Topological role of cytochrome P450 2D6 active site residues

Recent reports have identified Phe120, Asp301, Thr309, and Glu216 as important residues in cytochrome P450 2D6 (CYP2D6) substrate binding and catalysis. Complementary homology models have located these amino acids within the binding pocket of CYP2D6 and in the present study we have used aryldiazenes to test these models and gain further insight in the role these amino acids have in maintaining the integrity of the active site cavity. When Phe120 was replaced to alanine, there was a significant increase in probe migration to pyrrole nitrogens C and D, in agreement with homology models which have located the phenyl side-chain of Phe120 above these two pyrrole rings. No changes in topology were observed with the D301Q mutant, supporting claims that in this mutant the electrostatic interactions with the B/C-loop are largely maintained and the loop retains its native orientation. The T309V mutation resulted in significant topological alteration suggesting that, in addition to its potential role in dioxygen activation, Thr309 plays an important structural role within the active site crevice. Replacement of Ile106 with Glu, engineered to cause electrostatic repulsion with Glu216, had a profound topological effect in the higher region within the active site cavity and impaired the catalytic activity towards CYP2D6 probe substrates.     

Molecular modelling of human CYP2D6 and molecular docking of a series of ajmalicine- and quinidine-like inhibitors

3D-models were created and refined for CYP2D6 and for its complexes with ajmalicine and quinidine. The influence of the conformation of the enzyme active site on its interaction with ligands was evaluated by performing three series of molecular docking on selected ajmalicine- and quinidine-like inhibitors. The results suggested that the experimental binding values of ajmalicine- and quinidine-like inhibitors better fit with the energetic terms derived from their interaction with structures of CYP2D6 obtained by, respectively, optimizing the ajmalicine/CYP2D6 and the quinidine/CYP2D6 complexes, rather than exploiting the 3D-strucure of the enzyme not subjected to a ligand-induced conformational change. It suggests the relevance of induced-fit phenomena in the biological system of interest.     

Residues glutamate 216 and aspartate 301 are key determinants of substrate specificity and product regioselectivity in cytochrome P450 2D6

Cytochrome P450 2D6 (CYP2D6) metabolizes a wide range of therapeutic drugs. CYP2D6 substrates typically contain a basic nitrogen atom, and the active-site residue Asp-301 has been implicated in substrate recognition through electrostatic interactions. Our recent computational models point to a predominantly structural role for Asp-301 in loop positioning (Kirton, S. B., Kemp, C. A., Tomkinson, N. P., St.-Gallay, S., and Sutcliffe, M. J. (2002) Proteins 49, 216-231) and suggest a second acidic residue, Glu-216, as a key determinant in the binding of basic substrates. We have evaluated the role of Glu-216 in substrate recognition, along with Asp-301, by site-directed mutagenesis. Reversal of the Glu-216 charge to Lys or substitution with neutral residues (Gln, Phe, or Leu) greatly decreased the affinity (K(m) values increased 10-100-fold) for the classical basic nitrogen-containing substrates bufuralol and dextromethorphan. Altered binding was also manifested in significant differences in regiospecificity with respect to dextromethorphan, producing enzymes with no preference for N-demethylation versus O-demethylation (E216K and E216F). Neutralization of Asp-301 to Gln and Asn had similarly profound effects on substrate binding and regioselectivity. Intriguingly, removal of the negative charge from either 216 or 301 produced enzymes (E216A, E216K, and D301Q) with elevated levels (50-75-fold) of catalytic activity toward diclofenac, a carboxylate-containing CYP2C9 substrate that lacks a basic nitrogen atom. Activity was increased still further (>1000-fold) upon neutralization of both residues (E216Q/D301Q). The kinetic parameters for diclofenac (K(m) 108 microm, k(cat) 5 min(-1)) along with nifedipine (K(m) 28 microm, k(cat) 2 min(-1)) and tolbutamide (K(m) 315 microm, k(cat) 1 min(-1)), which are not normally substrates for CYP2D6, were within an order of magnitude of those observed with CYP3A4 or CYP2C9. Neutralizing both Glu-216 and Asp-301 thus effectively alters substrate recognition illustrating the central role of the negative charges provided by both residues in defining the specificity of CYP2D6 toward substrates containing a basic nitrogen.     

Crystal structure of human cytochrome P450 2D6

Cytochrome P450 2D6 is a heme-containing enzyme that is responsible for the metabolism of at least 20% of known drugs. Substrates of 2D6 typically contain a basic nitrogen and a planar aromatic ring. The crystal structure of human 2D6 has been solved and refined to 3.0A resolution. The structure shows the characteristic P450 fold as seen in other members of the family, with the lengths and orientations of the individual secondary structural elements being very similar to those seen in 2C9. There are, however, several important differences, the most notable involving the F helix, the F-G loop, the B'helix, beta sheet 4, and part of beta sheet 1, all of which are situated on the distal face of the protein. The 2D6 structure has a well defined active site cavity above the heme group, containing many important residues that have been implicated in substrate recognition and binding, including Asp-301, Glu-216, Phe-483, and Phe-120. The crystal structure helps to explain how Asp-301, Glu-216, and Phe-483 can act as substrate binding residues and suggests that the role of Phe-120 is to control the orientation of the aromatic ring found in most substrates with respect to the heme. The structure has been compared with published homology models and has been used to explain much of the reported site-directed mutagenesis data and help understand the metabolism of several compounds.     

Impact of incorporating the 2C5 crystal structure into comparative models of cytochrome P450 2D6

Cytochrome P450 2D6 (CYP2D6) metabolizes approximately one third of the drugs in current clinical use. To gain insight into its structure and function, we have produced four different sets of comparative models of 2D6: one based on the structures of P450s from four different microorganisms (P450 terp, P450 eryF, P450 cam, and P450 BM3), another on the only mammalian P450 (2C5) structure available, and the other two based on alternative amino acid sequence alignments of 2D6 with all five of these structures. Principal component analysis suggests that inclusion of the 2C5 crystal structure has a profound effect on the modeling process, altering the general topology of the active site, and that the models produced differ significantly from all of the templates. The four models of 2D6 were also used in conjunction with molecular docking to produce complexes with the substrates codeine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP); this identified Glu 216 [in the F-helix; substrate recognition site (SRS) 2] as a key determinant in the binding of the basic moiety of the substrate. Our studies suggest that both Asp 301 and Glu 216 are required for metabolism of basic substrates. Furthermore, they suggest that Asp 301 (I-helix, SRS-4), a residue thought from mutagenesis studies to bind directly to the basic moiety of substrates, may play a key role in positioning the B'-C loop (SRS-1) and that the loss of activity on mutating Asp 301 may therefore be the result of an indirect effect (movement of the B'-C loop) on replacing this residue.     

Role of glutamic acid 216 in cytochrome P450 2D6 substrate binding and catalysis

Human cytochrome P450 (P450) 2D6 is an important enzyme involved in the metabolism of drugs, many of which are amines or contain other basic nitrogen atoms. Asp301 has generally been considered to be involved in electrostatic docking with the basic substrates, on the basis of previous modeling studies and site-directed mutagenesis. Substitution of Glu216 with a residue other than Asp strongly attenuated the binding of quinidine, bufuralol, and several other P450 2D6 ligands. Catalytic activity with the substrates bufuralol and 4-methoxyphenethylamine was strongly inhibited by neutral or basic mutations at Glu216 (>95%), to the same extent as the substitution of Asn at Asp301. Unlike the Asp301 mutants, the Gln216 mutant (E216Q) retained 40% enzyme efficiency with the substrate spirosulfonamide, devoid of basic nitrogen, suggesting that the substitutions at Glu216 affect binding of amine substrates more than other catalytic steps. Attempts to induce catalytic specificity toward new substrates by substitutions at Asp301 and Glu216 were unsuccessful. Collectively, the results provide evidence for electrostatic interaction of amine substrates with Glu216, and we propose that both of these acidic residues plus at least another residue(s) is (are) involved in binding the repertoire of P450 2D6 ligands.     

Binding of bufuralol, dextromethorphan, and 3,4-methylenedioxymethylamphetamine to wild-type and F120A mutant cytochrome P450 2D6 studied by resonance Raman spectroscopy

Cytochrome P450 2D6 (CYP2D6) is one of the most important drug-metabolizing enzymes in humans. Resonance Raman data, reported for the first time for CYP2D6, show that the CYP2D6 heme is found to be in a six-coordinated low-spin state in the absence of substrates, and it is perturbed to different extents by bufuralol, dextromethorphan, and 3,4-methylenedioxymethylamphetamine (MDMA). Dextromethorphan and MDMA induce in CYP2D6 a significant amount of five-coordinated high-spin heme species and reduce the polarity of its heme-pocket, whereas bufuralol does not. Spectra of the F120A mutant CYP2D6 suggest that Phe120 is involved in substrate-binding of dextromethorphan and MDMA, being responsible for the spectral differences observed between these two compounds and bufuralol. These differences could be explained postulating a different substrate mobility for each compound in the CYP2D6 active site, consistently with the role previously suggested for Phe120 in binding dextromethorphan and MDMA.     

Altered spin state equilibrium in the T309V mutant of cytochrome P450 2D6: a spectroscopic and computational study

Cytochrome P450 2D6 (CYP2D6) is one of the most important cytochromes P450 in humans. Resonance Raman data from the T309V mutant of CYP2D6 show that the substitution of the conserved I-helix threonine situated in the enzyme’s active site perturbs the heme spin equilibrium in favor of the six-coordinated low-spin species. A mechanistic hypothesis is introduced to explain the experimental observations, and its compatibility with the available structural and spectroscopic data is tested using quantum-mechanical density functional theory calculations on active-site models for both the CYP2D6 wild type and the T309V mutant. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Alois Bonifacio, André R. Groenhof and Peter H. J. Keizers contributed equally to this work.     

Structural diversity within the mononuclear and binuclear active sites of N-acetyl-D-glucosamine-6-phosphate deacetylase

NagA catalyzes the hydrolysis of N-acetyl-d-glucosamine-6-phosphate to d-glucosamine-6-phosphate and acetate. X-ray crystal structures of NagA from Escherichia coli were determined to establish the number and ligation scheme for the binding of zinc to the active site and to elucidate the molecular interactions between the protein and substrate. The three-dimensional structures of the apo-NagA, Zn-NagA, and the D273N mutant enzyme in the presence of a tight-binding N-methylhydroxyphosphinyl-d-glucosamine-6-phosphate inhibitor were determined. The structure of the Zn-NagA confirms that this enzyme binds a single divalent cation at the beta-position in the active site via ligation to Glu-131, His-195, and His-216. A water molecule completes the ligation shell, which is also in position to be hydrogen bonded to Asp-273. In the structure of NagA bound to the tight binding inhibitor that mimics the tetrahedral intermediate, the methyl phosphonate moiety has displaced the hydrolytic water molecule and is directly coordinated to the zinc within the active site. The side chain of Asp-273 is positioned to activate the hydrolytic water molecule via general base catalysis and to deliver this proton to the amino group upon cleavage of the amide bond of the substrate. His-143 is positioned to help polarize the carbonyl group of the substrate in conjunction with Lewis acid catalysis by the bound zinc. The inhibitor is bound in the alpha-configuration at the anomeric carbon through a hydrogen bonding interaction of the hydroxyl group at C-1 with the side chain of His-251. The phosphate group of the inhibitor attached to the hydroxyl at C-6 is ion paired with Arg-227 from the adjacent subunit. NagA from Thermotoga maritima was shown to require a single divalent cation for full catalytic activity.     

Why is quinidine an inhibitor of cytochrome P450 2D6? The role of key active-site residues in quinidine binding

We have previously shown that Phe(120), Glu(216), and Asp(301) in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolizing enzyme (Paine, M. J., McLaughlin, L. A., Flanagan, J. U., Kemp, C. A., Sutcliffe, M. J., Roberts, G. C., and Wolf, C. R. (2003) J. Biol. Chem. 278, 4021-4027 and Flanagan, J. U., Maréchal, J.-D., Ward, R., Kemp, C. A., McLaughlin, L. A., Sutcliffe, M. J., Roberts, G. C., Paine, M. J., and Wolf, C. R. (2004) Biochem. J. 380, 353-360). We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge at either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (K(d)) for quinidine binding to the wild-type enzyme and the E216D and D301E mutants were 0.25-0.50 microm. The amide substitution of Glu(216) or Asp(301) resulted in 30-64-fold increases in the K(d) for quinidine. The double mutant E216Q/D301Q showed the largest decrease in quinidine affinity, with a K(d) of 65 microm. Alanine substitution of Phe(120), Phe(481),or Phe(483) had only a minor effect on the inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolize quinidine. E216F produced O-demethylated quinidine, and F120A and E216Q/D301Q produced both O-demethylated quinidine and 3-hydroxyquinidine metabolites. Homology modeling and molecular docking were used to predict the modes of quinidine binding to the wild-type and mutant enzymes; these were able to rationalize the experimental observations.     

Functional Expression of House Fly (Musca domestica) Cytochrome P450 CYP6D1 in Yeast (Saccharomyces cerevisiae)

Cytochrome P450 CYP6D1 from the house fly is important in the detoxication of xenobiotics and in resistance to pyrethroid insecticides. In house fly microsomes CYP6D1 requires cytochrome b₅ for the metabolism of some substrates, such as benzo[a]pyrene, but does not require cytochrome b₅ for the metabolism of other substrates such as methoxyresorufin. To examine the molecular mechanisms involved in its metabolism of pyrethroids and other substrates, a system for the heterologous expression of CYP6D1 in the yeast Saccharomyces cerevisiae was developed. Heterologous CYP6D1 can be inducibly expressed by culture in media with galactose as the sole carbon source, and is successfully inserted into the yeast microsomes. CYP6D1 is enzymatically active, as measured by methoxyresorufin-O-demethylation, indicating that CYP6D1 is able to interact with yeast P450 reductase. However, CYP6D1 expression did not result in measurable benzo[a]pyrene hydroxylation, suggesting that CYP6D1 cannot interact with yeast cytochrome b₅, or that there is insufficient cytochrome b₅ in the yeast microsomes to support this CYP6D1-mediated activity. Some suggestions are made for improving the yeast microsomal oxidoreductase environment in order to optimize CYP6D1 function.     

Inhibition of human cytochrome P450 1A2 by flavones: A molecular modeling study

Cytochrome P450 1A2 metabolizes a number of important drugs, procarcinogens, and endogenous compounds. Several flavones, a class of phytochemicals consumed in the human diet, have been shown to differentially inhibit human P450 1A2-mediated methoxyresorufin demethylase. A molecular model of this P450 was constructed in order to elucidate the molecular basis of the P450-flavone interaction. Flavone and its 3,5,7-trihydroxy and 3,5,7-trimethoxy derivatives were docked into the active site to assess their mode of binding. The site is hydrophobic and includes several residues that hydrogen bond with substituents on the flavone nucleus. The binding interactions of these flavones in the modeled active side are consistent with their relative inhibitory potentials, namely 3,5,7-trihydroxylflavone > flavone >3,5,7-trimethoxylflavone, toward P450 1A2-mediated methoxyresorufin demethylation.     

High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy: II. Validation of a direct injection/on-line guard cartridge extraction–tandem mass spectrometry method for CYP2D6 inhibition assessment

A highly efficient direct injection/on-line guard cartridge extraction–tandem mass spectrometry (DI/GCE–MS–MS) method has been validated for high-throughput evaluation of cytochrome P450 (CYP) 2D6 inhibition potential using human hepatic microsomes and 96-well microtiter plates. Microsomal incubations were terminated with formic acid, centrifuged, and the resulting supernatants were injected for DI/GCE–MS–MS analysis. Due to the novel use of an extremely short C₁₈ guard cartridge, this method exhibits several advantages, such as no sample preparation, excellent on-line extraction, short run time (2.5 min), and minimized source contamination and performance deterioration. The DI/GCE–MS–MS method demonstrates acceptable accuracy and precision for the quantification of dextrorphan, a marker metabolite of dextromethorphan mediated by CYP2D6, in microsomal incubations. The CYP2D6 inhibition assay has been validated using quinidine as a known selective inhibitor of the isoform. The IC₅₀ value (0.20 μM) measured by the new method is in good agreement with the literature value (0.22 μM).     

Inhibition of human cytochrome P450 1A2 by flavones: A molecular modeling study

Cytochrome P450 1A2 metabolizes a number of important drugs, procarcinogens, and endogenous compounds. Several flavones, a class of phytochemicals consumed in the human diet, have been shown to differentially inhibit human P450 1A2-mediated methoxyresorufin demethylase. A molecular model of this P450 was constructed in order to elucidate the molecular basis of the P450-flavone interaction. Flavone and its 3,5,7-trihydroxy and 3,5,7-trimethoxy derivatives were docked into the active site to assess their mode of binding. The site is hydrophobic and includes several residues that hydrogen bond with substituents on the flavone nucleus. The binding interactions of these flavones in the modeled active side are consistent with their relative inhibitory potentials, namely 3,5,7-trihydroxylflavone > flavone >3,5,7-trimethoxylflavone, toward P450 1A2-mediated methoxyresorufin demethylation.     

Structural insight into the altered substrate specificity of human cytochrome P450 2A6 mutants

Human P450 2A6 displays a small active site that is well adapted for the oxidation of small planar substrates. Mutagenesis of CYP2A6 resulted in an increased catalytic efficiency for indole biotransformation to pigments and conferred a capacity to oxidize substituted indoles (Wu, Z.-L., Podust, L.M., Guengerich, F.P. J. Biol. Chem. 49 (2005) 41090-41100.). Here, we describe the structural basis that underlies the altered metabolic profile of three mutant enzymes, P450 2A6 N297Q, L240C/N297Q and N297Q/I300V. The Asn297 substitution abolishes a potential hydrogen bonding interaction with substrates in the active site, and replaces a structural water molecule between the helix B'-C region and helix I while maintaining structural hydrogen bonding interactions. The structures of the P450 2A6 N297Q/L240C and N297Q/I300V mutants provide clues as to how the protein can adapt to fit the larger substituted indoles in the active site, and enable a comparison with other P450 family 2 enzymes for which the residue at the equivalent position was seen to function in isozyme specificity, structural integrity and protein flexibility.     

Essential requirements for substrate binding affinity and selectivity toward human CYP2 family enzymes

A detailed analysis of substrate selectivity within the cytochrome P450 2 (CYP2) family is reported. From a consideration of specific interactions between drug substrates for human CYP2 family enzymes and the putative active sites of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1, it is likely that the number and disposition of hydrogen bond donor/acceptors and aromatic rings within the various P450 substrate molecules determines their enzyme selectivity and binding affinity, together with directing their preferred routes of metabolism by the CYP2 enzymes concerned. Although many aliphatic residues are present in most P450 active sites, it would appear that their main contribution centers around hydrophobic interactions and desolvation processes accompanying substrate binding. Molecular modeling studies based on the recent CYP2C5 crystal structure appear to show close agreement with site-directed mutagenesis experiments and with information on substrate metabolism and selectivity within the CYP2 family.     

Molecular Modeling of Cytochrome P450 2B1: Mode of Membrane Insertion and Substrate Specificity

A molecular model of a mammalian membrane-bound cytochrome P450, rat P450 2B1, was constructed in order to elucidate its mode of attachment to the endoplasmic reticulum and the structural basis of substrate specificity. The model was primarily derived from the structure of P450BM-3, which as a class II P450 is the most functionally similar P450 of known structure. However, model development was also guided by the conserved core regions of P450cam and P450terp. To optimally align the P450 2B1 and P450BM-3 sequences, multiple alignment was performed using sequences of five P450s in the II family, followed by minor adjustments on the basis of secondary structure predictions. The resulting P450 2B1 homology model structure was refined by molecular dynamics heating, equilibration, simulation, and energy minimization. The model suggests that the F–G loop serves as both a hydrophobic membrane anchor and entrance channel for hydrophobic substrates from the membrane to the P450 active site. To assess the mode of substrate binding, benzphetamine, testosterone, and benzo[a]pyrene were docked into the active site. The hydrophobic substrate-binding pocket is consistent with the preferences of this P450 toward hydrophobic substrates, while the presence of an acidic Glu-105 in this pocket is consistent with the preference of this P450 for the cationic substrate benzphetamine. This model is thus consistent with several known experimental properties of this P450, such as membrane attachment and substrate selectivity.     

Effect of carbonate anion on cytochrome P450 2D6-mediated metabolism in vitro: the potential role of multiple oxygenating species

Studies were designed to investigate various anions and their effects on cytochrome P450 2D6-mediated metabolism in vitro. Incubations were initially performed in buffered phosphate, carbonate, sulfate, and acetate solutions (50mM, pH 7.4), with CYP2D6 substrates dextromethorphan, 7-methoxy-4-(aminomethyl)-coumarin (MAMC), (S,S)-3-[3-(methylsulfonyl)phenyl]-1-propylpiperidine hydrochloride [(-)-OSU6162], and amitriptyline. Dextromethorphan and MAMC O-dealkylation activity in buffered carbonate was approximately 25 and 38%, respectively, relative to phosphate, while activity in sulfate and acetate buffers displayed minor differences. In contrast, N-dealkylation reactions for both (-)-OSU6162 and amitriptyline were unaffected by the presence of carbonate, and the other anions tested. Subsequent kinetic studies revealed that the basis of reduced turnover of dextromethorphan was primarily a V(max) effect, as the V(max) for the rate was 16.9 and 5.6 pmol/min/pmol P450 in phosphate and carbonate, respectively. Interestingly, similar rates of dextromethorphan O-demethylation in phosphate and carbonate were observed when reactions were supported by cumene hydroperoxide (CuOOH). Furthermore, it was observed that while CuOOH could equally support dextromethorphan O-demethylation compared to NADPH, amitriptyline N-demethylation was only minimally supported. Finally, intramolecular kinetic isotope effect (KIE) experiments with amitriptyline-d3 in CuOOH-supported reactions yielded a k(H)/k(D) of 5.2, substantially higher than in phosphate and carbonate supported by NADPH (k(H)/k(D)=1.5). Overall, results suggest that carbonate disrupts the relative ratios of the potential P450 oxygenating species, which differentially catalyze O- and N-dealkylation reactions mediated by CYP2D6.     

The roles of amino acid residues at positions 216 and 219 in the structural stability and metabolic functions of rat cytochrome P450 2D1 and 2D2

We examined the effects of the mutual substitution of amino acid residues at positions 216 and 219 between rat CYP2D1 and CYP2D2 on their microsomal contents and enzymatic functions using a yeast cell expression system and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) as a substrate. CYP2D1 has amino acid residues, leucine and valine, at positions of 216 and 219, respectively, whereas CYP2D2 has phenylalanine and aspartic acid at the same positions. In reduced carbon monoxide-difference spectroscopic analysis, the substitution of Asp-219 of CYP2D2 by valine markedly increased a peak at 450 nm and concomitantly decreased a peak at 420 nm, while the replacement of Phe-216 of CYP2D2 with leucine gave no observable change. The double substitution of Phe-216 and Asp-219 by leucine and valine, respectively, yielded a typical CYP spectrum. The substitution of Val-219 of CYP2D1 by aspartic acid decreased the CYP content to one-half, whereas the replacement of Leu-216 with phenylalanine did not have any effect. The double substitution of Leu-216 and Val-219 of CYP2D1 by phenylalanine and aspartic acid, respectively, diminished the CYP content by 90%. CYP2D1 catalyzed both 5-MeO-DIPT N-deisopropylation and O-demethylation at relatively low levels, while CYP2D2 catalyzed 5-MeO-DIPT O-demethylation efficiently. The substitution of the amino acid at position 216 substantially increased 5-MeO-DIPT oxidation activities of the two CYP2D enzymes. The replacement of the amino acid at position 219 increased the 5-MeO-DIPT O- and N-dealkylation activities of CYP2D1, whereas it decreased the 5-MeO-DIPT O-demethylation activity of CYP2D2. These results indicate that amino acid residues at positions 216 and 219 have important roles in the enzymatic functions of rat CYP2D1 and CYP2D2.     

CYP3A4 and CYP2D6 inhibitory activities of Indonesian medicinal plants

Thirty samples of Indonesian medicinal plants were analyzed for their capacity to inhibit in vitro metabolism by human cytochrome P450 3A4 (CYP3A4) and CYP2D6 with a radiometric assay. The MeOH-soluble fractions of 25 samples, prepared from water extracts, demonstrated inhibitory activity more than 50% on the metabolism mediated by CYP3A4, and 21 samples on the metabolism mediated by CYP2D6. Among the MeOH-soluble fractions, Piper nigrum leaf showed the highest inhibitory activity against CYP3A4 (91.7%), and Punica granatum against CYP2D6 (98.1%). The water extracts of which MeOH-soluble fraction showed inhibitory activity more than 70% were fractionated with EtOAc. From the EtOAc-soluble fractions, Curcuma heyneana (67.0%), Pi. cubeba (75.0%), Pi. nigrum fruit (84.0%), Pi. nigrum leaf (85.8%), and Zingiber aromaticum (75.3%) demonstrated inhibitory activity more than 50% on the metabolism mediated by CYP3A4, but only Pi. nigrum fruit (72.8%) and Pi. nigrum leaf (69.1%) showed strong inhibitory activity against CYP2D6. For samples that showed more than 70% inhibition, their IC(50) values were determined. The most potent inhibitory activity against CYP3A4 (IC(50) value of 25 microg/ml) was found for the extract of Pi. nigrum leaf, while that of Catharanthus roseus showed the most potent inhibitory effect against CYP2D6 (IC(50) value of 11 microg/ml). These results should indicate once more the possibility of potential medicinal plant-drug interactions.