MEDCH5271/2/2017Non-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-oxideisfirstdescribedbyDr.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

ONN

NADP

H, NADPH

OHOH

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

FADFADH2

NADP

NADP

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

FMO5–BayerVilligerReac<ons•  Ra<onalizedbytheac<veenzymespeciesbeingthehydroperoxyanion–FAD-O-O-.

Regula<onofFMOExpression/Ac<vity• FMOproteinexpressionisnotsignificantlyinducedbypretreatmentofanimalswithcommonP450inducerssuchaspolycyclichydrocarbonsoran<convulsantdrugslikephenobarbital.

• HighlevelsofAhreceptor-dependentinduc<onofFMO2andFMO3mRNAhasbeenobservedinmice,butFMOacXvityincreasedmodestly.

• FMOac<vitycanbemodulatedbysexsteroidsandglucocor<coids.

-estradiol/progesteroneupregulateFMO2inrabbitlung,-glucocor<coidsinducesFMO2inrabbitkidney,-testosteronesuppressesFMO3inmalemice.

CommonFMOReac<ons

NH

N

NH

N

O

O

Ribosyl

O

O H

FAD-OOH: FMO active oxygenating species

N

NH

N

NH

N

O

O

Ribosyl

N

O OH

• MicrosomalFMOsoxidizesubstratescontainingnucleophilicnitrogen,sulfur,phosphorousandseleniumatoms.

• TheprototypicalFMOreac<onisN-oxygena<onofater<aryamine,e.g.trimethylamine,N,N-dimethylaniline,totherespec<veN-oxidemetabolite,asshownbelow.

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.XanthineistheprototypicsubstarteforXOandallopurinolisaselec<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–renaltoxicityduetoinsolublelactammetabolite

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

SubstrateprobesforAOSubstrateprobe-Pthalazine

Internalstandard

Substrateprobe–DACA

HumanlivercytosolKmis4-8µMVmis2-4nmol/mg/min

BarrandJones,DMD(2011,2013)

‘Diagnos<c’chemicalinhibitorsforAOMenadione–classicAOinhibitor,butP450inhibitorandcytotoxictohepatocytes

Raloxifene–verypotent(lownM),uncompe<<veAOinhibitor,MBIforCYP3A4,soavoidinhepatocytes

Hydralazine–selec<ve<me-dependentinhibitorofAO.Lowpotency(Ki~80µM),butcanuseat25µMinhepatocytestoes<matefrac<onmetabolizedbyAOwithnoeffectonP450s(Strelevitzetal.,DMD2012). Vanillin–alterna<vealdehydesubstrate,

alsoa‘classicalAOinhibitor

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.PolymorphismsinhumanAO:CommoncodingSNPsatN1135S(13-26%)andH1297R(5-6%).Thesearepossiblerapidmetabolizers(Hartmannetal.,DMD,2012).

Cataly<cMechanism

•  Ac<vesitecontainstheexoXccofactor-molybdopterin.

•  Substrates(RH)reactattheMo(VI)centre.

•  Catalysisisini<atedbybase-assisted,nucleophilicakackoftheMoVI–OHgroupontheelectrondeficientcarbonoftheN-Cbond,withconcertedhydridetransfertotheMo=Sgroup.

•  HydrolysisoftheMo-O-Cbondbywaterreleasesoxidizedsubstrate(ROH).

o

MAO:GeneralCharacteris<cs

ExamplesofcommonlyusedMAOsubstrates

PrimaryamineneurotransmikersaretypicallyMAO-AsubstratesXenobio<cs,includingenvironmentaltoxins,e.g.MPTPareopenMAO-BsubstratesHowever,molecularsizeisadeterminant,withMAO-Atypicallypreferringlargermolecules

MAOcatalyzesoxidaXvedeaminaXon(cleavageofC-Nbond)toformaldehydes

Oxida<onofAminestoIminesbyMAO-boundFAD

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)

ALDHCataly<cMechanism

•  Cataly<ccysteineresidue

•  Newoxygenatominacidmetabolitecomesfromwater

OO O

/FAD

CHEATSHEETFORNON-P450ENZYMES