Aldehyde oxidase and itsimportance in novel drugdiscovery: present and futurechallengesEnrico Garattini† & Mineko TeraoLaboratory of Molecular Biology, Istituto di Ricerche Farmacologiche “Mario Negri” IRCCS,
Milano, Italy
Introduction: Aldehyde oxidases (AOXs) are molybdo-flavoenzymes that oxi-
dize aromatic aldehydes into the corresponding carboxylic acids and hetero-
cycles into hydroxylated derivatives. AOXs have broad substrate specificity
and are present in the liver of humans and many experimental animals.
These enzymes play an important role in Phase I metabolism of drugs and
xenobiotics of toxicological interest.
Areas covered: Preclinical studies on the AOX-dependent metabolism of new
drug candidates are problematic. Furthermore, there is a general lack of
reliable in silico methodologies to predict whether a new organic molecule
is an AOX substrate. In vitro systems, for the design of high- or medium-
throughput screening tests, to identify AOX substrates have many limitations.
In vivo studies on AOX-dependent metabolism in animal models, on the other
hand, are difficult because the complement of liver AOXs in humans and
popular experimental animals is different. The authors discuss the possible
ways to overcome all these problems.
Expert opinion: The significance of AOXs as drug-metabolizing enzymes is
increasing, as the current strategies of organic synthesis designed to avoid
cytochrome P450 (CYP450)-dependent metabolism tend to enrich for new
chemical structures efficiently oxidized by these enzymes. There is need
for reliable methods to screen for, predict, and validate AOX-dependent
metabolism of new drug candidates.
Keywords: aldehyde oxidase, drug discovery, drug metabolism, environmental pollutants,
molybdo-enzymes
Expert Opin. Drug Discov. [Early Online]
1. Introduction
Mammalian aldehyde oxidases (AOXs, EC 1.2.3.1) are cytosolic molybdo-flavoenzymes (MOFEs), a group of proteins that require a flavin adeninedinucleotide (FAD) and a molybdopterin [molybdenum cofactor (MoCo)] for theircatalytic activity [1-12]. The broad substrate specificity of mammalian AOXs, theability of the enzymes to oxidize different types of heterocycles [13-16], which oftenconstitute the building blocks of new pharmacophores, and the presence of highlevels of human AOX1 in the liver make this class of enzymes extremely interestingin the drug metabolism and drug discovery fields. Indeed, the role played by humanAOX1 in inactivating and activating xenobiotics of medical and toxicologicalinterest is rapidly emerging. In many instances, it is clear that human AOX1, ratherthan the much better known cytochrome P450 (CYP450)-dependent monooxyge-nases, plays a key role in Phase I metabolism of registered drugs and drugcandidates. In other instances, the enzyme contributes to drug metabolism via the
oxidation of metabolic intermediates generated by otherenzymes such as CYP450 itself [17] or monoamine oxidase(MAO) [18,19].In this Expert Opinion, we intend to provide a brief over-
view of the significance that AOXs have in the biotransforma-tion of synthetic organic compounds, focusing on a numberof specific and unresolved problems that may be encounteredduring the preclinical development of new drug candidates.A brief introduction to the structural and enzymatic charac-teristics of this family of enzymes is provided. This is followedby a brief discussion on the evolution of the AOX family ofenzymes in vertebrates with a comparison of the numberand types of AOXs present in humans and animal species of
relevance for the preclinical development of new drug candi-dates. The core of the article discusses a number of shortcom-ings in studies aimed at defining the biotransformation oforganic compounds by AOXs that ranges from the lackof in silico systems to predict AOX-dependent oxidation ofnewly synthesized compounds to the absence of optimal prox-ies of the human situation in terms of experimental animals tobe used for in vivo studies.
2. Strict conservation of the structures ofAOX proteins and relative genes andspecies-specific variations in the number ofisoenzymatic forms
Similar to xanthine oxidoreductase (XOR) [20-23], the othermember of mammalian MOFEs, the catalytically activeform of AOXs is a homodimer, consisting of two identical150 kDa subunits, as illustrated in Figure 1A by the structuralmodel of mouse AOX3 [24]. Each AOX and XOR subunit is asmall oxidoreductive chain consisting of three distinct regionsand four redox centers. The 20 kDa amino-terminal domaincontains two nonidentical 2Fe/2S centers and the bindingsite for FAD is present in the 40 kDa intermediate domain,while the 90 kDa carboxy-terminal domain consists of theMoCo binding site, which lies in close proximity to the sub-strate pocket [3,10,24]. The similarity between AOXs andXOR is not limited to this, since the primary and secondarystructures [25-27] of the two proteins are also extremely con-served, as underscored by the recent crystallization of the firstvertebrate AOX, that is, mouse AOX3 [24]. A major differencebetween AOXs and XOR is represented by the substrate spec-ificity of the two types of enzymes. The specificity of mamma-lian AOXs is very broad and it is not limited to organicmolecules containing an aldehyde functionality, as it wouldbe suggested by the name of these enzymes [2,3,28-31]. Indeed,AOXs oxidize a wide array of compounds, which includesaza- and oxo-heterocyclic structures. A simplified and generalscheme of the enzymatic reaction catalyzed by AOXs is shownin Figure 1B. At present, the physiological function and theendogenous substrate(s) of AOXs are unknown, althoughknockout animals generated for mouse AOX4 indicate thatall-trans retinaldehyde, the precursor of all-trans retinoicacid, and the vitamin A active metabolite may represent aphysiological intermediate the enzyme acts on [32]. A furtherphysiological substrate of AOXs is represented by the activespecies of vitamin B6, pyridoxal [3,30]. In contrast, XOR activ-ity is limited to the oxidation of hypoxanthine into xanthineand xanthine into uric acid, making XOR the key enzymein the last steps of the catabolic pathway of purines [33-36].
The number of AOXs in vertebrates varies from one to fouraccording to the species considered [10]. The two extremes arerepresented by mice and humans. The mouse is characterizedby four isoenzymes (AOX1, AOX3, AOX4, and AOX3L1),while humans possess a single enzyme, which is the orthologof mouse AOX1. Each mouse isoenzyme is encoded by a
Article highlights.
. Mammalian AOXs are cytosolic MOFEs, a group ofproteins that require FAD and a molybdopterin for theircatalytic activity. Different animal species arecharacterized by a different complement of active AOXgenes, which vary from one in humans (AOX1) to fourin mice (Aox1, Aox3, Aox4, and Aox3l1). The broadsubstrate specificity of mammalian AOXs, the ability ofthe enzymes to oxidize different types of heterocycles,which often constitute the building blocks of newpharmacophores, and the presence of high levels ofhuman AOX enzymatic activity in the liver make thisclass of enzymes interesting in the drug metabolism anddrug discovery fields.
. AOX-dependent biotransformation of new drugcandidates is an emerging problem, as new strategies ofchemical synthesis aimed at reducingCYP450-dependent metabolism tend to enrich forpharmacophores, which are AOX substrates and areinactivated by this enzymatic system. This calls for thedevelopment of new approaches to predict and testAOX-dependent metabolism particularly during thepreclinical development of new drugs.
. In silico methodologies that can be used to predictwhether a new organic molecule is a potential AOXsubstrate are required. The situation is likely to changebecause of the recent availability of the crystalcoordinates and the structure of the first mammalianAOX, mouse AOX3.
. Robust in vitro systems allowing the design ofappropriate high- or medium-throughput screening teststo identify AOX substrates are required. Current effortsare focusing on the development of new technologiesfor the expression and purification of human AOX1 andother mammalian AOXs with high catalytic activity.
. In vivo studies on AOX-dependent metabolism in animalmodels are highly problematic, as the complement ofliver AOXs in humans and popular experimental animalsis different. Although not optimal, the current bestproxies of the human situation in terms of liver AOXexpression are represented by the guinea pig and theRhesus monkey. Studies aimed at humanizing the profileof AOX gene expression are urgently needed.
This box summarizes the key points contained in the article.
E. Garattini & M. Terao
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distinct gene and the four murine loci cluster on a small por-tion of chromosome 1c1 at a short distance from one another(Figure 2). Clustering of the AOX1 genes on small portions ofsingle chromosomes is a characteristic of all the mammalianspecies [10]. The murine Aox1, Aox3, Aox4, and Aox3l1 genesare characterized by very conserved exon/intron structures,which supports the concept that they originated from anancestral AOX precursor via a series of asynchronous geneduplication events during vertebrate evolution [10]. Thegenomes of the most ancient species of fishes contain a func-tional XOR, but they lack an AOX counterpart. The genome
of more recent species of fishes is characterized by the pres-ence of an AOX gene, indicating that the first AOX originatedin these vertebrates. All the vertebrate AOX genes so far iden-tified show a remarkable conservation of the exon/intronstructures and junctions, and this conservation extends tothe vertebrate XOR counterparts. Altogether, the two observa-tions support the concept that the first vertebrate AOXoriginated from a primitive duplication of the XOR ancestorgene, which occurred during the evolution of fishes. Furtherduplications of the primitive AOX gene are observed duringthe subsequent evolution of vertebrates. The most completeand updated reconstruction of AOXs’ evolutionary history isavailable in a recent publication of ours [10]. The first phaseof vertebrate evolution is dominated by a series of AOX geneduplication events, which often occurred in a lineage-specific fashion. The process of successive duplications leadingto the maximal extant number of AOX genes observed in mice,rats, and other mammals must have been completed inmarsupials, that is, long before the appearance of eutherian mam-mals. The multiplication of AOX genes is probably explained bynew functional necessities linked to the acquisition of distinctAOX enzymes characterized by tissue-specific expression (seebelow) or different substrate specificities.
The necessity of new functional AOX genes must havecome to an end during the subsequent evolution of mammals.In fact, the development of further mammalian species isdominated by a progressive reduction in the number of func-tional AOX genes. This process was accomplished by a seriesof species-specific deletions and pseudogenization events thatled to the disappearance or functional inactivation of all thegenes except AOX1 in humans.
The pseudogenization/deletion of the AOX3 gene is a veryfrequent event observed independently in at least four mam-malian lineages, resulting in many species which are devoidof the corresponding protein. Rodents are rather unique interms of AOX3 evolution, as these animals are generallycharacterized by the presence of an active gene with a fewexceptions that include the guinea pig (Figure 2). Pseudogeni-zation of the AOX4 gene is the second most frequent eventduring the evolution of mammals and it is highly representedin primates, consistent with the absence of a Harderian glandin these animals (see below). AOX3L1 is active in most mam-malian species except in all members of the hominoid lineageof primates. The absence of an AOX3L1 protein may berelated to differences in the olfactory system between primatesand other mammalian species, as expression of the enzymeseems to be limited to the olfactory mucosa [8]. In contrast,pseudogenization of AOX1, the most ancient gene of thefamily [10], is a rare event (dogs and cats; Figure 2), indicatingthat the protein exerts an important, though unknown,homeostatic function in mammals.
Most of the information acquired on the characteristics ofthe various AOX isoenzymatic forms has been acquired inthe mouse. In this species, the expression of AOX1, AOX3,AOX4, and AOX3L1 is relatively tissue and cell specific.
H2O2
Domain I20 kDA
Domain II40 kDA
FAD2x [2Fe-2S]
A.
B.
MoCo
Domain III90 kDA
Mo (VI)
FAD RH + H2O
Mo (IV)·RH
Mo (IV)
FADH2
FADH2·O2
Mo (IV)
O2
FAD
ROH
Figure 1. Structure and catalytic activity of aldehyde
oxidases. (A) A ribbon representation of the mouse
AOX3 crystal structure is illustrated (top) [24]. The right
section of the figure shows the structure of monomer A,
which is divided in the three different domains colored as in
the schematic representation drawn on the bottom. The
structure of the identical momomer B (left) is shown in gray
as the linker regions in monomer A. (B) The catalytic cycle of
AOXs is illustrated. A generic substrate RH is oxidized to the
corresponding ROH product by the enzymes with concomi-
tant reduction of the molybdenum center (Mo).FAD: Flavin adenine dinucleotide; Moco: Molybdenum cofactor;
Mo: Molybdenum.
Aldehyde oxidase and its importance in novel drug discovery
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The profile of AOX1 and AOX3 is largely overlapping andthe richest source is represented by the liver, although detect-able levels of the two enzymes are present in many other tis-sues [1,3]. AOX4 is represented predominantly in two organs.By far, the largest amounts of AOX4 are found in the Harder-ian gland, an important exocrine gland in lower mammalswhich is not present in primates and humans [1,3]. Significantlevels of AOX4 have also been found in the skin, wherethe enzyme is concentrated in the epidermal layer and thesebaceous glands [32]. AOX3L1 expression is restricted to theBowman’s gland, the main secretory organ of the nasal cav-ity [8]. Much less information is available for the only humanenzyme, AOX1, which is the bona fide ortholog of the corre-sponding isoenzyme in the mouse. The largest amounts ofhuman AOX1 have been measured in the hepatic organalthough significant amounts of the enzyme are also presentin many other body districts, with particular reference to thecortical component of the adrenal gland and the mammarygland. Detectable levels of the protein have also been reported
in the adipose tissue, where recent data suggest a role forAOX1 in the deposition of fat [37-39].
At present, there are no studies comparing the enzymaticproperties and the substrate specificities of the various mouseisoforms of AOX. By the same token, comparisons betweenhuman AOX1 and the other mouse AOXs, in terms of theseparameters, are equally unavailable. This kind of studies iscalled for as they are likely to provide insights into the reasonsas to why different animal species are endowed with adifferent complement of AOX enzymes.
3. The broad substrate specificity of AOXsand the importance of these enzymes in themetabolism of xenobiotics
As already mentioned, AOXs are enzymes capable of transform-ing a wide array of substrates, which is not limited to organiccompounds containing an aldehyde functionality [1-3,30,31].AOXs oxidize aldehyde groups to the corresponding carboxylic
H. sapiens (human)
P. troglodytes(chimpanzee)
M. mulatta(Rhesus monkey)
C. porcellus (guinea pig)
F. catus (cat)
C. lupus (dog)
S. scrofa (pig)
O. cuniculus (rabbit)
M. musculus (mouse)
R. norvegicus (rat)
AOX1 85 kb
AOX1 85 kb
AOX1 97 kb
AOX1 37 kb
AOX1* 69 kb
AOX1* 68 kb
AOX1 76 kb
AOX1 71 kb
AOX1 79 kb
AOX1 82 kb
AOX3* 52 kb
AOX3* 52 kb
AOX3* 84 kb
AOX3 129 kb
AOX3 89 kb
AOX3 91 kb
AOX4* 66 kb
AOX3* 99 kb
AOX3* 58 kb
AOX3* 54 kb
AOX4 63 kb
AOX4 32 kb
AOX4 54 kb
AOX4 58 kb
AOX4 63 kb
AOX3I1 87 kb
AOX4 76 kb
AOX3I1* 52 kb
AOX3I1 76 kb
AOX3I1* 53 kbCHR:2
CHR:2b
CHR:12
CHR:37
CHR:15
CHR:1
CHR:9
AOX3I1 75 kb
AOX3I1 85 kb
AOX3I1 77 kb
AOX3I1 102 kb
AOX3I1 90 kb
AOX4 38 kb AOX3I1 35 kb
Figure 2. AOX genes in humans and selected experimental animals of significance for drug metabolism studies. The figure
contains a schematic representation of the AOX genes in humans and selected primates or mammals of significance as
experimental models in drug metabolism studies. Orthologous genes are indicated by the same shadowing of the boxes
representing the genes and pseudogenes. Pseudogenes are crossed through and asterisked. When known, the chromosomal
location is shown on the right in bold.
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acid very efficiently, particularly when this functionality ispresent in the context of an aromatic compound. However,AOXs also oxidize various types of aromatic heterocycles,such aza-, oxo-, and sulfo-heterocycles. In addition, AOXsare capable of oxidizing iminium groups to the correspond-ing cyclic lactames. Iminium ions are generated during themetabolism of cyclic amines, such as pyrrolidines and piper-idines by MAO and CYP450, and these metabolic intermedi-ates function as substrates of AOXs. Finally, there is evidencein the literature, demonstrating that AOXs function not onlyas oxidases but also as reductases. In fact, AOXs catalyze thereduction of N-oxides, sulfoxides, nitro-compounds, and het-erocycles in certain conditions [40-42]. Nevertheless, the signif-icance of AOX reductase activity for the in vivo metabolismof drugs is questionable as it was observed only in vitro, inthe presence of appropriate electron donors [42,43]. The broadsubstrate specificity and the potential to oxidize and reduce anumber of organic compounds, along with the presence ofsignificant levels of enzymatic activity in the cytosolicfraction of the human liver have attracted a long-standinginterest on AOXs as drug-metabolizing systems. Indeed, as shownin Figure 3, several xenobiotics of medical and toxicologicalsignificance have been recognized as AOX substrates [14,19,43-53],such as the antitumor agents, methotrexate [54-57] and 6-mer-captopurine [58]; the antiestrogen, tamoxifen [18]; the antide-pressant, citalopram [17]; the norepinephrine--dopamineintake inhibitor, prolintane [59]; the cigarette component,nicotine; the main coffee ingredient, caffeine; and thesweetener, vanillin [30].
The potential of AOXs to oxidize heteroaryls is of particu-lar importance in the context of drug design and develop-ment, as these chemical groups are popular syntheticbuilding blocks in medicinal chemistry [60]. For instance,pyrrolidines and piperidines are very versatile pharmaco-phores, as they can bind and interact with different types ofpharmacological targets. In addition, they are often chosenas building blocks for new drug candidates because they arecharacterized by an electron-deficient nature, which makesthem resistant to metabolic inactivation by CYP450, a univer-sally recognized problem in terms of drug pharmacokinet-ics [13,14]. A common synthetic strategy to increase electrondeficiency and sensitivity to CYP450 metabolism consists ofreplacing a carbon in the aromatic ring with a nitrogen.Unfortunately, this makes the ring carbon atoms susceptibleto nucleophilic attack by AOXs, particularly at the level ofthe residues adjacent to the heterocyclic nitrogen(s) [14]. Thisand the implementation of other successful strategies in thedesign and synthesis of new drug candidates, which are poorCYP450 substrates, has resulted in a progressive increase ofmolecules acting as potential substrates of AOXs. The trendis accurately described in a recent article by Pryde et al. [14].In this publication, the authors used data from a largecollection of chemical compounds and calculated that the pro-portion of potential AOX substrates among the compoundsthat progressed to the market was 0.13. An almost fourfold
enrichment (0.45) was observed, if similar calculations weremade for compounds still under development.
The literature reviewed above indicate that AOX-dependentmetabolism is an emerging issue and a significant problem inthe design and synthesis of new drug candidates. From a medic-inal chemistry perspective, this has already resulted in the publi-cation of two studies exploring new approaches to chemicalsynthesis aimed at reducing the possibility that heterocycles actas efficient substrates of AOXs [13,60]. Using a series of imidazo[1,2-a]pyrimidine, Linton et al. investigated strategies based on1) the introduction of remote structural changes in the molecule;2) the evaluation of alternative heterocycles; 3) the incorporationof hindering residues either close to or right to the predicted siteof AOX oxidation [60]. Two other strategies were investigated byPryde et al. to avoid AOX-dependent metabolism in a series ofimidazopyrimidine analogs under development as potentagonists of the TLR7 receptor, which is an important pharmaco-logical target for the treatment of hepatitis C virus infections [13].As AOX-mediated oxidation of heterocycles, such as the pyri-dine ring, involves an initial nucleophilic attack at the carbonatom adjacent to the heteroatom, the authors explored the pos-sibility to block or remove this adjacent position. Both designstrategies proved successful in eliminating AOX metabolism [13].Thus, for organic compounds that are proven to be AOX sub-strates, the problem of AOX-dependent metabolism can beapproached effectively with the implementation of appropriatestrategies of chemical synthesis.
4. Toward the development of in silicomethods to predict the susceptibility of newdrug candidates to AOX-dependentmetabolism
Any synthetic approach used for the design of molecules thathave a low probability of being AOX substrates must be accom-panied by effective methods to predict and experimentallyvalidate the underlying hypotheses and assumptions. At presentand to the best of our knowledge, only a couple of in silicometh-ods capable of predicting the susceptibility of organic moleculesto the oxidation/reduction by AOXs have been described [61,62].The issue has been covered in more detail in a recent review arti-cle by Hutzler et al. [63]. Here, suffice it to say that the twocomputational methods are based on the chemical and electroniccharacteristics of established substrates of AOXs. However, thisgap is likely to be filled in the near future, as new informationon the primary/secondary structure of mammalian AOX pro-teins will become available and substrate/enzyme docking studieswill become possible. Progress in this direction has been madewith the recent publication of two articles [10,24].
The first study [24] reports on the crystallization andstructural characterization of mouse AOX3, the first enzymeof the family for which the crystal coordinates are available.The crystal structure of mouse AOX3 was solved to 2.9 A.As expected, deconvolution of the structure demonstrated
Aldehyde oxidase and its importance in novel drug discovery
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that the overall topologies of mouse AOX3 and mammalianXORs are similar. In fact, the AOX3 subunit consists of the pre-dicted and easily distinguishable 20-kDa amino-terminal,40-kDa intermediate, and 90-kDa carboxy-terminal domains.Most of the variations between AOX3 and XOR lay within theMoCo domain and the substrate pocket, and these differencesare likely to explain the differences in substrate specificitybetween the two enzymes. Detailed analysis of the AOX3 activecenter as well as docking and simulation studies showed thatthe different substrate and inhibitor specificities between AOXsand XORs are related to several ill-conserved residues presentnot only in the active site, but also in the active site funnel path-way. It is foreseen that the availability of the AOX3 crystal struc-ture will spur further computational studies aimed at developingalgorithms capable of predicting the access of defined organicmolecules to the substrate pocket of the enzymes.The second study reports on the evolution of vertebrate
AOXs and predicts the complete structures of 148 proteinsequences derived from 65 taxa. The alignment of all theseprimary structures permitted the determination of fingerprint
sequences for the three domains present in each of the fourvertebrate AOX isoenzymes: AOX1, AOX3, AOX4, andAOX3L1. The results obtained allowed the delineation ofamino acid residues specific to the substrate pocket of each iso-enzyme (Figure 4). The data presented lay the foundations tofurther computational and experimental studies aimed at defin-ing the molecular and structural determinants of possible differ-ences and similarities in substrate recognition by each vertebrateAOX isoenzyme. To gain insights into the specific and unre-solved issue as to whether human and mouse AOX1 have sim-ilar substrate selectivities and kinetics of enzymatic activity, itwould be of particular importance to define whether theAOX1-specific residues play a significant role in either process.
5. The necessity to develop new in vitrosystems to study AOX-dependentmetabolism
To define whether a new synthetic molecule is a substrate ofhuman AOX1, the availability and the selection of an
Figure 3. Drugs and xenobiotics metabolized by aldehyde oxidases. The panel illustrates the structures of selected drugs,
xenobiotics, or metabolic intermediates that are oxidized by AOXs. The dashed arrows indicate biotransformation of the drug
or xenobiotic by enzymes other than AOX.MAO: Monoamino oxidase; XOR: Xanthine oxidoreductase.
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appropriate and reliable cell-free or whole-cell in vitro assay isa prerequisite.
An early and popular approach to study AOX-dependentmetabolism in cell-free conditions is represented by humanliver cytosolic extracts, which do not contain significantamounts of contaminating CYP450. However, such prepara-tions have many limitations that prevent their use in thedevelopment of screening tests. Frozen cytosolic extractsfrom human liver are on the market, but they are generallyexpensive and their availability is limited. In addition, humanAOX1, like all the other AOXs, is a relatively unstable proteinand becomes very rapidly inactivated upon freezing-thawing(Mineko Terao (MT), unpublished observations). This resultsin low levels of AOX1 enzymatic activity and high batch-to-batch variability, making the use of these preparations problem-atic and unpredictable. Finally, the specificity of assays based onliver cytosolic extracts relies on the availability of selectiveinhibitors of human AOX1, such as isovanillin [19,51], whichcomplicates the assay design and the interpretation of the results.
A second option for a cell-free assay is represented by theuse of purified recombinant human AOX1. Although expres-sion of recombinant human AOX1 has been reported ininsect cells [64], most of the efforts in this direction have
been oriented toward bacterial systems. Expression of a cata-lytically active form of recombinant human AOX1 in modi-fied strains of Escherichia coli has been achieved [65,66] andone of the two described systems has been used to study theinfluence of some missense mutations determined in thehuman population on the activity of the enzyme [66].However, expression of recombinant MOFEs, includingAOXs, in E. coli is still problematic. In fact, E. coli host strainsneed to be genetically modified to synthesize the eukaryoticversion of MoCo, which differs from the prokaryotic formof the cofactor. In addition, assembly of the eukaryoticMoCo into the apoenzyme is still inefficient, which resultsin a relatively low proportion of catalytically active proteinin the preparations of recombinant human AOX1 [66]. Atthis stage, the use of simple in vitro assays based on purifiedrecombinant human AOX1 may not be advisable in screeningprograms aimed at identifying potential substrates of theenzyme, as the approach may lead to a high rate of falsenegatives, due to the generally low specific activities of thepurified preparations. Thus, further work aimed at optimizingthe production of recombinant AOX1 proteins not only ofhuman origin but also derived from animal species routinelyused in drug metabolism is called for [67,68]. The availability
Figure 4. Consensus sequences of the substrate pocket containing 90-kDa carboxy-terminal domain of mammalian AOX1,
AOX3, AOX4, and AOX3L1 proteins. An alignment of the consensus sequences corresponding to the substrate pocket
containing 90-kDa carboxy-terminal domain determined for mammalian AOX1, AOX3, AOX4, and AOX3L1 proteins. The
alignment was obtained after comparison of all the available primary structures and is based on 31 AOX1, 7 AOX3, 21 AOX4,
and 25 AOX3L1 sequences. The alignment position is indicated by the numbers shown on the right. All the boxed and
indicated residues are conserved across the species in at least one of the AOX isoforms. Amino acid residues specific to only
one of the four AOX isoforms are circled in black. When the residues are not conserved, they are indicated with an “X”.
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of recombinant mouse, rat, and monkey AOX1 is likely toallow the design of screening tests capable of predicting thedifferences in AOX-derived metabolism sometimes observedin humans and experimental animal species used for thepreclinical development of candidate drugs [14,64,69,70].The use of whole-cell systems to define potential
AOX1 substrates would be generally preferable in the contextof drug development programs. A complex experimental sys-tem like the whole cell takes into account the problems thatcannot be considered with cell-free assays, such as accessibilityof the substrate to AOXs. Indeed, accessibility to AOXs inthe intact cell is likely to be controlled by the permeabilityof the test organic molecule across the cytoplasmic membrane,the intracellular distribution, the selective accumulation inspecific organelles, and the metabolism by other enzymaticsystems (for instance, CYP450 and MAO). At present, themodels represented by common human hepatocytic cell lines,such as Hep3B and PLC/PRF/5 are not useful, as they do notsynthesize detectable levels of catalytically active AOX1 [71,72].Deficient synthesis of a catalytically active form of AOX is notlimited to the hepatocytic lineage, as this feature is observed inmany human and animal cell lines of different origin (MTand Enrico Garattini (EG), unpublished observations), oftenas a consequence of the inability of the cell to synthesize orto assemble MoCo into the apoprotein [73]. HumanHEK293 cells are an exception to this rule, containing an effi-cient machinery for the synthesis of MoCo. Genetically engi-neered cellular models expressing significant amounts ofdifferent AOXs will soon be available in this cellular model.In fact, we have recently engineered human the HEK293cell line for the conditional expression of human AOX1 aswell as mouse AOX1, AOX3, AOX4, and AOX3L1, using atetracycline (TET)-ON approach. A HEK-293 clone engi-neered for the expression of a TET repressor was transfectedwith plasmid constructs containing human or mouse AOXcomplementary DNAs (cDNAs) under the control of aTET-responsive regulatory element. After selection and isola-tion, clones expressing a catalytically active form of each of thefive AOXs upon treatment with TET were identified (Figure 5;MT and EG, unpublished results). Figure 5B shows the resultsobtained on a representative HEK293 clone overexpressingmouse AOX1 protein upon treatment with TET. It is clearthat this cellular model allows TET-dependent expression ofa catalytically active form of the protein, as determined by asensitive method based on the production of H2O2 uponAOX-dependent hydroxylation of the phthalazine substrate.The human AOX1-expressing clones are likely to represent atool of fundamental importance for the design of newscreening strategies. It is equally likely that the clones will beinstrumental in defining differences and similarities insubstrate specificities between the human and the rodentAOX isoenzymes. This will help in approaching the long-standing problem of the appropriateness of the murine modelin conducting preclinical studies of AOX-dependent drugmetabolism.
6. The choice of the best experimental modelto conduct preclinical in vivo studies on theAOX-dependent metabolism of drugcandidates
Before Phase I clinical studies, the development of new mole-cules requires an in vivo preclinical phase that must be per-formed in appropriate animal models. These studies arecommonly aimed at defining the therapeutic activity, the tox-icity, the plasma levels, the tissue distribution, and the metab-olism of drug candidates. For molecules that can be oxidizedby AOXs, this is a significant problem, as the pharmacody-namics, the pharmacokinetics, and the metabolism of suchcompounds may vary significantly in humans relative to pop-ular animal models. In fact, the profile of active AOX genes isdifferent in many mammals and this is particularly true in theliver, the most important organ in terms of drug metabolism.As already mentioned, human liver is characterized by thepresence of a single and active AOX isoenzyme, that is,AOX1. In contrast, the complement of the active liver AOXgenes present in many popular experimental models is vari-able. In mice, rats, rabbits, and Chinese hamsters, two hepaticenzymes, AOX1 and AOX3, have been identified and charac-terized. The predominant AOX form expressed in manymouse and rat strains is AOX3. Although, at present, a precisefigure of the relative proportion of the two enzymes in the liv-ers of mice and rats is not available, the semiquantitative dataobtained by Western blot analysis indicate that AOX3contributes the majority of the enzymatic activity present inthe liver cytosol of these two animal species [4,8,74]. This islikely to underly the reported differences in the profiles ofmetabolites observed in humans and mice or rats for anumber of drugs and drug candidates recognized as AOX sub-strates [14]. Two other experimental animals, cats and dogs [7,]
are characterized by pseudogenization and inactivation ofboth AOX1 and AOX3, predicting the absence of AOX enzy-matic activity in the corresponding hepatic tissues. In the caseof dogs, the prediction is confirmed by an old report demon-strating lack of detectable AOX activity in canine cytosolicfractions [70]. The best proxies for the human situation arepredicted to be represented by the guinea pig, the Rhesusmonkey, and the pig. In fact, the three species are endowedwith an active AOX1 gene and an inactive (pig and Rhesusmonkey) or deleted (guinea pig) AOX3 gene. Thus, the threetypes of experimental animals are predicted to synthesize thesole AOX1 isoenzyme in their liver, recapitulating what isobserved in the human counterpart [10].
On the basis of what is reported above and other practicalconsiderations, such as availability and costs, the experimentalmodel of the first choice to study the in vivo properties ofdrug candidates suspected to be metabolized by human liverAOX1 may be currently represented by the guinea pig. How-ever, it must be emphasize that the liver of this animalexpresses the AOX4 transcript. At present, it is unclearwhether this mRNA codes for an active protein, unlike what
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is observed in mice [10]. The second choice is represented byspecific mouse strains, like DBA2 and CBA, which are charac-terized by epigenetic silencing of the Aox3 gene and substan-tial expression of the sole Aox1 gene in the liver [74]. A finalalternative is represented by Rhesus monkeys, despite all thepractical limitations and restrictions associated with the labo-ratory use of primates.
Despite what has been discussed above, there are at leasttwo other pieces of evidence to be considered prior to select-ing the most appropriate experimental model to be used. Inhumans and the aforementioned experimental animals, thesynthesis of AOX1 protein is rather ubiquitous and certainly
not limited to the hepatic tissue. This extrahepatic localizationmay also be of significance for the metabolism of certaindrugs. For instance, appreciable amounts of AOX1 are pres-ent in human kidney and the organ is known to contributeto drug metabolism significantly [75]. There is evidence thatthe tissue distribution of AOX1 in humans, guinea pigs, androdents is different, which may represent an important con-founding factor at the basis of differences in the metabolismand pharmacodynamics of AOX1 substrates in humans andother mammalian models. In addition, while AOX1 is theonly active protein present in humans, AOX1 is not theonly enzyme produced by guinea pigs or DBA2 and CBAmice or Rhesus monkeys. Besides the already mentionedAOX3 isoform, rodents are endowed with two other proteins,AOX4 and AOX3L1. Guinea pigs are characterized by thethree active AOX1, AOX4, and AOX3L1 genes, whereas Rhe-sus monkeys express the AOX1 and AOX3L1 counterparts. Inrodents, the richest source of the AOX4 protein are the Har-derian gland followed by the skin, while the expression ofAOX3L1 is restricted to the Bowman’s gland in the nasalmucosa. At present, the tissue distribution of the AOX4 andAOX3L1 proteins in guinea pigs is unknown and we mustrely on data obtained at the mRNA level. The data availableindicate that the expression of the AOX4 transcript is ratherubiquitous, whereas the kidney, Harderian gland, andolfactory mucosa show the highest levels of AOX3L1 mRNA.
7. Conclusion
From the preceding updated overview of the relevant litera-ture, it is clear that human AOX1 is an enzyme of emerginginterest in drug development because of its ability to oxidizesome clinically used drugs and an increasing number ofdrug candidates. We limited our discussion to the relevancethat human AOX1 and other mammalian AOXs have in themetabolic inactivation of synthetic organic compounds,underscoring the necessity to develop methods that efficientlyreduce the probability of synthesizing AOX substrates. By thesame token, we stressed the necessity to develop better screen-ing methods capable of identifying unwanted AOX substrates.Finally, we enumerated the problems and limitationsassociated with the use of common experimental animals instudying AOX-dependent drug metabolism, always assumingthat AOX-dependent metabolism is a negative determinant interms of drug efficacy, as it results in increased clearance of thepharmacologically active species. However, it must be empha-sized that, in certain instances, AOX-dependent metabolism isa resource to be exploited rather than a problem to be avoidedin drug development programs. In this context, the recentlypublished link between AOXs and obesity [37] suggests thatthe enzyme may represent a molecular target for the develop-ment of new drugs. In addition, AOX-dependent metabolismhas been used for the activation of antitumor agents. Becauseof space limitations, these aspects of AOX relevance in drugmetabolism were not considered in this Expert Opinion and
A.
B.
Figure 5. TET-dependent expression of a catalytically active
form of mouse AOX1protein in HEK293 cells. (A) Schematic
representation of the enzymatic assay used to determine
AOX1 (mAOX1) catalyzes the oxidation of phthalazine
producing unstable superoxide anions that are spontaneously
degraded to H2O2. In the presence of horseradish peroxidase,
H2O2 reacts stoichiometrically with the Amplex red reagent
(INVITROGEN, Paisley, UK) to generate the red-fluorescent
oxidation product resorufin. Resorufin has absorption and
fluorescence emission maxima of approximately 571 and
585 nm, respectively. (B) The inset shows the expression level
of mAOX1 determined uponWestern blot analysis in cytosolic
extracts isolated from an mAOX1 overexpressing HEK293
representative clone after 48 h of treatment with vehicle
(dimethylsulfoxide (DMSO), --TET) or TET (1 µg/ml, +TET). The
enzymatic kinetics of phthalazine oxidation was followed up
to 60 min in cytosolic extracts (50 µg of protein).
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the reader is referred to another review article for an in-depthdiscussion of these issues [31].It is clear that after completion of the preclinical phase, a
new drug must enter the clinical phase of development. Estab-lishing that a new drug candidate is metabolically inactivatedby human AOX1 has important clinical implications that gobeyond potential pharmacokinetic problems. The existence ofpolymorphic sites in the coding regions of the human AOX1gene affecting the enzymatic activity of the encoded pro-tein [30,66] may be a source of unexpected toxicities in someindividuals. This is an added problem that should be takeninto account with the development of genetic or functionaltests capable of identifying patients at high risk of developingAOX-dependent types of toxicities. The recent developmentand application of noninvasive methods for the measurementof AOX1 activity in vivo [76] is the prerequisite for the identifi-cation of individuals carrying polymorphisms in the corre-sponding gene responsible for the inactivation/activation ofthe enzyme. These assays should be used in concert with genetictests to define individuals at risk for developing toxicity afterthe administration of drugs metabolized by AOX1.
8. Expert opinion
Vertebrate AOXs and the structurally related XORs are asmall group of proteins belonging to the family of MOFEs,which require FAD and a molybdopterin (MoCo) for theircatalytic activity. Both types of enzymes are characterized bytwo nonidentical 2Fe/2S: one FAD and one MoCo redoxcenters. The electrons flow from the substrate through theenzyme and reduce molecular oxygen. Different animal spe-cies are characterized by a different complement of activeAOX genes, which varies from one in humans (AOX1) tofour in mice (Aox1, Aox3, Aox4, and Aox3l1). In mammals,this is the consequence of two types of evolutionary events,that is, gene duplication and gene functional inactivationpredominantly via pseudogenization or deletion.AOXs metabolize a broad range of xenobiotics of medical
and toxicological interest. The main enzymatic reactions cata-lyzed by AOXs are the oxidation of aromatic aldehydes intothe corresponding carboxylic acids and the oxidation of aza-heterocycles into mono- or poly-hydroxylated derivatives.Aza-heterocycles are popular building blocks in the synthesisof new drug candidates. The significance of AOXs asdrug-metabolizing enzymes is increasing, as the currentstrategies of organic synthesis that are designed to avoidCYP450-dependent metabolism tend to enrich for new chem-ical structures with the potential to be efficiently oxidized byAOXs. Indeed, metabolic inactivation by human AOX1 hasbeen responsible for a series of drug failures in clinical trials.This is predominantly the result of the current difficulties inpredicting and validating the role played by AOXs in drugclearance during the preclinical phase of drug development.Preclinical studies focusing on the relevance of AOX-
dependent metabolism for new drug candidates are burdened
by a number of problems. First, there is a general lack of reli-able in silico methodologies that can be used to predictwhether a new organic molecule is a potential AOX substrate.The recent availability of the crystal coordinates of the firstmammalian AOX, that is, mouse AOX3, is likely to fosternew computational studies based on substrate dockingapproaches aimed at defining new predictive algorithms.However, it is of the utmost importance to multiply effortsaimed at obtaining the crystal coordinates of the humanAOX1 protein, as it is not known whether the structure ofAOX3 can be used as an appropriate proxy for the humanenzyme and an adequate model to be utilized in dockingexperiments. Second, robust in vitro systems allowing thedesign of appropriate high- or medium-throughput screeningtests to identify AOX substrates are not yet available. As thesimplest way to achieve this goal is to develop cell-free assaysbased on the use of purified recombinant proteins, a priorityis the development of new technologies for the expressionand purification of human AOX1and other mammalianAOXs with high catalytic activity. In fact, the availableprocedures developed in bacterial systems yield recombinantproteins with low catalytic activity, as a consequence of theinability to obtain efficient incorporation of the MoCo intothe AOX apoprotein. In this context, an equally appropriateoption is represented by the development of human cell linesengineered for the expression of catalytically active humanand murine AOXs that will allow the design of screeningtests based on whole-cell assays. Finally, in vivo studies onAOX-dependent metabolism in animal models is highlyproblematic, as the complement of liver AOXs in humans andpopular mouse or rat strains is different. At present, the bestproxies for the human situation in terms of liver AOX expres-sion are represented by the guinea pig and the Rhesus monkey.However, it must be emphasized that even these last two modelsare not optimal, given the expression of one (AOX3L1, Rhesusmonkey) or two (AOX4 and AOX3L1, guinea pig) AOX isoen-zymes that may contribute to the overall metabolism of a givenxenobiotic in other organs. Thus, a further priority in the field isrepresented by the identification and/or development of newanimal models mimicking or recapitulating the human situa-tion. With respect to this, metabolic studies using transplantsof human liver cells have already been published [77,78] and thedevelopment of Aox3 knockout animals or transgenic micecharacterized by liver-specific production of the humanAOX1 protein are under way in our laboratory.
Unequivocal establishment that a new drug candidate ismetabolically inactivated by human AOX1 has importantimplications for its clinical development and use. In fact, thehuman AOX1 gene is highly polymorphic and some inactivat-ing missense as well as nonsense polymorphic sites have beendescribed in the human population [30,66]. Such polymor-phisms are likely to result in reduced levels or in undetectableamounts of the encoded AOX1 protein and may explain thereported interindividual variability in AOX activity. Theymay be a source of unexpected toxicities in some individuals
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and the problem should be taken into account when Phase Iclinical trials are designed. Thus, we propose that a final prior-ity in the field is the development of robust diagnostic tests of afunctional or genetic nature to identify patient populations athigh risk of developing AOX-dependent types of toxicities.
Acknowledgments
The authors thank JN Fisher for critical reading of themanuscript and F Deceglie for the artwork.
Declaration of interest
E Garattini and M Terao are members of the permanent sci-entific staff of the Istituto di Ricerche Farmacologiche “MarioNegri,” a private nonprofit research institute. The authors fur-ther acknowledge the financial support of the Fondazione“Italo Monzino,” the Associazione Italiana per la Ricerca sulCancro (AIRC), and the Weizmann-Negri Foundation thatmade this work possible.
BibliographyPapers of special note have been highlighted as
either of interest (�) or of considerable interest(��) to readers.
1. Garattini E, Mendel R, Romao MJ, et al.
Mammalian molybdo-flavoenzymes, an
expanding family of proteins: structure,
genetics, regulation, function and
pathophysiology. Biochem J
2003;372(Pt 1):15-32.. An extensive overview on the structural
and enzymatic characteristics of
mammalian AOXs.
2. Garattini E, Fratelli M, Terao M. The
mammalian aldehyde oxidase gene
family. Hum Genomics
2009;4(2):119-30
3. Garattini E, Fratelli M, Terao M.
Mammalian aldehyde oxidases: genetics,
evolution and biochemistry. Cell Mol
Life Sci 2008;65(7-8):1019-48.. An updated overview on the structure
and enzymatic characteristics of
mammalian aldehyde oxidases.
The article contains also fundamental
information on the genetics and
evolution of mammalian AOXs.
4. Terao M, Kurosaki M, Saltini G, et al.
Cloning of the cDNAs coding for two
novel molybdo-flavoproteins showing
high similarity with aldehyde oxidase and
xanthine oxidoreductase. J Biol Chem
2000;275(39):30690-700
5. Terao M, Kurosaki M, Marini M, et al.
Purification of the aldehyde oxidase
homolog 1 (AOH1) protein and cloning
of the AOH1 and aldehyde oxidase
homolog 2 (AOH2) genes. Identification
of a novel molybdo-flavoprotein gene
cluster on mouse chromosome 1.
J Biol Chem 2001;276(49):46347-63
6. Terao M, Kurosaki M, Demontis S,
et al. Isolation and characterization of the
human aldehyde oxidase gene:
conservation of intron/exon boundaries
with the xanthine oxidoreductase gene
indicates a common origin. Biochem J
1998;332(Pt 2):383-93
7. Terao M, Kurosaki M, Barzago MM,
et al. Avian and canine aldehyde
oxidases. Novel insights into the biology
and evolution of molybdo-flavoenzymes.
J Biol Chem 2006;281(28):19748-61
8. Kurosaki M, Terao M, Barzago MM,
et al. The aldehyde oxidase gene cluster
in mice and rats. Aldehyde oxidase
homologue 3, a novel member of the
molybdo-flavoenzyme family with
selective expression in the olfactory
mucosa. J Biol Chem
2004;279(48):50482-98
9. Kurosaki M, Demontis S, Barzago MM,
et al. Molecular cloning of the
cDNA coding for mouse aldehyde
oxidase: tissue distribution and regulation
in vivo by testosterone. Biochem J
1999;341(Pt 1):71-80
10. Kurosaki M, Bolis M, Fratelli M, et al.
Structure and evolution of vertebrate
aldehyde oxidases: from gene duplication
to gene suppression. Cell Mol Life Sci
2012.. The most updated study on the
structure and evolution of vertebrate
AOXs. The work contains the
reconstruction of over 150 vertebrate
AOX genes. In addition, the article
reports on the cloning and structural
characterization of the AOX cDNasin
the Rhesus monkey and the
guinea pig.
11. Demontis S, Kurosaki M, Saccone S,
et al. The mouse aldehyde oxidase gene:
molecular cloning, chromosomal
mapping and functional characterization
of the 5¢-flanking region.
Biochim Biophys Acta
1999;1489(2-3):207-22
12. Calzi ML, Raviolo C, Ghibaudi E, et al.
Purification, cDNA cloning, and tissue
distribution of bovine liver aldehyde
oxidase. J Biol Chem
1995;270(52):31037-45. This is the first report on the complete
primary structure of a mammalian
AOX protein deduced from the
cloning of the corresponding
cDNA from bovine liver.
13. Pryde DC, Tran TD, Jones P, et al.
Medicinal chemistry approaches to avoid
aldehyde oxidase metabolism.
Bioorg Med Chem Lett
2012;22(8):2856-60
14. Pryde DC, Dalvie D, Hu Q, et al.
Aldehyde oxidase: an enzyme of
emerging importance in drug discovery.
J Med Chem 2010;53(24):8441-60.. The most significant review article
highlighting the emerging relevance of
AOXs in the metabolism of new drug
candidates from a chemical perspective.
15. Jones P, Pryde DC, Tran TD, et al.
Discovery of a highly potent series of
TLR7 agonists. Bioorg Med Chem Lett
2011;21(19):5939-43
16. Linton A, Kang P, Ornelas M, et al.
Systematic structure modifications of
imidazo[1,2-a]pyrimidine to reduce
metabolism mediated by Aldehyde
Oxidase (AO). J Med Chem
2011;54(21):7705-12.. A description of potential strategies of
chemical synthesis aimed at reducing
the probability of AOX-dependent
metabolism for
organic pharmacophores.
17. Rochat B, Kosel M, Boss G, et al.
Stereoselective biotransformation of the
selective serotonin reuptake inhibitor
citalopram and its demethylated
metabolites by monoamine oxidases in
human liver. Biochem Pharmacol
1998;56(1):15-23
18. Ruenitz PC, Bai X. Acidic metabolites of
tamoxifen. Aspects of formation and fate
Aldehyde oxidase and its importance in novel drug discovery
Expert Opin. Drug Discov. [Early Online] 11
Exp
ert O
pin.
Dru
g D
isco
v. D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsity
of
Gla
sgow
on
05/0
2/13
For
pers
onal
use
onl
y.
in the female rat. Drug Metab Dispos
1995;23(9):993-8
19. Obach RS, Huynh P, Allen MC,
Beedham C. Human liver aldehyde
oxidase: inhibition by 239 drugs.
J Clin Pharmacol 2004;44(1):7-19. This review article contains the most
extensive list of AOX inhibitors.
20. Nishino T. Purification of hepatic
xanthine dehydrogenase from chicken fed
a high-protein diet.
Biochim Biophys Acta 1974;341(1):93-8
21. Nishino T, Noda K, Tsushima K.
Structure of xanthine dehydrogenase
from chicken and rat liver: chemical
modification of NAD binding site with
5’-FSBA. Adv Exp Med Biol
1989;253B:173-8
22. Saito T, Nishino T, Massey V.
Differences in environment of FAD
between NAD-dependent and
O2-dependent types of rat liver xanthine
dehydrogenase shown by active site probe
study. J Biol Chem
1989;264(27):15930-5
23. Ichikawa M, Nishino T, Ichikawa A.
Subcellular localization of xanthine
oxidase in rat hepatocytes:
high-resolution immunoelectron
microscopic study combined with
biochemical analysis.
J Histochem Cytochem
1992;40(8):1097-103
24. Coelho C, Mahro M, Trincao J, et al.
The first mammalian aldehyde oxidase
crystal structure: insights into substrate
specificity. J Biol Chem
2012;287(48):40690-702.. The article reports on the
crystallization and deconvolution of
the crystal structure of the first
mammalian AOX, i.e., mouse AOX3.
25. Enroth C, Eger BT, Okamoto K, et al.
Crystal structures of bovine milk
xanthine dehydrogenase and xanthine
oxidase: structure-based mechanism of
conversion. Proc Natl Acad Sci USA
2000;97(20):10723-8.. The article reports on the
crystallization and deconvolution of
the crystal structure of the first
mammalian MOFE, i.e., bovine XOR.
26. Eger BT, Okamoto K, Enroth C, et al.
Purification, crystallization and
preliminary X-ray diffraction studies of
xanthine dehydrogenase and xanthine
oxidase isolated from bovine milk.
Acta Crystallogr D Biol Crystallogr
2000;56(Pt 12):1656-8
27. Abe Y, Okamoto K, Nishino T. Crystal
structures of heme binding protein
23 and xanthine dehydrogenase.
J Nippon Med Sch 2001;68(3):220-1
28. Beedham C, Critchley DJ, Rance DJ.
Substrate specificity of human liver
aldehyde oxidase toward substituted
quinazolines and phthalazines:
a comparison with hepatic enzyme from
guinea pig, rabbit, and baboon.
Arch Biochem Biophys
1995;319(2):481-90
29. Beedham C. Molybdenum hydroxylases:
biological distribution and
substrate-inhibitor specificity.
Prog Med Chem 1987;24:85-127
30. Garattini E, Terao M. The role of
aldehyde oxidase in drug metabolism.
Expert Opin Drug Metab Toxicol
2012;8(4):487-503.. The authors review the literature to
highlight the fact that aldehydes are
not the only AOX substrates as aza
and oxo-heterocycles that represent the
scaffold of many drugs are also
oxidized efficiently by these enzymes.
The overview contains information and
views which are complementary to
those contained in the present
Expert opinion.
31. Garattini E, Terao M. Increasing
recognition of the importance of
aldehyde oxidase in drug development
and discovery. Drug Metab Rev
2011;43(3):374-86.. The review article reports on the
importance of AOXsin drug
development and discovery, discussing
many aspects and issues that are not
covered in the present Expert Opinion.
32. Terao M, Kurosaki M, Barzago MM,
et al. Role of the molybdoflavoenzyme
aldehyde oxidase homolog 2 in the
biosynthesis of retinoic acid: generation
and characterization of a knockout
mouse. Mol Cell Biol 2009;29(2):357-77. The generation and characterization of
the first knock-out mouse for a
mammalian AOX gene, i.e., AOX4, are
described in this article. The study
demonstrates that AOX4 knock-out
animals are viable, although they have
a local deficit in retinoic
acid metabolism.
33. Nishino T, Okamoto K, Eger BT,
Pai EF. Mammalian xanthine
oxidoreductase - mechanism of transition
from xanthine dehydrogenase to xanthine
oxidase. FEBS J 2008;275(13):3278-89
34. Terao M, Kurosaki M, Zanotta S,
Garattini E. The xanthine oxidoreductase
gene: structure and regulation.
Biochem Soc Trans 1997;25(3):791-6
35. Kurosaki M, Zanotta S, Li Calzi M,
et al. Expression of xanthine
oxidoreductase in mouse mammary
epithelium during pregnancy and
lactation: regulation of gene expression
by glucocorticoids and prolactin.
Biochem J 1996;319(Pt 3):801-10
36. Cazzaniga G, Terao M, Lo Schiavo P,
et al. Chromosomal mapping, isolation,
and characterization of the mouse
xanthine dehydrogenase gene. Genomics
1994;23(2):390-402
37. Weigert J, Neumeier M, Bauer S, et al.
Small-interference RNA-mediated knock-
down of aldehyde oxidase 1 in
3T3-L1 cells impairs adipogenesis and
adiponectin release. FEBS Lett
2008;582(19):2965-72.. The study demonstrates that human
AOX1 is involved in the differentiation
of adipocytes as well as lipid synthesis
and deposition.
38. Sigruener A, Buechler C, Orso E, et al.
Human aldehyde oxidase 1 interacts with
ATP-binding cassette transporter-1 and
modulates its activity in hepatocytes.
Horm Metab Res 2007;39(11):781-9
39. Graessler J, Fischer S. The dual substrate
specificity of aldehyde oxidase 1 for
retinal and acetaldehyde and its role in
ABCA1 mediated efflux.
Horm Metab Res 2007;39(11):775-6
40. Kitamura S, Tatsumi K. Involvement of
liver aldehyde oxidase in the reduction of
nicotinamide N-oxide. Biochem Biophys
Res Commun 1984;120(2):602-6
41. Kitamura S, Tatsumi K. Reduction of
tertiary amine N-oxides by liver
preparations: function of aldehyde
oxidase as a major N-oxide reductase.
Biochem Biophys Res Commun
1984;121(3):749-54
42. Dick RA, Kanne DB, Casida JE.
Substrate specificity of rabbit aldehyde
oxidase for nitroguanidine and
nitromethylene neonicotinoid
insecticides. Chem Res Toxicol
2006;19(1):38-43
E. Garattini & M. Terao
12 Expert Opin. Drug Discov. [Early Online]
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ert O
pin.
Dru
g D
isco
v. D
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oade
d fr
om in
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ahea
lthca
re.c
om b
y U
nive
rsity
of
Gla
sgow
on
05/0
2/13
For
pers
onal
use
onl
y.
43. Obach RS. Potent inhibition of human
liver aldehyde oxidase by raloxifene.
Drug Metab Dispos 2004;32(1):89-97
44. Beedham C, Miceli JJ, Obach RS.
Ziprasidone metabolism, aldehyde
oxidase, and clinical implications.
J Clin Psychopharmacol
2003;23(3):229-32
45. Dalvie D, Xiang C, Kang P, Zhou S.
Interspecies variation in the metabolism
of zoniporide by aldehyde oxidase.
Xenobiotica 2012; Epub ahead of print
46. Obach RS, Walsky RL. Drugs that
inhibit oxidation reactions catalyzed by
aldehyde oxidase do not inhibit the
reductive metabolism of ziprasidone
to its major metabolite,
S-methyldihydroziprasidone: an
in vitro study. J Clin Psychopharmacol
2005;25(6):605-8
47. Zientek M, Jiang Y, Youdim K,
Obach RS. In vitro-in vivo correlation
for intrinsic clearance for drugs
metabolized by human aldehyde oxidase.
Drug Metab Dispos 2010;38(8):1322-7
48. Critchley DJ, Rance DJ, Beedham C.
Biotransformation of carbazeran in
guinea pig: effect of hydralazine
pretreatment. Xenobiotica
1994;24(1):37-47
49. Panoutsopoulos GI, Kouretas D,
Beedham C. Contribution of aldehyde
oxidase, xanthine oxidase, and aldehyde
dehydrogenase on the oxidation of
aromatic aldehydes. Chem Res Toxicol
2004;17(10):1368-76
50. Panoutsopoulos GI, Beedham C.
Metabolism of isovanillin by aldehyde
oxidase, xanthine oxidase, aldehyde
dehydrogenase and liver slices.
Pharmacology 2005;73(4):199-208
51. Panoutsopoulos GI, Beedham C.
Enzymatic oxidation of vanillin,
isovanillin and protocatechuic aldehyde
with freshly prepared Guinea pig liver
slices. Cell Physiol Biochem
2005;15(1-4):89-98
52. Panoutsopoulos GI. Contribution of
aldehyde oxidizing enzymes on the
metabolism of 3,4-dimethoxy-2-
phenylethylamine to
3,4-dimethoxyphenylacetic acid by guinea
pig liver slices. Cell Physiol Biochem
2006;17(1-2):47-56
53. Panoutsopoulos GI. Metabolism of
2-phenylethylamine to phenylacetic acid,
via the intermediate phenylacetaldehyde,
by freshly prepared and cryopreserved
guinea pig liver slices. In Vivo
2004;18(6):779-86
54. Moriyasu A, Sugihara K, Nakatani K,
et al. In vivo-in vitro relationship of
methotrexate 7-hydroxylation by
aldehyde oxidase in four different strain
rats. Drug Metab Pharmacokinet
2006;21(6):485-91
55. Kuroda T, Namba K, Torimaru T, et al.
Species differences in oral bioavailability
of methotrexate between rats and
monkeys. Biol Pharm Bull
2000;23(3):334-8
56. Kitamura S, Sugihara K, Nakatani K,
et al. Variation of hepatic methotrexate
7-hydroxylase activity in animals and
humans. IUBMB Life 1999;48(6):607-11
57. Jordan CG, Rashidi MR, Laljee H, et al.
Aldehyde oxidase-catalysed oxidation of
methotrexate in the liver of guinea-pig,
rabbit and man. J Pharm Pharmacol
1999;51(4):411-18
58. Rashidi MR, Beedham C, Smith JS,
Davaran S. In vitro study of
6-mercaptopurine oxidation catalysed by
aldehyde oxidase and xanthine oxidase.
Drug Metab Pharmacokinet
2007;22(4):299-306
59. Vickers S, Polsky SL. The
biotransformation of nitrogen containing
xenobiotics to lactams. Curr Drug Metab
2000;1(4):357-89
60. Linton A, Kang P, Ornelas M, et al.
Systematic structure modifications of
imidazo[1,2-a]pyrimidine to reduce
metabolism mediated by aldehyde
oxidase (AO). J Med Chem
2011;54(21):7705-12
61. Torres RA, Korzekwa KR,
McMasters DR, et al. Use of density
functional calculations to predict the
regioselectivity of drugs and molecules
metabolized by aldehyde oxidase.
J Med Chem 2007;50(19):4642-7
62. Jones JP, Korzekwa KR. Predicting
intrinsic clearance for drugs and drug
candidates metabolized by aldehyde
oxidase. Mol Pharm
2013; Epub ahead of print.. The article proposes a new in silico
method to predict the metabolism of
organic compounds by AOXs.
63. Hutzler JM, Yang YS, Albaugh D, et al.
Characterization of aldehyde oxidase
enzyme activity in cryopreserved human
hepatocytes. Drug Metab Dispos
2012;40(2):267-75
64. Zhang X, Liu HH, Weller P, et al. In
silico and in vitro pharmacogenetics:
aldehyde oxidase rapidly metabolizes a
p38 kinase inhibitor.
Pharmacogenomics J 2011;11(1):15-24
65. Alfaro JF, Joswig-Jones CA, Ouyang W,
et al. Purification and mechanism of
human aldehyde oxidase expressed in
Escherichia coli. Drug Metab Dispos
2009;37(12):2393-8
66. Hartmann T, Terao M, Garattini E,
et al. The impact of single nucleotide
polymorphisms on human aldehyde
oxidase. Drug Metab Dispos
2012;40(5):856-64
67. Schumann S, Terao M, Garattini E,
et al. Site directed mutagenesis of amino
acid residues at the active site of mouse
aldehyde oxidase AOX1. PLoS One
2009;4(4):e5348
68. Mahro M, Coelho C, Trincao J, et al.
Characterization and crystallization of
mouse aldehyde oxidase 3: from mouse
liver to Escherichia coli heterologous
protein expression. Drug Metab Dispos
2011;39(10):1939-45.. The article provides details on an
E. coli based methodology to obtain
protein preparations of catalytically
active recombinant AOXs.
69. Klecker RW, Cysyk RL, Collins JM.
Zebularine metabolism by aldehyde
oxidase in hepatic cytosol from humans,
monkeys, dogs, rats, and mice: influence
of sex and inhibitors. Bioorg Med Chem
2006;14(1):62-6
70. Beedham C, Bruce SE, Critchley DJ,
et al. Species variation in hepatic
aldehyde oxidase activity. Eur J Drug
Metab Pharmacokinet 1987;12(4):307-10
71. Sahi J, Khan KK, Black CB. Aldehyde
oxidase activity and inhibition in
hepatocytes and cytosolic fractions from
mouse, rat, monkey and human.
Drug Metab Lett 2008;2(3):176-83
72. Neumeier M, Weigert J, Schaffler A,
et al. Aldehyde oxidase 1 is highly
abundant in hepatic steatosis and is
downregulated by adiponectin and
fenofibric acid in hepatocytes in vitro.
Biochem Biophys Res Commun
2006;350(3):731-5
73. Falciani F, Terao M, Goldwurm S, et al.
Molybdenum(VI) salts convert the
xanthine oxidoreductase apoprotein into
Aldehyde oxidase and its importance in novel drug discovery
Expert Opin. Drug Discov. [Early Online] 13
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the active enzyme in mouse
L929 fibroblastic cells. Biochem J
1994;298(Pt 1):69-77
74. Vila R, Kurosaki M, Barzago MM, et al.
Regulation and biochemistry of mouse
molybdo-flavoenzymes. The DBA/
2 mouse is selectively deficient in the
expression of aldehyde oxidase
homologues 1 and 2 and represents a
unique source for the purification and
characterization of aldehyde oxidase.
J Biol Chem 2004;279(10):8668-83
75. Lohr JW, Willsky GR, Acara MA. Renal
drug metabolism. Pharmacol Rev
1998;50(1):107-41
76. Sugihara K, Tayama Y, Shimomiya K,
et al. Estimation of aldehyde oxidase
activity in vivo from conversion ratio of
N1-methylnicotinamide to pyridones,
and intraspecies variation of the enzyme
activity in rats. Drug Metab Dispos
2006;34(2):208-12
77. Sanoh S, Nozaki K, Murai H, et al.
Prediction of human metabolism of
FK3453 by aldehyde oxidase using
chimeric mice transplanted with human
or rat hepatocytes. Drug Metab Dispos
2012;40(1):76-82
78. Sanoh S, Horiguchi A, Sugihara K, et al.
Prediction of in vivo hepatic clearance
and half-life of drug candidates in
human using chimeric mice with
humanized liver. Drug Metab Dispos
2012;40(2):322-8.. This is the first report of a humanized
murine model to study
AOX-dependent drug metabolism.
AffiliationEnrico Garattini† & Mineko Terao†Author for correspondence
