MEDCH5271/20/2017Non-P450OxidasesandOxygenasesFlavin-ContainingMonooxygenases(microsomalFMOs)MolybdenumHydroxylases(cytosolicAOandXO)MonoamineOxidases(mitochondrialMAO-A,MAO-B)AldehydeDehydrogenases(cytosolicandmitochondrialALDH)
RendicandGuengerich,Chem.Res.Tox.(2015)
Usefulliterature• Fo<,RSandDalvieDK.CytochromeP450andNon–CytochromeP450
Oxida<veMetabolism.DrugMetab.Dispos.44:1229(2016).• KruegerSKandWilliamsDE.Mammalianflavin-containing
monooxygenases:structure/func<on,gene<cpolymorphismsandroleindrugmetabolism.PharmacolTher2005;106:357-387.
• Fenemaetal.TrimethylamineandTrimethylamineN-Oxide,aFlavin-ContainingMonooxygenase3(FMO3)-MediatedHost-MicrobiomeMetabolicAxisImplicatedinHealthandDisease.DrugMetab.Dispos.44:1839(2016).
• Hutzleretal.,Strategiesforacomprehensiveunderstandingofmetabolismbyaldehydeoxidase.ExpertOpin.DrugMetab.Toxicol.2013(2):153-68.
• YoudimMB,EdmondsonDEandTiptonKF.Thetherapeu<cpoten<alofmonoamineoxidaseinhibitors.NatureRev.Neurosci.7:295(2006).
• KoppakaVetal.Aldehydedehydrogenaseinhibitors.PharmacolRev.2012Jul;64(3):520-39.
Why should we care about non-P450 pathways? • P450-mediated metabolism dominates oxidative metabolic clearance of drugs, and so, multiple co-administered drugs can be competing for the same clearance pathways, leading potentially to serious drug-drug interactions.
• Genetic polymorphisms that reduce P450 function are well documented and can also cause serious drug-gene interactions, at least for low therapeutic index drugs.
• For both reasons, designing away from P450-dependent clearance mechanisms could be considered a useful strategy during early drug discovery.
• Moreover, as pharmaceutical companies have tried to design ligands for increasingly more complicated biological targets, there has been an inevitable increase in the lipophilicity and molecular volumes of new chemical entities, which have been associated with safety failures in the clinic.
• Therefore, replacement of easily incorporated carbocyclic rings (benzene, naphthyl) rings with heteroaromatic rings (pyrimidine, pthalazine, etc) has become an increasingly employed strategy to reduce lipophilicity.
• This has had the effect of switching away from P450, and towards aldehyde oxidase, as the main metabolic enzyme, for many newer drugs.
Outline• History/General Enzyme Characteristics
• Structure/Catalytic Mechanism
• Multiplicity/Regulation
• Substrates and Reaction Pathways
• In Vitro Methodologies for;
- differentiating FMO-mediated from P450-mediated Catalysis
- identifying AO- versus XO-dependent catalysis
- discriminating between MAO-A and MAO-B catalysis
FMOHistory1960s Alivermicrosomalenzymesystem(E.C.1.14.13.8)that
u<lizesNADPHandmolecularoxygentoconvertN,N’-dimethylanilinetoN,N-dimethylanilineN-oxidefirstdescribedbyDr.DanielZieglerandcolleagues.
1970s ‘Ziegler’senzyme’,purifiedfromhogliver,showntocontain
flavin,butnoheme,therebydis<nguishingthisflavin-containingmonooxygenase(FMO)fromthemicrosomalhemoprotein-containingcytochromeP450s.[1980s–‘lung’FMO]
1990s FMONomenclatureCommikeenameshogliverFMOasFMO1andthe‘lung’formasFMO2.Otherformswith<60%sequenceiden<tyarenamedwithascendingarabicnumerals,FMO3,4,5inallmammals.
N
CH3
CH3
N
CH3
O
CH3
FMO
ComparisonofGeneralProper<es:FMOvsP450 FMO P450
Func?on Monooxygenase Monooxygenase
Reducingcofactor NADPH NADPH
CellularLoca?on Microsomal Microsomal
Size 60-65kDa 50-60kDa
Substrates OxidizesatN,S,P N,S,PandC
Prosthe?cgroup FAD Heme
S+O2+NADPH+H+àSO+H2O+NADP+
FMOCataly<cCycle
O
NN
NADP
H, NADPH
OHO
OO
ON
NH
N
NH
O
ON
NH
N
NH
O
OHN
NH
N
NH
O
ON
NH
N
N
O2 NADPH2O
FAD-OOH FAD-OH
FADFADH
NADP
NADP
H
2
AspectsofFMOCatalysis• Thereac<onmechanismisordered,i.e.NADPH,oxygenand
oxidizablesubstrateaddtotheenzymebeforeanyoftheproductsleave.
• Theenzyme-boundhydroperoxyflavin[FAD-OOH]isaverystable,albeitrela<velyweakoxidant.
• Releaseofwaterand/orNADP+isbelievedtobetherate-limi<ngstep.
• FMOcanoxidizeprac<callyanysopnucleophilethattheenzyme’sFAD-OOHac<vecenterencounters.
• Ingeneral,unchargedandsingleposi<velychargedsubstratescangainaccess,inpreferencetonega<velychargedandmul<pleposi<velychargedcompounds.
FMOMul<plicity
HumanChromosome1
FMO7P-11P
• Fiveac<vegenes,FMO1-FMO5,arepresentinmostspecies.
• FMO6isinac<veduetoalterna<vesplicing.
• FMO1,FMO2andFMO3arethebestcharacterizedenzymes
FMO1:GeneralCharacteris<cs
• ThemajorityofourknowledgeoftheFMOenzymesisderivedfromdetailedbiochemicalstudiesconductedinthe1970sand‘80sonhogliverFMO1-thefirstFMOtobepurified.
• FMO1ischaracterizedbyitspromiscuoussubstratespecificity,whichisthewidestofalloftheFMOisoforms.
• FMO1isthedominantformoftheenzymeintheliverofmanyexperimentalanimalspecies,butisnotexpressedinadulthumanliver.
• Inhumans,liverFMO1isafetalenzyme,butisalsoexpressedatrela<velyhighlevelsinadultkidney.
FMO2:GeneralCharacteris<cs
• Generallyfoundathighestlevelsinlung<ssueofexperimentalspecies.
• FMO2isalsocharacterizedbyitsunusualthermostability.
• Structure-func<ondataobtainedwithphenothiazinesandrabbitFMO2suggesttheC4a-hydroperoxyflavinliesabout6-8angstromsbelowtheenzymesurfaceinachannelnomorethan8angstromsindiameter.
• Gene<cpolymorphismsinhumans,commonlyQ472X,rendershumanFMO2inac<veinallbutasmallpropor<onofthepopula<on.
FMO3:GeneralCharacteris<cs
• ThemajorFMOenzymepresentinadulthumanliver~50pmol/mg.
• Possessesreasonablybroadsubstratespecificity,butismoreselec<vethanFMO1;i.e.FMO1selec<vity<FMO3<FMO2.
• Trimethylamine(TMA)istheclassicalendogenoussubstrate.
• In1otrimethylaminuria(TMAU),affectedindividualscannotmetabolizeTMAandsoexcretetheodoriferouscompoundintheirurine,sweatandbreathduetoTMAUmutaXonsintheFMO3gene.
FMO4andFMO5:GeneralCharacteris<cs• FMO4appearstobeubiquitouslyexpressedatlowlevelsina
varietyof<ssues,butlikleisknownofitssubstratespecificity,duelargelytodifficul<esinexpressionoftherecombinantenzyme.
• FMO5isamajorFMOformpresentinhumanliver(~30pmol/mg),butexhibitslikleornocataly<cac<vitytowardscommonFMOsubstrates.ActsasaBayer-Villigerase!
Laietal.,DMD,2011Fioren<nietal.,ACSChemBiol,2016
CommonFMOMetabolites• MicrosomalFMOsoxidizesubstratescontainingnucleophilicnitrogen,sulfur,phosphorousandseleniumatoms.
• Commonmetabolitesaresulfoxides(S-oxides)generatedbyFMOfromalkylarylsulfides.
• TheprototypicalFMOreac<onisN-oxygena<onofater<aryamine,e.g.trimethylamine,N,N-dimethylaniline,totherespec<veN-oxidemetabolite.
• N-oxidesandS-oxidesarehighlypolarmetabolitescontaincoordinatecovalentbondsbetweentheoxygenandheteroatom.
• N.B.BothFMOandP450cancatalyzeforma<onofN-oxidesandS-oxidesandsotoolshavebeendevelopedtodiscriminatebetweenthesetwoenzyma<cprocessesinbiological<ssues.
OtherN-containingDrugSubstrates
• WhileFMOhasthecapacitytometabolizeavastrangeofamine-containingcompounds,asignificantcontribu<ontothemetabolicclearanceofdrugsapprovedintheUSismuchmorelimited.
• Examplesinclude;rani?dine,benzydamine,itopride,olanzapine,pargyline,xanomelineandchlorpheniramine.
O
N
H3C
CH3
S
HN
NH
N+
H3C
O
O-
FMO-catalyzedN-oxygena<on• FMOscanalsoconvert1oand2oaminestohydroxylamine,nitroneand
oximemetabolites.
• Ingeneral,theseFMOreac<onsareconsidereddetoxifica<onpathways.
• However,somesecondaryhydroxylamines,e.g.thosederivedfrom3,3-iminodipropionitrileandN-deacetylketoconazole,havebeenassociatedwithneurotoxicityandhepatotoxicity,respec<vely.
• Thismayoccurviareac<vespeciesderivedfromthenitronemetabolite(seeFig2inFo<andDalvie,2016).
H2C NH2
H2C NH
R1
R H2C N
R1
R
OH
HC N
R1
R
O
R H2C NHR
OH
HC NR
OH
FMO-CatalyzedS-Oxygena<on• FMOscatalyzesulfoxideforma<onfromsulfidesand(less
efficiently)sulfoneforma<onfromsulfoxides,
• OtherS-containingdrugsubstratesinclude;cime<dine,methimazole,ethionamideandSM-12502.
CH2CO2H
H
F
S
O
H3C
CH2CO2H
H
F
S
CH2CO2H
H
F
S
H3C O
O
H3C
Sulindac sulfide Sulindac Sulindac sulfone
FMO-catalyzedBioac<va<on• MostcommonlyassociatedwithmetabolismofS-containingsubstrates,
e.g.thioureas,thatcanbeconvertedsequen<allytoreac<vesulfenicandsulfinicacids.
• Classicalsubstratesac<vatedinthisfashionarethehepatotoxins,
thioacetamideandthiobenzamide.
• Adrugexampleisethionamide,whichrequiresbioac<va<onbyaM.tuberculosisFMOforitsan<tubercularac<vity,whereasorgantoxici<esmaybeduetohumanFMO-dependent(off-target)bioac<va<on.
R NH C
S
NH2 R N C
S
NH2R N C
S
NH2
OH OHO
thiourea sulfenic acid sulfinic acid
H2N S
N
CH3
HN S
N
CH3
OH
HN S
N
CH3
OH
O
ToolsforIden<fyingFMOCatalysisinMicrosomes
• P450scancatalyzeessen<allyallthesamereac<onsasFMO.
• Maingoalistodifferen<ateFMOac<vityfromthatofP450inmicrosomes.
• Approachisto(A)selec<velyinhibitP450or,(B)selec<velyac<vateorinac<vateFMOac<vity.
Diagnos<cinhibitorsforFMO?• NoinhibitoryanXbodieshavegeneratedagainstany
FMOisoform.• Nomechanism-basedinhibitorsofFMOhavebeen
described.• Reversibleinhibitorsincludealternatesubstrates,suchas
methimazole.• However,methimazoleisnotaspecificinhibitorfor
FMO.• Otherpoten<allyisoform-selec<vealternatesubstrates
areimipramine(FMO1)andtrimethylamine(FMO3).
S
NHNCH3
(A)Selec<veinhibi<onofP450
1. UseaP450reductasean<body.- AllP450susethesamereductasetotransfer
electronsfromNADPH.2. Useamechanism-basedinhibitoroftheP450s,e.g.1-
aminobenzotriazole(ABT)isapaninhibitorofmicrosomalP450,butnotFMO.- ReliesonP450-mediatedconversionofABTto
benzyne,whichthenreactswithP450heme.
(B)Selec<veinhibi<onofFMO
1. ExploitthermolabilityofFMOs(exceptFMO2) -Heatmicrosomesat45oCintheabsenceofNADPHfor~2minselec<velyinac<vatesFMO1andFMO3.
2. ExploitFMOsrela<veinsensi<vitytonon-ionicdetergents. -Treatmentofmicrosomeswith0.2%LubrolorEmulgenwillinac<vateP450s,butnotFMOs.
MolybdenumHydroxylases
Twoforms:Xanthineoxidase(XO)andaldehydeoxidase(AO)areseparategeneproductsexhibi<ng~50%aminoacidhomology.Thereisonlyonegeneforeachenzymeinhumans-XORandAOX1,respec<vely.
MolybdenumHydroxylaseComplex
Theenzymeshaveacomplextri-par<te(flavoprotein)structure,comprisingtwoiden<calsubunitsof~145KDaeach.Theenzymecomplextypicallyshukleselectronsfromsubstrates(i.e,thesubstrategetsoxidizedintheprocess)toanelectronacceptor,usuallyoxygen,genera<ngROS.N-oxides,sulfoxides,aroma<cnitrocompoundscanalsobereduced.
RH+H2O ROH+2e-+2H+
e.g.SuperoxideanionradicalHydroxylradical
SubstrateSpecificity–Guaninederiva<ves
Inthemain,XOandAOtargetsp2-hybridizedcarbonatomsrenderedelectron-deficientbyanitrogenatomtowhichtheyarelinkedbyadoublebond,-CH=N.ThemajorsubstratesforXOandAOarenitrogenheterocycles.XanthineistheprototypicsubstrateforXOandallopurinolisaselec<veXOinhibitor.XOandAObioac<vate6-deoxyguanineprodrugsforthean<-viral,penciclovir.Thisstrategywashelpfulbecausetheac<veagentswerepoorlybioavailable.XOalsometabolizesthean<cancerdrug,6-MP(minorpathway)
N
N N
N
H2N
OH
OH
HN
N N
N
H2N
OH
OH
O
6-Deoxypenciclovir Penciclovir
AO/XO
N
NNH
N
OH
N
N
N
N
OH
N
NNH
N
OH
N
N
N
N
OH
N
NNH
N
OH
XO XO/AO
XO
Hypoxanthine Xanthine Uric acid
Allopurinol Oxypurinol
HO
OH
HO
Addi<onalheterocyclicsubstratesforAO
N
NN
N
COCH3
CH2CH3
CN
NH
NN
N
COCH3
CH2CH3
CN
O
Zaleplon 5-Oxo-Zaleplon
AO
Carbazeran–<5%oralbioavailability,rapidmetabolism
SGX523–renaltoxicityduetoinsoluble(!)lactammetabolite
InvivoprobeforXO?
Short-ac<nghypno<c
MetabolismofIminiumionsbyAO
N
R
N
R
N
R
N
R
O
O
N
R
AO
Iminium ion
AOalsoplaysanimportantroleinthedetoxifica<onofpoten<allyreac<veiminiumionsthatcanbegeneratedbyP450orMAOopenfromcyclicter<aryamines.Lactammetabolitesaretheresult,e.g.AO-catalyzedforma<onofthenico<nemetabolite,co<nine,thatisformedinbysequen<alP450/AOmetabolism.
MetabolismofAldehydesbyAOAOcancatalyzetheoxida<onofaldehydestocarboxylicacids,andbothendogenous(e.g.re<naldehyde)andxenobio<caldehydes(below)maybesubstrates.Aswithiminiumions,theseprocessescanbesequen<al.Note:AO-catalyzedoxida<onofaldehydesinvivomaynotbeahighlysignificantprocess.OtherenzymeslikeALDHsandP450smayhaveahigheraffinityforcertainaldehydes.
Reduc<veMetabolismbyAO(maynothappeninvivo!)AOandXOcancatalyzereduc<vemetabolism(invitro)inthepresenceofagoodelectrondonorsuchasxanthine(XO)and2-hydroxypyrimidineorN-methylnico<namide(bothAO),andintheabsenceofair.
Ringscissions
Twoand6-electronreducXons!ProdrugacXvaXon
SubstrateandinhibitorprobesforAO
Pthalazine–substrateprobe
Hydralazine–selec<ve<me-dependentinhibitorofAO.Lowpotency(Ki~80µM),butcanuseat25µMinhepatocytestoes<matefrac<onmetabolizedbyAOwithnoeffectonP450s(Strelevitzetal.,DMD2012).
Raloxifene–verypotent(lownM),uncompe<<veAOinhibitor,MBIforCYP3A4,soavoidinhepatocytes
VanillinandMenadioneareother‘classical’AOinhibitors
Inter-species,inter-organandinter-individualvariability
Generaldogma:AOac<vityishighinhumansandmonkeys,lowinrodents,andabsentindogs.Caveats:Largestraindifferenceinratsandmice.Genderdifferencesinmice,withmalesexhibi<ng3-4xac<vityoffemales.HumanlivercytosolicacXviXes:Effortstoscaleinvitroac<vitytoinvivotypicallyunder-predictAO-mediatedclearance.
- Isthisduetoenzymelabilityaperprocessing?
- Tosignificantextra-hepa<cAOcatalysis?
- Orperhapstothepresenceofdietaryinhibitorsinprocessedhumanlivercytosols?
- NB:XOac<vitylowinprocessedhumanliverduetoinclusionofallopurinolinperfusionsolu<ons.
Cataly<cMechanism
• Ac<vesitecontainstheexoXccofactor-molybdopterin.
• Substrates(RH)reactattheMo(VI)centre.
• Catalysisisini<atedbybase-assisted,nucleophilicakackoftheMoVI–OHgroupontheelectrondeficientcarbonoftheN-Cbond,withconcertedhydridetransfertotheMo=Sgroup.
• HydrolysisoftheMo-O-Cbondbywaterreleasesoxidizedsubstrate(ROH).
o
ExamplesofcommonlyusedMAOsubstrates
PrimaryamineneurotransmikersaretypicallyMAO-AsubstratesXenobio<cs,includingenvironmentaltoxins,e.g.MPTPareopenMAO-BsubstratesHowever,molecularsizeisadeterminant,withMAO-Atypicallypreferringlargermolecules
MAOcatalyzesoxidaXvedeaminaXon(cleavageofC-Nbond)toformaldehydes
Metabolismofxenobio<csbyMAO• Not very many examples,
- drugs with moieties resembling the indoleamine moiety of serotonin (5HT) e.g. sumitriptan, citalopram, and MPTP - a neurotoxin bioactivated by MAO-B in the brain.
HN
NH2
HO
O
F
NCH3
CH3
NC
Serotonin
Citalopram
• NOVA–TheCaseofTheFrozenAddicthkp://openvault.wgbh.org/catalog/V_474CF2C8A20B4173988486AC4C605A3C
Diagnos<cMAOInhibitors
• Crystalstructuresareavailableforthetwoenzymestohelpfurtherra<onalizeligandselec<vi<es(Milczeketal.,FEBSJ(2011).
Mechanism-basedinac<va<onofMAObyacetylenes
CovalentlyboundtotheN-5posiXononFADBindietal.,J.MedChem,2004
ALDH3A1isexpressedathighlevelsintumorsand,formthatoftheenzymethat,alongwithALDH1A1/5A1,maydeterminecellularresponsetothean<cancerdrug,cyclophosphamide(CP)