Twenty-five years of Research on Cytochromes P450 2B: To ...

Goals <strong>of</strong> Predictive DrugMetabolism• Overcome metabolic lability <strong>of</strong> a newchemical entity (NCE)• Avoid drug interactions caused by anNCE• Predict functional consequences <strong>of</strong>SNP's (individuals, populations)• Predict pharmacokinetics <strong>of</strong> an NCE

Overview1. Species, strain, and individualdifferences in P450 2B function2. Conformational plasticity <strong>of</strong> cytochromesP450 2B3. Multiplicity <strong>of</strong> cytochromes P450 3A invarious species4. Cooperativity <strong>of</strong> human P450 3A45. Lessons learned

Species, Strain, and IndividualDifferences in P450 2B Function• Mechanism-based inactivation• Steroid hydroxylation• PCB oxidation

P450 2B Enzymes1. Highly inducible by phenobarbital in a number<strong>of</strong> species (human, rat, rabbit, mouse, dog)2. Among the first P450s purified andcharacterized (LM2, PB-B/P450b)3. Metabolize angular, medium-sized neutral orbasic compounds4. Exhibit marked species and strain differencesin function

14 C (cpm)Halpert, J. (1981). Covalent modification <strong>of</strong> lysine during the suicide inactivation<strong>of</strong> rat liver cytochrome P-450 by chloramphenicol. Biochem. Pharmacol.30:875-881.Halpert, J., Miller, N., and Gorsky, L. (1985). On the mechanism <strong>of</strong> theinactivation <strong>of</strong> the major phenobarbital-inducible isozyme <strong>of</strong> rat livercytochrome P-450 by chloramphenicol. J. Biol. Chem. 260:8397-8403.TIME (min)• Chloramphenicol is converted by P450 2B1 to an oxamyl chloride, whichbinds to the ε-amino group <strong>of</strong> one or more lysine residues in the enzyme.• The modified enzyme is unable to undergo enzymatic reduction by NADPHcytochromeP450 reductase.

Halpert, J., Balfour, C., Miller, N.E., Morgan, E.T., Dunbar, D., and Kaminsky,L.S. (1985) Isozyme-selectivity <strong>of</strong> the inhibition <strong>of</strong> rat liver cytochromes P-450by chloramphenicol in vivo. Mol. Pharmacol. 28:290-296.• Chloramphenicol inactivates three (2B1, 2C6, 2C11) <strong>of</strong> eightmajor rat liver microsomal cytochrome P450 enzymes.

Halpert, J., Jaw, J.-Y., Balfour, C., and Kaminsky, L.S. (1990). Selectiveinactivation by chlor<strong>of</strong>luoroacetamides <strong>of</strong> the major phenobarbital-inducibleform(s) <strong>of</strong> rat liver cytochrome P-450. Drug Metab. Dispos. 18:168-174.• N-(2-p-nitrophenethyl)chlor<strong>of</strong>luoroacetamide is aselective inactivator <strong>of</strong> P450 2B1 in vitro and in vivo.

% 16 -Hydroxylase ActivityKedzie, K.M., Balfour, C.A., Escobar, G.Y., Grimm, S.W., He, Y.-A., Pepperl, D.,Regan, J., Stevens, J.C., and Halpert, J.R. (1991). Molecular basis for afunctionally unique cytochrome P450IIB1 variant. J. Biol. Chem. 266:22515-22521.WMIIB11616• A Gly-478 to Ala substitution in P450 2B1 from Wistar-Munich ratsis responsible for the low androgen 16 β-hydroxylase activity andlack <strong>of</strong> susceptibility to inactivation by N-(2-p-nitrophenethyl)-chlor<strong>of</strong>luoroacetamide.

245-HCB Metabolism(% <strong>of</strong> Control)Duignan, D.B., Sipes, I.G., Leonard, T.B., and Halpert, J. (1987). Purificationand characterization <strong>of</strong> the dog hepatic cytochrome P-450 isozymeresponsible for the metabolism <strong>of</strong> 2,2', 4,4', 5,5'-hexachlorobiphenyl. Arch.Biochem. Biophys. 255:290-303.• Canine cytochrome P450 2B11 is responsible for the hydroxylation <strong>of</strong>245-HCB in liver microsomes from control and phenobarbital-treatedanimals.• The high constitutive expression and activity <strong>of</strong> this enzyme account forthe unique ability <strong>of</strong> dogs to eliminate 245-HCB in vivo.

Spatzenegger, M., Wang, Q., He, Y.Q., Wester, M.R., Johnson, E.F. and Halpert,J.R. (2001). Amino acid residues critical for differential inhibition <strong>of</strong> CYP2B4,CYP2B5 and CYP2B1 by phenylimidazoles. Mol. Pharmacol. 59:475-484.Inhibition <strong>of</strong> CYP2B4, 2B5 and 2B1 byphenylimidazole derivativesCompoundsIC 50CYP2B4 CYP2B5 CYP2B11-Phenylimidazole 0.90 4.2 0.591-(4-Chlorophenyl)imidazole 0.12 15.6 0.08µM4-Phenylimidazole 0.49 8.4 0.264-(4-Chlorophenyl)imidazole 0.04 3.8 0.07• Selective inhibition <strong>of</strong> P450 2B4 vs. 2B5 can be rationalized withthe help <strong>of</strong> homology models based on P450 2C5.• Differences in the determinants <strong>of</strong> inhibition are caused byresidue-residue interactions as well as residue-inhibitor contacts.

% P450 ExtractedScott, E.E., Spatzenegger, M., and Halpert, J.R. (2001). A truncation <strong>of</strong>2B subfamily cytochromes P450 yields increased expression levels,increased solubility, and decreased aggregation while retaining function.Arch. Biochem. Biophys. 395:57-68.2B1dH 2B4dH 2B6dH 2B11dH2B P450• P450 2B4 is an excellent candidate for crystallization trials.

Conformational Plasticity <strong>of</strong> P4502B Enzymes• X-ray crystallography <strong>of</strong> rabbit P450 2B4• Hydrogen-deuterium mass exchangestudies <strong>of</strong> 2B4• X-ray crystallography <strong>of</strong> human P450 2B6

• protein engineering:highly concentrated proteinsolution(+ salt, detergent, DTT,EDTA, buffer . . .)+Control <strong>of</strong> Stability/Solubility1 10 20 30 4902B4 MALLLAVLLA FLAGLLLLLF RGHPKAHGRL PPGP . . . AR2B4dH MA KKTSSKGKL PPGP . . . ARHHHHUnderline indicates alteration from the native sequence.• salt• detergentprecipitant(EtOH, Na citrate pH 5.5)Crystallizationprotein + precipitantprecipitant sitting dropvapor diffusion• The engineering and purification approaches elaborated for P4502C5 enable 2B4 crystallization.

Cytochrome P450 2B4 Dimermolecule 1molecule 2• In the absence <strong>of</strong> exogenous ligand, P450 2B4 crystallizedas a dimer, which allowed trapping <strong>of</strong> an open form <strong>of</strong> theenzyme and helps explain ligand access to the active site.

Location <strong>of</strong> Active Site Residues in the 2B4 OpenConformation vs. a Closed 2B5 Structureactive siteresidues2B42C5-DMZ

Goal: Substrate/Inhibitor-Bound ComplexApproaches to Discourage Homodimer Formation:1. Protein engineering: H226Y2. Choose soluble, high-affinity ligand:3. Form protein-ligand complex early by adding inhibitor prior toprotein concentrationClNN

Comparison <strong>of</strong> 2B4 StructuresB'-C helicesF'-G helicesCytochrome P450 2B42B4(H226Y)-4(4Cl)PI

Conclusions: Conformational Shift MayAccommodate Sequential Substrate Binding andCatalysiscleft open for substratebinding/metabolite release2B4cleft closed for regio- andstereoselective metabolism2B4(H226Y)-4(4Cl)PI

P450 PlasticityDefinition• Involves the large-scale motions required forsubstrate access and product egress• Includes the reshaping <strong>of</strong> the active site to adaptto ligands <strong>of</strong> different geometryQuestions• Do the large conformational changes inferredfrom crystal structures occur in solution?• What are the implications <strong>of</strong> plasticity forprediction <strong>of</strong> ligand binding?

Imidazole Inhibitors <strong>of</strong> P450 2B41-BI1-PBI1-TIClotrimazoleNNNCl1-BPINNNNNNN1-CPI1-BHIBifonazole1-(Dibiphenyl)MeINClNCl4-CPINHNNNNNNN

Comparison <strong>of</strong> Five 2B4 Structures

Pivoting <strong>of</strong> the F-G Helix Casette toAccommodate the VariousInhibitors

DX-MS Setup (Exchange)• Remove aliquot for timepoint• Quench on ice (0°C)• Aliquot replicates• Place all vials in dry ice t<strong>of</strong>lash freeze• Store samples in -80°CfreezerGarcia, Pantazatos, and Villarreal. Assay and drug developmenttechnologies (2004) 2(1): 81-91.

DX-MS Setup (MS)Samples quickly thawed to 0°C from -80°C.Garcia, Pantazatos, and Villarreal. Assay and drug developmenttechnologies (2004) 2(1): 81-91.

B’-C RegionF-G Cassette2B4dH Exchange (1000s)B’-C RegionF-G Cassette2B4 No Ligand2B4 + 4-CPIB’-C RegionF-G CassetteB’-C RegionF-G Cassette2B4 + 1-PBIDifference in # <strong>of</strong> DeuteronsDifference (4-CPI vs. No L)

Oxidation by Human P450 2B6 <strong>of</strong>Antiplatelet Drugs

CYP2B4:Ticlopidine Complex Ribbon DiagramNH 2TiclopidineAF’G’CB’HGKIEFJDCOOH

Ticlopidine Bound in the Active Site <strong>of</strong> P450 2B4in the Opposite Orientation Predicted from 2B6I209S210F206I101V477F297F115I114A298E301V367I363T302

Structure <strong>of</strong> Ticlopidine and the % Differencein Paramagnetic Relaxation Rate Relative tothe Slowest Proton

Crystallization <strong>of</strong> t-BPA Labeled2B4• Proposed mechanism <strong>of</strong> formation <strong>of</strong> tBPA-2B4 adduct (Zhang, et al. (2009), Mol Pharm)• Collaboration with Hollenberg Lab (University<strong>of</strong> Michigan)

Molecular ReplacementE301T302• Maps generatedusing 2B4-1-CPIas a MR searchmodel• E301 is pointedout <strong>of</strong> active site.• Density appearsto agree withdocking <strong>of</strong> tBPAby Zhang, et al.

Induced-fit (1) vs. ConformationalSelection (2) Models <strong>of</strong> P450 2B4DX-MS suggests that an open conformation <strong>of</strong> ligand-freeenzyme predominates in solution. However, we recentlysolved a structure <strong>of</strong> a closed ligand-free form <strong>of</strong> P450 2B4.

Properties <strong>of</strong> Human P450 2B6– Major catalyst <strong>of</strong> oxidation <strong>of</strong> bupropion,cyclophosphamide, and efavirenz– Highly polymorphic (Q172H and K262R <strong>of</strong>particular importance)– Represents 1-5% <strong>of</strong> total hepatic P450– Along with CYP3A5 one <strong>of</strong> the few humandrug metabolizing P450s yet to be crystallized– Low thermal stability

%4020Engineering Cytochrome P450 2B6 for EnhancedExpression and Stability0P450P450nmol/LRat 2B1dH 800 - 1200Rabbit 2B4dH 300 - 500Human 2B6dH 50 - 75Dog 2B11dH 400 -600P450 (HS, LS, P420)ActivityT m( C) T 50( C)2B1dH 61.3 0.2 52.7 0.32B4dH 58.9 0.3 50.8 0.42B6dH 48.2 0.2 45.2 0.42B11dH 51.4 0.3 48.4 0.2% P450% Activity100806040200030 35 40 45 50 55 60 65 70 7530 35 40 45Temperature ( O C)10080604020% Activity030 35 40 45 50 55 60 65Temperature ( o C)10030 35 40 452B1dH802B4dH2B6dH2B11dH604020x column 5 vs y column 5x column 6 vs y column 6x column 7 vs y column 7x column 8 vs y column 82B1dH2B4dH2B6dH2B11dHx column 5 vs y column 5x column 6 vs y column 6x column 7 vs y column 7x column 8 vs y column 8TemperaTempe

Approaches to EnableCrystallization <strong>of</strong> Human P450 2B6• N-terminal modification and use <strong>of</strong>chaperones• Internal mutations to improve stability(Y226H) and solubility (K262R)• Different CM-resin• Replacement <strong>of</strong> glycerol by sucrose afterchromatography• Inclusion <strong>of</strong> facial amphiphiles during trials

P450 2B6:4-CPI Complex Structure (2 Å)

Structural Alignment <strong>of</strong> P450 2B4and 2B6 4-CPI Complexes2B6 = Green; 2B4 = MagentaAF’G’CGHFGIB’F’G’AIEFHCDThe RMSD in a C α overlay <strong>of</strong> 2B6 and 2B4 is only 0.65 Å.

Only residue 363 and orientation <strong>of</strong> E301 differ between the active sites.2B6 Active Site Density/2B6 and2B4 Active Site OverlayV104I209F206V477F297I101F115I114A298E301V367L363/I363T3022Fo-Fc map contoured at 1-σ

Summary1. P450 2B4 exhibits remarkableconformational flexibility with imidazoles<strong>of</strong> different size and shape as evidencedby X-ray crystallography and solutionmethods.2. Despite ~100 amino acid differences, theX-ray crystal structures <strong>of</strong> the 4-CPIcomplexes <strong>of</strong> P450 2B4 and 2B6 arealmost identical.3. How do we reconcile these twoseemingly contradictory findings?

AcknowledgmentsDr. Chris ChinDr. Sean GayYou-ai HeYou Qun HeDr. Santosh KumarDr. Hong LiuDr. Keiko MaekawaDr. B.K. MuralidharaDr. Art RobertsDr. Emily ScottDr. Manish ShahDr. Margit SpatzeneggerLing SunDr. Jyothi TalakadDr. Qinmi WangDr. Mark WhiteDr. Ross WildermanDr. Yonghong ZhaoDr. Eric JohnsonDr. Dave StoutDr. Mike WesterDr. Qinghai ZhangDr. Sheng LiDr. Tong LiuDr. Virgil WoodsDr. Paul HollenbergDr. Haoming ZhangES003619 (JRH)ES006676 (JRH)GM31001 (EFJ)GM59229 (CDS)CA016954 (PFH)

Multiplicity <strong>of</strong> P450 3A enzymes• Purification <strong>of</strong> multiple rat P450 3Aenzymes• cDNA cloning <strong>of</strong> two canine P450 3Aenzymes

Graves, P.E., Kaminsky, L.S., and Halpert, J. (1987). Evidence forfunctional and structural multiplicity <strong>of</strong> pregnenolone-16 -carbonitrileinduciblecytochrome P450 isozymes in rat liver microsomes.Biochemistry 26:3887-3894.Treatment 9,10-DehydrowarfarinR-10-OHwarfarinAdione6 -OHTAOcomplexNone 0.04 0.22 0.20 0.11PCN 0.21 1.26 1.03 0.77PCN +CAP0.11 2.64 0.46 0.13• Chloramphenicol exerts differential effects on four enzymeactivities considered diagnostic <strong>of</strong> the single known PCNinducibleP450 in female rat liver microsomes.

Halpert, J.R. (1988). Multiplicity <strong>of</strong> steroid-inducible cytochromes P450in rat liver microsomes. Arch. Biochem. Biophys. 263:59-68.• Rat liver microsomes contain three steroid-inducible P450 forms thatcross-react immunologically. One can be distinguished from the othertwo on SDS-PAGE gels, by Ouchterolony double immunodiffusion, andby amino terminal sequence analysis.

Fraser, D.R., He, Y.Q., Harlow, G.R., and Halpert, J.R. (1999). Use <strong>of</strong>chimeric enzymes and site-directed mutagenesis for identification <strong>of</strong>three key residues responsible for differences in steroid hydroxylationbetween canine cytochromes P-450 3A12 and 3A26. Mol. Pharmacol.55:241-247.• Heterologously expressed P450 3A12 and 3A26 can account forthe two proteins in dog liver microsomes recognized by antibodiesto a purifed PB-inducible canine liver P450 (PBD-1).

Complications Presented by CYP3A4• Sigmoidal steady-state kinetics.aflatoxin B 1 , amitriptyline,carbamazepine, diazepam,17 -estradiol, progesterone,testosterone.• Stimulation by some substrates <strong>of</strong> the oxidation <strong>of</strong> othersubstrates.-naphth<strong>of</strong>lavone, diazepam, progesterone, quinidine,testosterone.• Partial or no inhibition in the presence <strong>of</strong> two substrates.erythromycin and testosterone, midazolam andterfenadine, aflatoxin B 1 and -naphth<strong>of</strong>lavone,progesterone and 7-benzyloxy-4-trifluoromethylcoumarin.

Domanski, T.L,, He, Y.-A., Khan, K.K., Roussel, F., Wang, Q., andHalpert, J.R. (2001). Phenylalanine and tryptophan scanningmutagenesis <strong>of</strong> CYP3A4 substrate recognition site residues and effecton substrate oxidation and cooperativity. Biochemistry 40:10150-10106.•The differential effects <strong>of</strong> site-specific substitutions onmultiple P450 3A4 activities and heterotropic activation byANF suggest the presences <strong>of</strong> three subpockets within asingle large active site.

Multiple Conformers as an Alternative toMultiple Binding Sites within One P450MoleculeInitial concept: P450 isrepresented by twoconformers with differentsubstrate specificity anddifferent response uponsubstrate binding (Koleyet al., 1995, 1997).Challenge: Static distribution <strong>of</strong> the enzymebetween two populations suggested by the modelhas no obvious basis.

Our Understanding <strong>of</strong> the Mechanisms<strong>of</strong> P450 3A4 CooperativityP450 3A4 has two binding sites for substrates revealinghomotropic cooperativity (e.g. 1-PB).Interaction with the high affinity binding site triggers aconformational transition, which promotes binding atthe second, low affinity site and subsequent spinshift.This second binding event is likely also accompanied by aconformational change.P450 3A4 in solution and in the membrane is representedby a mixture <strong>of</strong> (at least) two persistent conformerswith different affinities for substrates and differentposition <strong>of</strong> spin equilibrium.This conformational heterogeneity is stabilized by theoligomerization <strong>of</strong> the enzyme.ANF can modulate the partitioning <strong>of</strong> these conformers.

• Lack <strong>of</strong> Substrate Inhibition in a Monomeric Form <strong>of</strong> HumanCytosolic Sult2A1 (Abstract 967.5)I.T. Cook, University <strong>of</strong> Alabama at BirminghamProtein-Protein Interactions and Modulation <strong>of</strong> DrugMetabolism, Wednesday 9:00 AM,Anaheim Convention Center, Room 210AB• IntroductionT. Tracy, University <strong>of</strong> Minnesota• Effect <strong>of</strong> P450-P450 Complex Formation on MonooxygenaseFunctionW. L. Backes, Louisiana State University School <strong>of</strong> Medicine• Microsomal Monooxygenase as a Multienzyme System: ExploringProtein-Protein Interactions <strong>of</strong> Cytrochromes P450D. Davydov, University <strong>of</strong> California, San Diego• UGT-CYP Protein Interactions: Role <strong>of</strong> Conjugating Enzymes inModulating Oxidative Enzyme ActivityY. Ishii, Kyushu University

AcknowledgmentsDr. Dmitri DavydovNadia DavydovaDr. Harshica FernandoDr. Jessica RumfeldtDr. Elena SinevaDr. Tamara TsalkovaDr. Steve SligarGM054995 (JRH)GM 31756, GM33775 (SGS)

Lessons Learned from Colleagues• Do what you should do not just what youcan do now (EFJ, RMP).• It’s better to be scared than bored (DCL).• Always look out for the next generation(MJC, RWE, BSM, PFH, AYHL, MRW,POM)

Some Members <strong>of</strong> the P450 Superfamily