MEDCH 527 1/25/2013. Non P450 Oxygenases. Flavin Containing Monooxygenases. Molybdenum Hydroxylases (cytosolic AO and XO)

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1 MEDCH 527 1/25/2013 on P450 Oxygenases Flavin Containing Monooxygenases (microsomal FMOs) Molybdenum Hydroxylases (cytosolic AO and XO) Monoamine Oxidases (mitochondrial MAO A, MAO B) Aldehyde Dehydrogenases (cytosolic and mitochondrial ALDHs)

2 Useful literature Krueger SK and Williams DE. Mammalian flavin containing monooxygenases: structure/ polymorphisms and role in drug metabolism. Pharmacol Ther 2005; 106: Phillips IR and Shephard EA. Flavin containing monooxygenases: disease and drug response. TIPS 2008; 29: Pryde et al., Aldehyde oxidase: an enzyme of emerging importance in drug discovery. J. Med. Chem. 2010: 53: Hutzler et al., Strategies for a comprehensive understanding of metabolism by aldehyde oxidase. Expert Opin. Drug Metab. Toxicol Early on line. Youdim MB, Edmondson DE and Tipton KF, The therapeu@c poten@al of monoamine oxidase inhibitors. ature Rev. eurosci. 7:295, Kappako et al., Aldehyde dehydrogenase inhibitors. Pharmacol Rev Jul;64(3):

3 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 become that appear to be 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 metabolism and towards, especially, aldehyde oxidase as the main metabolic enzyme.

4 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

5 FMO History 1960s A liver microsomal enzyme system (E.C ) that u@lizes ADPH and molecular oxygen to convert, dimethylaniline to, dimethylaniline oxide is described by Dr. Daniel Ziegler and colleagues. CH 3 CH 3 CH 3 FMO O CH s Ziegler s enzyme, purified from hog liver, shown to contain flavin, but no heme, thereby dis@nguishing this flavin containing monooxygenase (FMO) from the microsomal hemoproteincontaining cytochrome P450s. [1980s lung FMO] 1990s FMO omenclature Commiiee names hog liver FMO as FMO1 and the lung form as FMO2. Other forms with <60% sequence iden@ty are named with ascending arabic numerals, FMO3, 4, 5 im all mammals.

6 Comparison of General FMO vs P450 FMO P450 FuncCon Monooxygenase Monooxygenase S + O 2 + ADPH + H + SO + H 2 O + ADP + Reducing cofactor ADPH ADPH Cellular LocaCon Microsomal Microsomal Size kda kda Substrates Oxidizes at,s,p,s,p and C ProstheCc group FAD Heme

7 FMO Cycle ADP + ADP + ADP +

8 Aspects of FMO Catalysis The mechanism is ordered, i.e. ADPH, oxygen and oxidizable substrate add to the enzyme before any of the products leave. The enzyme bound hydroperoxyflavin [FAD OOH] is a very stable, albeit rela@vely weak oxidant. Release of water and/or ADP + is believed to be the ratelimi@ng step. FMO can oxidize prac@cally any soo nucleophile that the enzyme s FADOOH ac@ve center encounters. In general, uncharged and single posi@vely charged substrates can gain access, in preference to nega@vely charged and mul@ple posi@vely charged compounds.

9 FMO Structure Crystal structures available only for soluble yeast and bacterial forms of the enzyme that have 20 30% sequence similarity to microsomal FMO. All, however, use ADPH as cofactor, and possess conserved cofactor binding sequence and an FMO FMO structure has two domains with FAD (green) and ADPH (red) bound near the interface.

10 FMO Center Substrate bound Asn 91 (conserved in all FMOs) may stabilize the C4a hydroperoxide through polar 4a ADP+ may also stabilize by shielding the hydroperoxide from solvent

11 FMO Five genes, FMO1 FMO5, are present in most species. FMO6 is due to splicing. Human Chromosome 1 FMO7P 11P FMO1, FMO2 and FMO3 are the best characterized enzymes

12 FMO1: General The majority of our knowledge of the FMO enzymes is derived from detailed biochemical studies conducted in the 1970s and 80s on hog liver FMO1 the first FMO to be purified. FMO1 is characterized by its thermolability and promiscuous substrate specificity, which is the widest of all of the FMO isoforms. FMO1 is the dominant form of the enzyme in the liver of most experimental animal species, but is not expressed in adult human liver. In humans, liver FMO1 is a fetal enzyme, but is also expressed at rela@vely high levels in adult kidney.

13 FMO2: General Generally found at highest levels in of experimental species. FMO2 is also characterized by its unusual thermostability. Structure data obtained with phenothiazines and rabbit FMO2 suggest the C4a hydroperoxyflavin lies about 6 8 angstroms below the enzyme surface in a channel no more than 8 angstroms in diameter. Enzyme is currently the focus of crystalliza@on efforts. Gene@c polymorphisms in humans, commonly Q472X, renders human FMO2 inac@ve in all but a small propor@on of the popula@on.

14 FMO3: General The major FMO enzyme present in adult human liver. Possesses reasonably broad substrate specificity, but is more than FMO1; i.e. FMO1 < FMO3 < FMO2. Trimethylamine (TMA) is the classical endogenous substrate. In 1 o trimethylaminuria (TMAU), affected individuals cannot metabolize TMA and so excrete the odoriferous compound in their urine, sweat and breath due to TMAU muta@ons in the FMO3 gene.

15 FMO4 and FMO5: General FMO4 appears to be ubiquitously expressed at low levels in a variety but liile is known of its substrate specificity, due largely to difficul@es in expression of the recombinant enzyme. FMO5 is a major FMO form present in human liver, but exhibits liile or no cataly@c ac@vity towards common FMO substrates.

16 of FMO FMO protein expression is not significantly induced by pretreatment of animals with common P450 inducers such as polycyclic hydrocarbons or drugs like phenobarbital. High levels of Ah receptor dependent of FMO2 and FMO3 mra has been observed in mice, but FMO acsvity increased modestly. FMO can be modulated by sex steroids and estradiol/progesterone up regulate FMO2 in rabbit lung, induces FMO2 in rabbit kidney, testosterone suppresses FMO3 in male mice.

17 FMO Microsomal FMOs oxidize substrates containing nucleophilic nitrogen, sulfur, phosphorous and selenium atoms. The prototypical FMO is of a ter@ary amine, e.g. trimethylamine,, dimethylaniline, to the respec@ve oxide metabolite, as shown below. FAD-OOH: FMO active oxygenating species Ribosyl O Ribosyl O H O O H O H OH O H O H

18 Examples of Containing Drugs Metabolized Predominantly by FMO BEZYDAMIE - ~20% of dose excreted in urine as -oxide O CH 3 CH 3 MOCLOBEMIDE - ~30% of dose excreted in urine as -oxide Cl COHCH 2 CH 2 O ITOPRIDE - ~75% of dose excreted in urine as -oxide H 3 CO H 3 CO COHCH 2 OCH 2 CH 2 CH 3 CH 3

19 Other containing Drug Substrates While FMO has the capacity to metabolize a vast range of amine containing compounds, a significant contribu@on to the metabolic clearance of drugs approved in the US is much more limited. Examples include; ranicdine, olanzapine, pargyline, xanomeline and chlorpheniramine. H 3 C O S H + O - CH 3 H 3 C H O

20 FMO catalyzed FMOs can also convert 1 o and 2 o amines to hydroxylamine, nitrone and oxime metabolites. OH OH R H 2 C H 2 R H 2 C H R HC R H 2 C H R H 2 C OH R HC O R 1 R 1 R 1 In general, these FMO reac@ons are considered detoxifica@on pathways. However, some secondary hydroxylamines, e.g. those derived from 3,3 iminodipropionitrile and deacetyl ketoconazole, have been associated with neurotoxicity and hepatotoxicity, respec@vely.

21 FMO Catalyzed S Oxygena@on FMOs catalyze sulfoxide forma@on from sulfides and (less efficiently) sulfone forma@on from sulfoxides, H 3 C O H 3 C O S H 3 C S O S H H H F F F CH 2 CO 2 H CH 2 CO 2 H CH 2 CO 2 H Sulindac sulfide Sulindac Sulindac sulfone Other S containing drug substrates include; cime@dine, methimazole, ethionamide and SM

22 FMO catalyzed Most commonly associated with metabolism of S containing substrates, e.g. thioureas, that can be converted sequen@ally to reac@ve sulfenic and sulfinic acids. S S OH O S OH R H C H 2 R C H 2 R C H 2 thiourea sulfenic acid sulfinic acid Classical substrates ac@vated in this fashion are the hepatotoxins, thioacetamide and thiobenzamide. A drug example is ethionamide, which requires bioac@va@on by a M. tuberculosis FMO for its an@tubercular ac@vity, whereas organ toxici@es may be due to human FMO dependent (off target) bioac@va@on. OH OH H 2 S H S H S O CH 3 CH 3 CH 3

23 Tools for FMO Catalysis in Microsomes P450s can catalyze all the same as FMO. Main goal is to FMO from that of P450 in microsomes. Approach is to (A) inhibit P450 or, (B) or FMO

24 inhibitors for FMO? o inhibitory an@bodies have generated against any FMO isoform. o mechanism based inhibitors of FMO have been described. Reversible inhibitors include alternate substrates, such as methimazole. S H CH 3 However, methimazole is not a specific inhibitor for FMO. Other poten@ally isoform selec@ve alternate substrates are imipramine (FMO1) and trimethylamine (FMO3).

25 (A) of P Use a P450 reductase an@body. All P450s use the same reductase to transfer electrons from ADPH. 2. Use a mechanism based inhibitor of the P450s, e.g. 1 aminobenzotriazole (ABT) is a pan inhibitor of microsomal P450, but not FMO. Relies on P450 mediated conversion of ABT to benzyne, which then reacts with P450 heme.

26 (B) of FMO 1. Exploit thermolability of FMOs (except FMO2) Heat microsomes at 45oC in the absence of ADPH for ~2 min FMO1 and FMO3. 2. Exploit FMOs to non ionic detergents. Treatment of microsomes with 0.2% Lubrol or Emulgen will P450s, but not FMOs.

27 Molybdenum Hydroxylases Xanthine oxidase (XOR; XO) and aldehyde oxidase (AOX1, AO) are separate gene products ~50% amino acid homology. There is only one gene for each in humans. The enzymes have a complex tripar@te structure, comprising two iden@cal subunits of ~145 KDa each. The enzyme complex typically shuiles electrons from substrates (i.e, the substrate gets oxidized in the process) to an electron acceptor, usually oxygen, (although XD can use AD +). oxides, sulfoxides, aroma@c nitro compounds and some others can also be reduced. RH + H 2 O ROH + 2e +2H +

28 Substrate Specificity Guanine In the main, XO and AO target sp 2 hybridized carbon atoms rendered electron deficient by a nitrogen atom to which they are linked by a double bond, i.e. CH=. OH H XO OH HO OH H XO/AO Hypoxanthine Xanthine Uric acid OH OH H OH The major substrates for XO and AO are nitrogen heterocycles. Xanthine is the prototypic substrate for XO and allopurinol is a selecsve XO inhibitor. XO and AO bioac@vate 6 deoxy guanine prodrugs for the an@ viral, penciclovir. This strategy was helpful because the ac@ve agents were poorly bioavailable. H 2 OH OH 6-Deoxypenciclovir XO Allopurinol AO/XO HO H 2 Oxypurinol O H Penciclovir OH OH XO also metabolizes the an@cancer drug, 6 MP (minor pathway).

29 heterocyclic substrates for AO In vivo probe for XO? Carbazeran <5% oral bioavailability, rapid metabolism COCH 3 COCH 3 CH 2 CH 3 CH 2 CH 3 AO C O H C Zaleplon Short ac@ng hypno@c 5-Oxo-Zaleplon SGX 523 renal toxicity due to insoluble lactam metabolite

30 Metabolism of Iminium ions by AO AO also plays an important role in the of iminium ions that are ooen generated by P450 or MAO from cyclic tersary amines. R AO R O Lactam metabolites are the result, e.g. AO catalyzed forma@on of the nico@ne metabolite, co@nine, that is formed in by sequen@al P450/AO metabolism. R R Iminium ion R O

31 Metabolism of Aldehydes by AO AO can catalyze the of aldehydes to carboxylic acids, and both endogenous (e.g. and aldehydes (below) may be substrates. As with iminium ions, these processes can be ote: AO catalyzed of aldehydes may not be a highly significant process in vivo. Other enzymes like ALDHs and P450s likely play a role.

32 Metabolism by AO (may not happen in vivo!) AO and XO can catalyze metabolism (in vitro) in the presence of a good electron donor such as xanthine (XO) and 2 hydroxypyrimidine or methylnico@namide (both AO), and in the absence of air. Ring scissions Prodrug acsvason Two and 6 electron reducsons!

33 Substrate probes for AO Substrate probe Pthalazine Internal standard Human liver cytosol Km is 4 8 µm Vm is 2 4 nmol/mg/min Substrate probe DACA Barr and Jones, DMD (2011, 2013)

34 chemical inhibitors for AO Menadione classic AO inhibitor, but P450 inhibitor too, and cytotoxic to hepatocytes. Raloxifene very potent (low nm), AO inhibitor, MBI for CYP3A4, so avoid in hepatocytes. dependent inhibitor of AO. Low potency (Ki ~80 µm), but can use at 25 µm in hepatocytes to metabolized by AO with no effect on P450s (Strelevitz et al., DMD 2012). Vanillin (aldehyde) substrate, also a classical AO inhibitor.

35 Inter species, inter organ and inter individual variability General dogma: AO is high in humans and monkeys, low in rodents, and absent in dogs. Caveats: Large strain difference in rats and mice. Gender differences in mice, with males 3 4x of females. Human liver cytosolic Efforts to scale in vitro to in vivo typically under predict AOmediated clearance. Is this due to enzyme lability aoer processing or to significant AO catalysis? ote: High AO in the human Polymorphisms in human AO: Common coding SPs at 1135S (13 26%) and H1297R (5 6%). These are possible rapid metabolizers (Hartmann et al., DMD, 2012).

36 Mechanism site contains the cofactor molybdopterin. Substrates (RH) react at the Mo (VI) centre. Catalysis is by base assisted, nucleophilic aiack of the Mo VI OH group on the electron deficient carbon of the C bond, with concerted hydride transfer to the Mo=S group. Hydrolysis of the Mo O C bond by water releases oxidized substrate (ROH).

37 MAO: General

38 Examples of common MAO substrates Primary amine neurotransmi[ers are typically MAO A substrates Xenobio@cs, including environmental toxins, e.g. MPTP, are ooen MAO B substrates However, molecular size is a determinant, with MAO A typically preferring larger molecules MAO catalyzes oxidasve deaminason (cleavage of C bond) to form aldehydes

39 of Amines to Imines by MAO bound FAD

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42 Metabolism of drugs by MAO ot very many examples, drugs with resembling the indoleamine moiety of 5HT see citalopram (previous an SSRI.

43 MAO Inhibitors

44 Crystal structures reveal that the MAO A has one large substrate cavity (550 A 3, whereas MAO B s ac@ve site is divided into two, with (400 A 3 substrate cavity and a 290 A 3 entrance cavity gated by Tyr326 and Ile199. Milczek et al., FEBS J (2011)

45 Mechanism based of MAO by acetylenes Covalently bound to the 5 posison on FAD Bindi et al., J.Med Chem, 2004

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47 ALDH3A1 is expressed at high levels in tumors and, form that of the enzyme that, alongwith ALDH1A1/5A1, may determine cellular response to the drug, cyclophosphamide (CP)