Human Arylacetamide Deacetylase Research Articles

The implication of fragment-centric mapping strategy to explore the first selective inhibitor against target protein

Introduction: Identification of the first selective inhibitor, also called “hit molecules, " is crucial for developing drugs against a protein target. However, the crystal structures of protein-ligand complexes are usually not resolved in time due to the process's time-consuming and costly nature. However, it does not prevent scientists from understanding the binding modes’ urgent advantages and drawbacks of protein-ligand interaction to guide the optimization of hit molecules. Methods: Here, we have developed a pocket-centric computational strategy to facilitate a comprehensive understanding of the hit molecules against the protein target. Results: The results show that the pocket-centered mapping method not only allows for accurate prediction of the native docking pose and in-depth analysis of the binding mode but also has the potential of rapidly identifying partially unoccupied, unutilized, but targetable pockets to afford optimized hit molecules. We tested the strategy on the first selective inhibitor, epigallocatechin gallate (EGCG), against human arylacetamide deacetylase (AADAC). Molecular dynamics simulation and MM/PBSA binding energy calculation are used to verify the efficacy of the strategy. Conclusion: The results show that the pocket-centered mapping method not only allows for accurate prediction of the native docking pose and in-depth analysis of the binding mode but also has the potential of rapidly identifying partially unoccupied, unutilized, but targetable pockets to afford optimized hit molecules.

In vitro deacetylation of N-acetylserotonin by arylacetamide deacetylase.

Arylacetamide deacetylase (AADAC) is a deacetylation enzyme present in the mammalian liver, gastrointestinal tract, and brain. During our search for mammalian enzymes capable of metabolizing N-acetylserotonin (NAS), AADAC was identified as having the ability to convert NAS to serotonin. Both human and rodent recombinant AADAC proteins can deacetylate NAS in vitro, although the human AADAC shows markedly higher activity compared with rodent enzyme. The AADAC-mediated deacetylation reaction can be potently inhibited by eserine in vitro. In addition to NAS, recombinant hAADAC can deacetylate melatonin (to form 5-methoxytryptamine) and N-acetyltryptamine (NAT) (to form tryptamine). In addition to the in vitro deacetylation of NAS by the recombinant AADAC proteins, liver (mouse and human) and brain (human) extracts were able to deacetylate NAS; these activities were sensitive to eserine. Taken together, these results demonstrate a new role for AADAC and suggest a novel pathway for the AADAC-mediated metabolism of pineal indoles in mammals.

PPARα regulates the expression of human arylacetamide deacetylase involved in drug hydrolysis and lipid metabolism

Human arylacetamide deacetylase (AADAC) hydrolyzes various drugs containing an acetyl group, such as ketoconazole and rifampicin. Knowledge about the role of human AADAC in drug metabolism is accumulating, but the regulatory mechanism of its expression has not been elucidated. In mice, it has been suggested that Aadac expression may be regulated by peroxisome proliferator-activated receptor α (Pparα). This study examined whether human AADAC is regulated by PPARα, which widely regulates the expression of lipid metabolism-related genes. In human hepatoma Huh-7 cells, AADAC mRNA and protein levels were significantly increased by treatment with fenofibric acid and WY-14643, PPARα ligands. Knockdown and overexpression of PPARα resulted in decreased and increased expression of AADAC, respectively. Luciferase assays revealed that the direct repeat 1 (DR1) at −193/−181 in the AADAC promoter region is responsible for transactivation by PPARα. Chromatin immunoprecipitation assays revealed the binding of PPARα to DR1. Thus, it was demonstrated that human AADAC is regulated by PPARα through binding to DR1. Oil red O staining showed that overexpression of AADAC in Huh-7 cells suppressed lipid accumulation after treatment with free fatty acids. The suppression was restored by treatment with diisopropyl fluorophosphate, an AADAC inhibitor. The WY-14643-mediated suppression of lipid accumulation was restored by AADAC knockdown. These results suggested that AADAC has a role in suppressing cellular lipid accumulation. In conclusion, this study demonstrated the regulation of human AADAC by PPARα and its significance in lipid accumulation.

Differences in Hydrolase Activities in the Liver and Small Intestine between Marmosets and Humans.

For drug development, species differences in drug-metabolism reactions present obstacles for predicting pharmacokinetics in humans. We characterized the species differences in hydrolases among humans and mice, rats, dogs, and cynomolgus monkeys. In this study, to expand the series of such studies, we attempted to characterize marmoset hydrolases. We measured hydrolase activities for 24 compounds using marmoset liver and intestinal microsomes, as well as recombinant marmoset carboxylesterase (CES) 1, CES2, and arylacetamide deacetylase (AADAC). The contributions of CES1, CES2, and AADAC to hydrolysis in marmoset liver microsomes were estimated by correcting the activities by using the ratios of hydrolase protein levels in the liver microsomes and those in recombinant systems. For six out of eight human CES1 substrates, the activities in marmoset liver microsomes were lower than those in human liver microsomes. For two human CES2 substrates and three out of seven human AADAC substrates, the activities in marmoset liver microsomes were higher than those in human liver microsomes. Notably, among the three rifamycins, only rifabutin was hydrolyzed by marmoset tissue microsomes and recombinant AADAC. The activities for all substrates in marmoset intestinal microsomes tended to be lower than those in liver microsomes, which suggests that the first-pass effects of the CES and AADAC substrates are due to hepatic hydrolysis. In most cases, the sums of the values of the contributions of CES1, CES2, and AADAC were below 100%, which indicated the involvement of other hydrolases in marmosets. In conclusion, we clarified the substrate preferences of hydrolases in marmosets. SIGNIFICANCE STATEMENT: This study confirmed that there are large differences in hydrolase activities between humans and marmosets by characterizing marmoset hydrolase activities for compounds that are substrates of human CES1, CES2, or arylacetamide deacetylase. The data obtained in this study may be useful for considering whether marmosets are appropriate for examining the pharmacokinetics and efficacies of new chemical entities in preclinical studies.

Epicatechin gallate and epigallocatechin gallate are potent inhibitors of human arylacetamide deacetylase

Recently, in addition to carboxylesterases (CESs), we found that arylacetamide deacetylase (AADAC) plays an important role in the metabolism of some clinical drugs. In this study, we screened for food-related natural compounds that could specifically inhibit human AADAC, CES1, or CES2. AADAC, CES1, and CES2 activities in human liver microsomes were measured using phenacetin, fenofibrate, and procaine as specific substrates, respectively. In total, 43 natural compounds were screened for their inhibitory effects on each of these enzymes. Curcumin and quercetin showed strong inhibitory effects against all three enzymes, whereas epicatechin, epicatechin gallate (ECg), and epigallocatechin gallate (EGCg) specifically inhibited AADAC. In particular, ECg and EGCg showed strong inhibitory effects on AADAC (IC50 values: 3.0 ± 0.5 and 2.2 ± 0.2 μM, respectively). ECg and EGCg also strongly inhibited AADAC-mediated rifampicin hydrolase activity in human liver microsomes with IC50 values of 2.2 ± 1.4 and 1.7 ± 0.4 μM, respectively, whereas it weakly inhibited p-nitrophenyl acetate hydrolase activity, which is catalyzed by AADAC, CES1, and CES2. Our results indicate that ECg and EGCg are potent inhibitors of AADAC.

Hydrolase activities of cynomolgus monkey liver microsomes and recombinant CES1, CES2, and AADAC

The cynomolgus monkey is a nonhuman primate that is often used for pharmacokinetic and toxicokinetic studies of new chemical entities. Species differences in drug metabolism are obstacles for the extrapolation of animal data to humans. This study aimed to characterize hydrolase activities for typical compounds by cynomolgus monkey liver microsomes and recombinant monkey carboxylesterases (CES1 and CES2) and arylacetamide deacetylase (AADAC) compared with the activities in humans. To estimate the contribution of each hydrolase, the ratios of the expression level of each hydrolase in the liver microsomes and recombinant systems were used. For almost all of the tested human CES1 substrates, hydrolase activities in cynomolgus monkey liver microsomes tended to be lower than those in human liver microsomes, and recombinant cynomolgus monkey CES1 showed catalytic activity, but not for all substrates. For human CES2 substrates, hydrolase activities in cynomolgus monkey liver were higher than those in human liver microsomes, and recombinant monkey CES2 was responsible for their hydrolysis. Among human AADAC substrates, phenacetin was mainly hydrolyzed by monkey AADAC, whereas indiplon and ketoconazole were hydrolyzed by AADAC and other unknown enzymes. Flutamide was hydrolyzed by monkey CES2, not by AADAC. Rifamycins were hardly hydrolyzed in monkey liver microsomes. In conclusion, this study characterized the hydrolase activities of cynomolgus monkeys compared with those in humans. The findings would be helpful for pharmacokinetic or toxicokinetic studies of new chemical entities whose main metabolic pathway is hydrolysis.

Carboxylesterase Activities and Protein Expression in Rabbit and Pig Ocular Tissues.

Hydrolytic reactions constitute an important pathway of drug metabolism and a significant route of prodrug activation. Many ophthalmic drugs and prodrugs contain ester groups that greatly enhance their permeation across several hydrophobic barriers in the eye before the drugs are either metabolized or released, respectively, via hydrolysis. Thus, the development of ophthalmic drug therapy requires the thorough profiling of substrate specificities, activities, and expression levels of ocular esterases. However, such information is scant in the literature, especially for preclinical species often used in ophthalmology such as rabbits and pigs. Therefore, our aim was to generate systematic information on the activity and expression of carboxylesterases (CESs) and arylacetamide deacetylase (AADAC) in seven ocular tissue homogenates from these two species. The hydrolytic activities were measured using a generic esterase substrate (4-nitrophenyl acetate) and, in the absence of validated substrates for rabbit and pig enzymes, with selective substrates established for human CES1, CES2, and AADAC (d-luciferin methyl ester, fluorescein diacetate, procaine, and phenacetin). Kinetics and inhibition studies were conducted using these substrates and, again due to a lack of validated rabbit and pig CES inhibitors, with known inhibitors for the human enzymes. Protein expression levels were measured using quantitative targeted proteomics. Rabbit ocular tissues showed significant variability in the expression of CES1 (higher in cornea, lower in conjunctiva) and CES2 (higher in conjunctiva, lower in cornea) and a poor correlation of CES expression with hydrolytic activities. In contrast, pig tissues appear to express only CES1, and CES3 and AADAC seem to be either low or absent, respectively, in both species. The current study revealed remarkable species and tissue differences in ocular hydrolytic enzymes that can be taken into account in the design of esterase-dependent prodrugs and drug conjugates, the evaluation of ocular effects of systemic drugs, and in translational and toxicity studies.

Role of Human Arylacetamide Deacetylase (AADAC) on Hydrolysis of Eslicarbazepine Acetate and Effects of AADAC Genetic Polymorphisms on Hydrolase Activity.

Human arylacetamide deacetylase (AADAC) plays a role in the detoxification or activation of drugs and is sometimes involved in the incidence of toxicity by catalyzing hydrolysis reactions. AADAC prefers compounds with relatively small acyl groups, such as acetyl groups. Eslicarbazepine acetate, an antiepileptic drug, is a prodrug rapidly hydrolyzed to eslicarbazepine. We sought to clarify whether AADAC might be responsible for the hydrolysis of eslicarbazepine acetate. Eslicarbazepine acetate was efficiently hydrolyzed by human intestinal and liver microsomes and recombinant human AADAC. The hydrolase activities in human intestinal and liver microsomes were inhibited by epigallocatechin gallate, a specific inhibitor of AADAC, by 82% and 88% of the control, respectively. The hydrolase activities in liver microsomes from 25 human livers were significantly correlated (r = 0.87, P < 0.001) with AADAC protein levels, suggesting that the enzyme AADAC is responsible for the hydrolysis of eslicarbazepine acetate. The effects of genetic polymorphisms of AADAC on eslicarbazepine acetate hydrolysis were examined by using the constructed recombinant AADAC variants with T74A, V172I, R248S, V281I, N366K, or X400Q. AADAC variants with R248S or X400Q showed lower activity than wild type (5% or 21%, respectively), whereas those with V172I showed higher activity than wild type (174%). Similar tendencies were observed in the other four substrates of AADAC; that is, p-nitrophenyl acetate, ketoconazole, phenacetin, and rifampicin. Collectively, we found that eslicarbazepine acetate is specifically and efficiently hydrolyzed by human AADAC, and several AADAC polymorphic alleles would be a factor affecting the enzyme activity and drug response. SIGNIFICANCE STATEMENT: This is the first study to clarify that arylacetamide deacetylase (AADAC) is responsible for the activation of eslicarbazepine acetate, an antiepileptic prodrug, to eslicarbazepine, an active form, in the human liver and intestines. In addition, we found that several AADAC polymorphic alleles would be a factor affecting the enzyme activity and drug response.

Strain and sex differences in drug hydrolase activities in rodent livers

Carboxylesterase (CES) 1, CES2, and arylacetamide deacetylase (AADAC) are the major drug hydrolases in humans, and they have different substrate preferences. Because rodents are widely used in preclinical studies, we aimed to clarify the extent of the species, strain, and sex differences in hydrolase activity in rats and mice. Hydrolase activities for 24 compounds were evaluated in Fischer 344, Sprague-Dawley, and Wistar-Imamichi rat liver microsomes (RLM) and Balb/c, C3H/He, C57BL/6J, and ddY mouse liver microsomes (MLM) by comparing the results with the activities in human liver microsomes (HLM). Imidapril hydrolase activities in RLM from all strains were substantially higher than those in MLM and HLM, whereas oseltamivir was hardly hydrolyzed in rodents, although both are specific substrates of CES1 in humans. In rats, males tended to show higher hydrolase activities for most human CES1 substrates than females. Hydrolase activities for irinotecan and procaine, which are CES2 substrates in humans, tended to be higher in RLM and MLM than in HLM. Rifamycins, substrates of human AADAC, were not hydrolyzed in RLM and MLM. The results of this study provide important information about the species, strain, and sex differences in hydrolase activities in rats and mice.

Arylacetamide Deacetylase Enzyme: Presence and Interindividual Variability in Human Lungs.

Human arylacetamide deacetylase (AADAC) is a single microsomal serine esterase involved in the hydrolysis of many acetyl-containing drugs. To date, the presence and activity of the AADAC enzyme in human lungs has been scarcely examined. We investigated its gene and protein expression as well as interindividual variations in AADAC activities in a large number of human lungs (n = 25) using phenacetin as a selective substrate. The kinetic parameters K m and V max were determined. Our findings highlighted a high interindividual variability in both AADAC mRNA levels and hydrolysis activities. Furthermore, for the first time we demonstrated the presence of the AADAC protein in various lung samples by means of immunoblot analysis. As a comparison, phenacetin hydrolysis was detected in pooled human liver microsomes. Lung activities were much lower than those found in the liver. However, similar K m values were found, which suggests that this hydrolysis could be due to the same enzyme. Pulmonary phenacetin hydrolysis proved to be positively correlated with AADAC mRNA (*P < 0.05) and protein (*P < 0.05) levels. Moreover, the average values of AADAC activity in smokers was significantly higher than in nonsmoker subjects (*P < 0.05), and this might have an important role in the administration of some drugs. These findings add more information to our knowledge of pulmonary enzymes and could be particularly useful in the design and preclinical development of inhaled drugs. SIGNIFICANCE STATEMENT: This study investigated the presence and activity of the AADAC enzyme in several human lungs. Our results highlight high interindividual variability in both AADAC gene and protein expression as well as in phenacetin hydrolysis activity. These findings add more information to our knowledge of pulmonary enzymes and could be particularly useful in the design and preclinical development of inhaled drugs.

Arylacetamide Deacetylase Is Involved in Vicagrel Bioactivation in Humans.

Vicagrel, a structural analog of clopidogrel, is now being developed as a thienopyridine antiplatelet agent in a phase II clinical trial in China. Some studies have shown that vicagrel undergoes complete first-pass metabolism in human intestine, generating the hydrolytic metabolite 2-oxo-clopidogrel via carboxylesterase-2 (CES2) and subsequently the active metabolite H4 via CYP450s. This study aimed to identify hydrolases other than CES2 that are involved in the bioactivation of vicagrel in human intestine. This study is the first to determine that human arylacetamide deacetylase (AADAC) is involved in 2-oxo-clopidogrel production from vicagrel in human intestine. In vitro hydrolytic kinetics were determined in human intestine microsomes and recombinant human CES and AADAC. The calculated contribution of CES2 and AADAC to vicagrel hydrolysis was 44.2 and 53.1% in human intestine, respectively. The AADAC-selective inhibitors vinblastine and eserine effectively inhibited vicagrel hydrolysis in vitro. In addition to CES2, human intestine AADAC was involved in vicagrel hydrolytic activation before it entered systemic circulation. In addition, simvastatin efficiently inhibited the production of both 2-oxo-clopidogrel and active H4; further clinical trials are needed to determine whether the hydrolytic activation of vicagrel is influenced by coadministration with simvastatin. This study deepens the understanding of the bioactivation and metabolism properties of vicagrel in humans, which can help further understand the bioactivation mechanism of vicagrel and the variations in the treatment responses to vicagrel and clopidogrel.

Difference in substrate specificity of carboxylesterase and arylacetamide deacetylase between dogs and humans

Carboxylesterase (CES) and arylacetamide deacetylase (AADAC) are the major enzymes responsible for the hydrolysis of various clinical drugs. Our recent study demonstrated that the identity of the responsible hydrolase can be roughly surmised based on the chemical structures of compounds in humans. Dogs are used for preclinical studies in drug development, but the substrate specificities of dog CES and AADAC remain to be clarified. The purpose of this study is to characterize their substrate specificities. We prepared recombinant dog CES1, CES2, and AADAC. p-Nitrophenyl acetate, a general substrate for esterases, was hydrolyzed by dog CES1 and AADAC, while it was not hydrolyzed by CES2. CES2 protein was not substantially detected in the recombinant system or in the dog liver and intestinal microsomes by Western blot using anti-human CES2 antibodies. In silico analyses demonstrated slight differences in the three-dimensional structures of dog CES2 and human CES2, indicating that dog CES2 might be unstable or inactive. By evaluating the hydrolase activities of 22 compounds, which are known to be substrates of human CES and/or AADAC, we found that the activities of dog recombinant CES1 and AADAC as well as dog tissue preparations for nearly all compounds were lower than those of human enzymes. The dog enzymes that were responsible for the hydrolysis of most compounds corresponded to the human enzymes, but the following differences were observed: oseltamivir, irinotecan, and rifampicin were not hydrolyzed in the dog liver or by any of the recombinant esterases and procaine, a human CES2 substrate, was hydrolyzed by dog CES1. In conclusion, the present study could provide new finding to facilitate our understanding of species differences in drug hydrolysis, which can facilitate drug development and drug safety evaluation.

Human arylacetamide deacetylase hydrolyzes ketoconazole to trigger hepatocellular toxicity

Ketoconazole (KC), an antifungal agent, rarely causes severe liver injury when orally administered. It has been reported that KC is mainly hydrolyzed to N-deacetyl ketoconazole (DAK), followed by the N-hydroxylation of DAK by flavin-containing monooxygenase (FMO). Although the metabolism of KC has been considered to be associated with hepatotoxicity, the responsible enzyme(s) remain unknown. The purpose of this study was to identify the responsible enzyme(s) for KC hydrolysis in humans and to clarify their relevance to KC-induced toxicity. Kinetic analysis and inhibition studies using human liver microsomes (HLM) and recombinant enzymes revealed that human arylacetamide deacetylase (AADAC) is responsible for KC hydrolysis to form DAK, and confirmed that FMO3 is the enzyme responsible for DAK N-hydroxylation. In HLM, the clearance of KC hydrolysis occurred to the same extent as DAK N-hydroxylation, which indicates that both processes are not rate-limiting pathways. Cytotoxicity of KC and DAK was evaluated using HepaRG cells and human primary hepatocytes. Treatment of HepaRG cells with DAK for 24h showed cytotoxicity in a dose-dependent manner, whereas treatment with KC did not show due to the low expression of AADAC. Overexpression of AADAC in HepaRG cells with an adenovirus expression system elicited the cytotoxicity of KC. Cytotoxicity of KC in human primary hepatocytes was attenuated by diisopropylfluorophosphate, an AADAC inhibitor. In conclusion, the present study demonstrated that human AADAC hydrolyzes KC to trigger hepatocellular toxicity.

Arylacetamide Deacetylase is Responsible for Activation of Prasugrel in Human and Dog.

Prasugrel, a thienopyridine anti-platelet agent, is pharmacologically activated by hydrolysis and hydroxylation. It is efficiently hydrolyzed in the intestine after oral administration, and the enzyme responsible for the hydrolysis in humans was demonstrated to be carboxylesterase (CES)2. Prasugrel hydrolase activity is detected in dog intestines, where CES enzymes are absent; therefore, this prompted us to investigate the involvement of an enzyme(s) other than CES. Human arylacetamide deacetylase (AADAC) is highly expressed in the small intestine, catalyzing the hydrolysis of several clinical drugs containing small acyl moieties. In the present study, we investigated whether AADAC catalyzes prasugrel hydrolysis. Recombinant human AADAC was shown to catalyze prasugrel hydrolysis with a CLint value of 50.0 ± 1.2 ml/min/mg protein with a similar Km value to human intestinal and liver microsomes, whereas the CLint values of human CES1 and CES2 were 4.6 ± 0.1 and 6.6 ± 0.3 ml/min/mg protein, respectively. Inhibition studies using various chemical inhibitors and the relative activity factor approach suggested that the contribution of AADAC to prasugrel hydrolysis in human intestine is comparable to that of CES2. In dog intestine, the expression of AADAC, but not CES1 and CES2, was confirmed by measuring the marker hydrolase activities of each human esterase. The similar Km values and inhibition profiles between recombinant dog AADAC and small intestinal microsomes suggest that AADAC is a major enzyme responsible for prasugrel hydrolysis in dog intestine. Collectively, we found that AADAC largely contributes to prasugrel hydrolysis in both human and dog intestine.

Comparison of substrate specificity among human arylacetamide deacetylase and carboxylesterases

Human arylacetamide deacetylase (AADAC) is an esterase responsible for the hydrolysis of some drugs, including flutamide, indiplon, phenacetin, and rifamycins. AADAC is highly expressed in the human liver, where carboxylesterase (CES) enzymes, namely, CES1 and CES2, are also expressed. It is generally recognized that CES1 prefers compounds with a large acyl moiety and a small alcohol or amine moiety as substrates, whereas CES2 prefers compounds with a small acyl moiety and a large alcohol or amine moiety. In a comparison of the chemical structures of known AADAC substrates, AADAC most likely prefers compounds with the same characteristics as does CES2. However, the substrate specificity of human AADAC has not been fully clarified. To expand the knowledge of substrates of human AADAC, we measured its hydrolase activities toward 13 compounds, including known human CES1 and CES2 substrates, using recombinant enzymes expressed in Sf21 cells. Recombinant AADAC catalyzed the hydrolysis of fluorescein diacetate, N-monoacetyldapsone, and propanil, which possess notably small acyl moieties, and these substrates were also hydrolyzed by CES2. However, AADAC could not hydrolyze another CES2 substrate, procaine, which possesses a moderately small acyl moiety. In addition, AADAC did not hydrolyze several known CES1 substrates, including clopidogrel and oseltamivir, which have large acyl moieties and small alcohol moieties. Collectively, these results suggest that AADAC prefers compounds with smaller acyl moieties than does CES2. The role of AADAC in the hydrolysis of drugs has been clarified. For this reason, AADAC should receive attention in ADMET studies during drug development.

Isolation and characterization of arylacetamide deacetylase in cynomolgus macaques.

Arylacetamide deacetylase (AADAC), a microsomal serine esterase, hydrolyzes drugs, such as flutamide, phenacetin and rifampicin. Because AADAC has not been fully investigated at molecular levels in cynomolgus macaques, the non-human primate species widely used in drug metabolism studies, cynomolgus AADAC cDNA was isolated and characterized. The deduced amino acid sequence, highly homologous (92%) to human AADAC, was more closely clustered with human AADAC than the dog, rat or mouse ortholog in a phylogenetic tree. AADAC was flanked by AADACL2 and SUCNR1 in the cynomolgus and human genomes. Moreover, relatively abundant expression of AADAC mRNA was found in liver and jejunum, the drug-metabolizing organs, in cynomolgus macaques, similar to humans. The results suggest molecular similarities of AADAC between cynomolgus macaques and humans.

Screening of specific inhibitors for human carboxylesterases or arylacetamide deacetylase.

Esterases catalyze the hydrolysis of therapeutic drugs containing esters or amides in their structures. Human carboxylesterase (CES) and arylacetamide deacetylase (AADAC) are the major enzymes that catalyze the hydrolysis of drugs in the liver. Characterization of the enzyme(s) responsible for drug metabolism is required in drug development and to realize optimal drug therapy. Because multiple enzymes may show a metabolic potency for a given compound, inhibition studies using chemical inhibitors are useful tools to determine the contribution of each enzyme in human tissue preparations. The purpose of this study was to find specific inhibitors for human CES1, CES2, and AADAC. We screened 542 chemicals for the inhibition potency toward hydrolase activities of p-nitrophenyl acetate by recombinant CES1, CES2, and AADAC. We found that digitonin and telmisartan specifically inhibited CES1 and CES2 enzyme activity, respectively. Vinblastine potently inhibited both AADAC and CES2, but no specific inhibitor of AADAC was found. The inhibitory potency and specificity of these compounds were also evaluated by monitoring the effects on hydrolase activity of probe compounds of each enzyme (CES1: lidocaine, CES2: CPT-11, AADAC: phenacetin) in human liver microsomes. Telmisartan and vinblastine strongly inhibited the hydrolysis of CPT-11 and/or phenacetin, but digitonin did not strongly inhibit the hydrolysis of lidocaine, indicating that the inhibitory potency of digitonin was different between recombinant CES1 and liver microsomes. Although we could not find a specific inhibitor of AADAC, the combined use of telmisartan and vinblastine could predict the responsibility of human AADAC to drug hydrolysis.

Indiplon Is Hydrolyzed by Arylacetamide Deacetylase in Human Liver

Human arylacetamide deacetylase (AADAC) catalyzes the hydrolysis of some clinically used drugs, but the information available on its substrates is limited. To increase our knowledge of the AADAC substrates, we examined whether AADAC catalyzes the hydrolysis of indiplon, which was initially developed as a hypnotic sedative drug. It has been reported that approximately 30-40% of the administered indiplon was hydrolyzed to deacetylindiplon in humans, but the enzyme responsible for this hydrolysis had not been identified. We detected high levels of indiplon hydrolase activity in human liver microsomes (HLMs), but the levels found in human liver cytosol and plasma were scarcely detectable. Recombinant AADAC showed a high level of indiplon hydrolase activity, whereas recombinant carboxylesterase 1 (CES1) and 2 (CES2) showed marginal activity. The indiplon hydrolase activity of HLM was potently inhibited by vinblastine, a potent inhibitor of AADAC and CES2, but it was not inhibited by digitonin and telmisartan, inhibitors of CES1 and CES2, respectively. In a panel of 24 individual HLM samples, the indiplon hydrolase activities were significantly correlated with the hydrolase activities of flutamide, phenacetin, and rifampicin, which are known AADAC substrates. An HLM sample with a homozygous AADAC*3 allele, which was previously found to exhibit decreased enzyme activity, showed the lowest indiplon hydrolase activity among the 24 tested samples. Collectively, we found that human AADAC is responsible for the hydrolysis of indiplon. Thus, we can add indiplon to the list of human AADAC substrates.

N-Glycosylation during translation is essential for human arylacetamide deacetylase enzyme activity

Human arylacetamide deacetylase (AADAC) can hydrolyze clinical drugs such as flutamide, phenacetin, and rifamycins. AADAC is a glycoprotein, but the role of glycosylation remains unclear. In the present study, we investigated the effect of glycosylation on AADAC enzyme activity. Immunoblot analysis of mutant AADACs that contained an asparagine (N, Asn) to glutamine (Q, Gln) substitution at either residue 78 or 282 (N78Q or N282Q) showed a different migration compared with the wild-type protein. A mutant AADAC that contained N to Q substitutions at both residue 78 and 282 (N78Q/N282Q) showed a similar migration to AADAC in human liver microsomes (HLM) treated with endoglycosidase H (Endo H), which produces deglycosylated proteins. This result indicated that AADAC was glycosylated at both N78 and N282. Mutant types of AADAC with the N282Q and the N78Q/N282Q substitutions showed dramatically lower phenacetin hydrolase activity than did the wild-type protein. The treatment of wild-type AADAC-expressing HuH-7 cells with tunicamycin, which produces unglycosylated protein, decreased AADAC enzyme activity. However, the treatment of the HLM with Endo H caused no decrease of AADAC activity. Thus, the oligosaccharide chain, per se, was not important for AADAC activity in the mature form. The mutant types of AADAC containing the N282Q and the N78Q/N282Q substitutions were not detected by immunoblotting analysis after non-reducing SDS-PAGE, suggesting that the glycosylation of AADAC at N282 was important for proper protein folding. Overall, this study found that the translational, but not post-translational, N-glycosylation of AADAC plays a crucial role in regulating AADAC enzyme activity.

Metabolic activation by human arylacetamide deacetylase, CYP2E1, and CYP1A2 causes phenacetin-induced methemoglobinemia

Phenacetin has been used as an analgesic antipyretic but has now been withdrawn from the market due to adverse effects such as methemoglobinemia and renal failure. It has been suggested that metabolic activation causes these adverse effects; yet, the precise mechanisms remain unknown. We previously demonstrated that human arylacetamide deacetylase (AADAC) was the principal enzyme catalyzing the hydrolysis of phenacetin. In this study, we assessed whether AADAC was involved in phenacetin-induced methemoglobinemia. A high methemoglobin (Met-Hb) level in the blood was detected 1h after administration of phenacetin (250mg/kg, p.o.) to male C57BL/6 mice. Pre-administration of tri-o-tolylphosphate, a general esterase inhibitor, was found to decrease the levels of Met-Hb and the plasma concentration of p-phenetidine, a hydrolyzed metabolite of phenacetin. An in vitro study using red blood cells revealed that incubation of phenacetin or p-phenetidine with human liver microsomes (HLM) increased the formation of Met-Hb. To identify the enzymes involved in the formation of Met-Hb, we used recombinant enzymes and HLM treated with inhibitors in the measurement of the formation of Met-Hb. High levels of Met-Hb were observed following incubation of human AADAC with either cytochrome P450 (CYP) 1A2 or CYP2E1. Furthermore, the increased Met-Hb formation by the incubation of HLM with phenacetin was significantly inhibited to 25.1±0.7% of control by eserine, a potent AADAC inhibitor. In conclusion, we found that the hydrolysis by AADAC and subsequent metabolism by CYP1A2 and CYP2E1 play predominant roles in phenacetin-induced methemoglobinemia.