-demethylation for CYP2B6, S-warfarin 7-hydroxylation for CYP2C9, dextromethorphan O-demethylation for CYP2D6, and midazolam 1-hydroxylation for CYP3A4. The available information indicates that we need to continue the search for better probe substrates for some enzymes. For CYP3A4-based drug interactions it may be necessary to evaluate two or more probe substrates. In many cases, the probe reaction represents a particular enzyme activity only under specific experimental conditions. Investigators must consider appropriateness of probe substrates and experimental conditions when conducting in vitro drug interaction studies and when extrapolating the results to in vivo situations.
Biochemistry, 1994
A unique characteristic of the CYP3A subfamily of cytochrome P450 enzymes is their ability to be activated by certain compounds. It is reported that CYP3A4-catalyzed phenanthrene metabolism is activated by 7,8-benzoflavone and that 7,8-benzoflavone serves as a substrate for CYP3A4. Kinetic analyses of these two substrates show that 7,8-benzoflavone increases the V,,, of phenanthrene metabolism without changing the K, and that phenanthrene decreases the V,,, of 7,8-benzoflavone metabolism without increasing the K m . These results suggest that both substrates (or substrate and activator) are simultaneously present in the active site. Both compounds must have access to the active oxygen, since neither phenanthrene nor 7,8-benzoflavone can competitively inhibit the other substrate. These data provide the first evidence that two different molecules can be simultaneously bound to the same P450 active site. Additionally, structure-activity relationship studies were performed with derivatives of 7,8-benzoflavone structure. The
Archives of Biochemistry and Biophysics, 1998
vation by progesterone but inhibition by a-NF. ᭧ 1998 Academic Press Sequence alignment of human cytochrome P450 3A4 Key Words: steroid hydroxylation; flavonoid; cooperwith bacterial enzymes of known structure has proativity; steady-state kinetics. vided a basis from which to predict residues involved in substrate oxidation. Substitutions were made at four residues (I301, F304, A305, and T309) predicted to be located within the highly conserved substrate Human cytochrome P450 3A4 metabolizes a wide recognition site 4. Site-directed mutants engineered array of both endogenous and exogenous compounds of to contain carboxy-terminal histidine tags were exvery diverse structure and is the most abundant cytopressed in Escherichia coli and purified on a metal chrome P450 in the liver. Its involvement in many drug affinity column. The integrity of each protein was asinteractions emphasizes the importance of understandsessed by SDS-polyacrylamide gel electrophoresis ing the basis of P450 3A4 specificity (1). Another unique and immunoblotting. Functional analysis was percharacteristic of the 3A subfamily is the phenomenon formed using progesterone and testosterone as substrates and a-naphthoflavone as an activator. In tes-of autoactivation by substrates and stimulation by flatosterone hydroxylase assays, all of the mutants dis-vonoids (2-4). Autoactivation is observed with subplayed rates of total product formation similar to strates such as aflatoxin and progesterone, resulting wild-type 3A4, with several mutants showing small difin sigmoidal kinetics. The mechanism of stimulation is ferences in specific products formed. However, with not well understood, but a number of hypotheses have progesterone as the substrate, mutants F304A, A305V, been proposed including the possibility that activators and T309A exhibited altered product ratios and/or bind to the same site as the substrate (5) or at a sepachanges in the rates of product formation. F304A and rate site, causing an allosteric effect on substrate bind-A305V also displayed altered flavonoid stimulation ing (6). A more recent report by Ueng et al. suggested that resulted in product ratios dramatically different that these two models for activation are not mutually from wild-type 3A4. Therefore, the kinetics of progesexclusive and differ only in the distance between the terone hydroxylation of these mutants and the wildsubstrate and activator binding sites (2). type enzyme were further assessed, and the data were To define the boundaries of the effector and substrate analyzed with the Hill equation. Results with wildbinding sites a structural analysis of P450 3A4 is retype 3A4 and F304A indicated that at high progesterquired. In the case of P450 family 2 enzymes, the disone concentrations, hydroxylation rates and product covery of variant enzymes of high structural relatratios are independent of the presence of a-NF. This edness but different functional properties has been suggests that progesterone may be equivalent to a-NF instrumental in unraveling structure-function relaas an activator. In contrast, A305V exhibited autoactitionships (7). However, the high degree of conservation of function among the members of the 3A family within 1 Supported by Grant GM54995 and Center Grant ES06694 from and across species precludes such an approach. Results the National Institutes of Health.
Key Residues Controlling Binding of Diverse Ligands to Human Cytochrome P450 2A Enzymes
Drug Metabolism and Disposition, 2009
Although the human lung cytochrome P450 2A13 (CYP2A13) and its liver counterpart cytochrome P450 2A6 (CYP2A6) are 94% identical in amino acid sequence, they metabolize a number of substrates with substantially different efficiencies. To determine differences in binding for a diverse set of cytochrome P450 2A ligands, we have measured the spectral binding affinities (K D ) for nicotine, phenethyl isothiocyanate (PEITC), coumarin, 2-methoxyacetophenone (MAP), and 8-methoxypsoralen. The differences in the K D values for CYP2A6 versus CYP2A13 ranged from 74-fold for 2-methoxyacetophenone to 1.1-fold for coumarin, with CYP2A13 demonstrating the higher affinity. To identify active site amino acids responsible for the differences in binding of MAP, PEITC, and coumarin, 10 CYP2A13 mutant proteins were generated in which individual amino acids from the CYP2A6 active site were substituted into CYP2A13 at the corresponding po-sition. Titrations revealed that substitutions at positions 208, 300, and 301 individually had the largest effects on ligand binding. The collective relevance of these amino acids to differential ligand selectivity was verified by evaluating binding to CYP2A6 mutant enzymes that incorporate several of the CYP2A13 amino acids at these positions. Inclusion of four CYP2A13 amino acids resulted in a CYP2A6 mutant protein (I208S/I300F/G301A/S369G) with binding affinities for MAP and PEITC much more similar to those observed for CYP2A13 than to those for CYP2A6 without altering coumarin binding. The structurebased quantitative structure-activity relationship analysis using COMBINE successfully modeled the observed mutant-ligand trends and emphasized steric roles for active site residues including four substituted amino acids and an adjacent conserved Leu 370 .
Elucidation of Distinct Ligand Binding Sites for Cytochrome P450 3A4 †
Biochemistry, 2000
Cytochrome P450 (P450) 3A4 is the most abundant human P450 enzyme and has broad selectivity for substrates. The enzyme can show marked catalytic regioselectivity and unusual patterns of homotropic and heterotropic cooperativity, for which several models have been proposed. Spectral titration studies indicated one binding site for the drug indinavir (M r 614), a known substrate and inhibitor. Several C-terminal aminated peptides, including the model morphiceptin (YPFP-NH 2 ), bind with spectral changes indicative of Fe-NH 2 bonding. The binding of the YPFP-NH 2 N-terminal amine and the influence of C-terminal modification on binding argue that the entire molecule (M r 521) fits within P450 3A4. YPFP-NH 2 was not oxidized by P450 3A4 but blocked binding of the substrates testosterone and midazolam, with K i values similar to the spectral binding constant (K s ) for YPFP-NH 2 . YPFP-NH 2 inhibited the oxidations of several typical P450 substrates with K i values 10-fold greater than the K s for binding YPFP-NH 2 and its K i for inhibiting substrate binding. The n values for cooperativity of these oxidations were not altered by YPFP-NH 2 . YPFP-NH 2 inhibited the oxidations of midazolam at two different positions (1′-and 4-) with 20-fold different K i values. The differences in the K i values for blocking the binding to ferric P450 3A4 and the oxidation of several substrates may be attributed to weaker binding of YPFP-NH 2 to ferrous P450 3A4 than to the ferric form. The ferrous protein can be considered a distinct form of the enzyme in binding and catalysis because many substrates (but not YPFP-NH 2 ) facilitate reduction of the ferric to ferrous enzyme. Our results with these peptides are considered in the context of several proposed models. A P450 3A4 model based on these peptide studies contains at least two and probably three distinct ligand sites, with testosterone and R-naphthoflavone occupying distinct sites. Midazolam appears to be able to bind to P450 3A4 in two modes, one corresponding to the testosterone binding mode and one postulated to reflect binding in a third site, distinct from both testosterone and R-naphthoflavone. The work with indinavir and YPFP-NH 2 also argues that room should be present in P450 3A4 to bind more than one smaller ligand in the "testosterone" site, although no direct evidence for such binding exists. Although this work with peptides provides evidence for the existence of multiple ligand binding sites, the results cannot be used to indicate their juxtaposition, which may vary through the catalytic cycle.
Archives of Biochemistry and Biophysics, 1998
Cytochromes P450 constitute a superfamily of hemo-The highly related rabbit cytochromes P450 2B4 and proteins that play a pivotal role in the metabolism of 2B5 differ in only 12 amino acid positions, but only 2B5 a wide variety of xenobiotics and endogenous comhas activity toward progesterone. Previously, simultapounds. Cytochromes P450 are classified on the basis neous site-directed mutagenesis of four key substrate of amino acid sequence into families and subfamilies, recognition site (SRS) residues (114, 294, 363, and 367) which often exhibit distinct substrate specificities (1). was shown to result in interconversion of the andro-In the absence of a three-dimensional structure for stenedione hydroxylase specificities of cytochrome mammalian cytochromes P450, site-directed mutagen-P450 2B4 and 2B5. However, the progesterone metaboesis and heterologous expression have become imlite profiles of the 2B4 quadruple mutant or of a quinportant methods to investigate structure-function retuple mutant in which residue 370 was also mutated lationships of P450 enzymes. This approach has into the 2B5 residue were not identical to that of P450 volved an intense effort to identify substrate contact 2B5. Therefore, single mutants of P450 2B5 at the reresidues and to understand the molecular basis for the maining seven positions were constructed, expressed alterations in substrate specificity that accompany rein Escherichia coli, and studied with progesterone as placement of particular side chains. the substrate. The single mutants at positions 120 and A number of investigators have identified amino acid 221, which are outside any known SRS, exhibited a differences that are principally responsible for characsignificant alteration in progesterone hydroxylation. teristic functional differences between closely related Based on these results, Ile-114, Arg-120, Ser-221, Serenzymes, such as those in P450 family 2 (2-22). Based 294, Ile-363, and Val-367 in cytochrome P450 2B4 were on sequence alignment of P450s from family 2 with replaced simultaneously with Phe, His, Pro, Thr, Val, bacterial P450 101 (P450cam), Gotoh (23) proposed the and Ala, respectively, from 2B5. This yielded a mutant existence of six putative substrate recognition sites with a very similar progesterone metabolite profile to (SRSs). 3 Almost all identified residues that are critical that of 2B5, although the total activity was lower. An for substrate specificity of P450 family 2 forms are loadditional substitution at residue 370 produced a mulcated in or near the SRSs. tiple mutant P450 2B4 I114F-R120H-S221P-S294T-I363V-V367A-T370M with very similar or identical sub-3 Abbreviations used: SRS, substrate recognition site; P450, cyto-strate specificity, regio-and stereospecificity and kichrome P450; DLPC, dilauroyl-L-3-phosphatidyl choline;-OH, hynetic properties to that of P450 2B5 wild type. ᭧ 1998
The Journal of Steroid Biochemistry and Molecular Biology, 2018
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. contrast, only the twofold decrease in the affinity of CYB5A/CYP21A2 interaction in the presence of P4 was caused by a slight increase in the koff value (the kon value of the complex did not change). This indicates a different format of the steroidal substrates effects expressed in a change in the stability of the CYB5A/CYPs complexes. Thus, it was found that P4 modulated the both kinetic and equilibrium constants of CYB5A/CYP17A1 and CYB5/CYP21A2 complex formation and complexes, while not affecting the CYB5A/CYP2C19 interaction (2C19 is the cytochrome P450 isoenzyme possessing broad substrate specificity), thereby indicating a specific influence of steroidal substrates on interactions involving steroidogenic CYPs. Our results are consistent with current understanding of the role of CYB5A as a regulator of cytochrome P450 activity in P450dependent monooxygenase system.
Cytochrome P450 Part 3: Impact of Drug-Drug Interactions
Journal of Pharmacy Practice and Research, 2009
The role of individual hepatic cytochrome P450 (CYP) enzymes in drug metabolism and the factors that modulate CYP activity are becoming increasingly well understood. These advances have resulted in a better understanding of drug-drug and drugfood interactions and an enhanced capacity to predict drug interactions that may occur with new drugs. This final article in the series describes the issues and principles that are important in identifying and assessing drug interactions that involve CYP enzymes.