Enzyme Kinetics, Pharmacokinetics, and Inhibition of Aldehyde Oxidase.

Aldehyde oxidase (AO) has emerged as an important drug metabolizing enzyme over the last decade. Several compounds have failed in the clinic because the clearance or toxicity was underestimated by preclinical species. Human AO is much more active than rodent AO, and dogs do not have functional AO. Metabolic products from AO-catalyzed oxidation are generally nonreactive and often they have much lower solubility. AO metabolism is not limited to oxidation as AO can also catalyze reduction of oxygen and nitrite. Reduction of oxygen leads to the reactive oxygen species (ROS) superoxide radical anion and hydrogen peroxide. Reduction of nitrite leads to the formation of nitric oxide with potential pharmacological implications. AO is also reported to catalyze the reductive metabolism of nitro-compounds, N-oxides, sulfoxides, isoxazoles, isothiazoles, nitrite, and hydroxamic acids. These reductive transformations may cause toxicity due to the formation of reactive metabolites. Moreover, the inhibition kinetics are complex, and multiple probe substrates should be used when assessing the potential for DDIs. Finally, AO appears to be amenable to computational predictions of both regioselectivity and rates of reaction, which holds promise for virtual screening.

Case Study 1: Practical Considerations with Experimental Design and Interpretation.

This chapter deals with practical considerations on key issues such as choosing an enzyme source, determining linear conditions, and choosing appropriate substrate and organic solvent concentrations. Practical solutions for working with limited resources and carrying out inhibition experiments are also addressed. Thus, after reading this chapter, the novice reader should have a better idea of how to design, develop, and interpret basic experiments using drug metabolism enzymes.

Strain-Dependent Variability of Early Discovery Small Molecule Pharmacokinetics in Mice: Does Strain Matter?

Drug discovery programs routinely perform pharmacokinetic (PK) studies in mice to prioritize lead compounds based on anticipated exposure-efficacy and exposure-toxicity relationships. Because of logistical and/or technical issues, the strain of mouse in early discovery PK studies may not always match the strain in toxicity or efficacy studies. This elicits the question do appreciable strain-dependent differences in PK parameters exist to an extent that would warrant conducting PK studies in a strain that matches efficacy and toxicity models? To understand the impact that strain may have on PK parameters, we selected eight marketed drugs with well characterized absorption, distribution, metabolism, and excretion properties and diverse structures to perform PK studies in three common mouse strains (Bagg Albino c, C57BL/6, and CD-1). Some statistical strain-dependent differences were observed; however, we found good general agreement of PK parameters between strains: 88%, 100%, 75%, 76%, 94%, and 88% of compounds were within twofold across strains for clearance, volume of distribution at steady state, t 1/2, C max, T max, and oral bioavailability, respectively. Overall, we recommend that an approach using a single strain of mouse is appropriate for discovery screening PK studies, provided that proper caution is exercised. SIGNIFICANCE STATEMENT: The mouse strain in discovery pharmacokinetic (PK) studies may not match the strain in efficacy and toxicology studies. Currently, there is a gap in the literature addressing whether differences in PK parameters across mouse strains exist such that multiple PK studies are warranted. The results from this study indicated that the PK properties of clinically used drugs between mouse strains are within an acceptable range such that single strain PK is appropriate.

Mechanistic Studies of Cytochrome P450 3A4 Time-Dependent Inhibition Using Two Cysteine-Targeting Electrophiles.

Experiments designed to identify the mechanism of cytochrome P450 inactivation are critical to drug discovery. Small molecules irreversibly inhibit P450 enzymatic activity via two primary mechanisms: apoprotein adduct formation or heme modification. Understanding the interplay between chemical structures of reactive electrophiles and the impact on CYP3A4 structure and function can ultimately provide insights into drug design to minimize P450 inactivation. In a previous study, raloxifene and N-(1-pyrene) iodoacetamide (PIA) alkylated CYP3A4 in vitro; however, only raloxifene influenced enzyme activity. Here, two alkylating agents with cysteine selectivity, PIA and pyrene maleimide (PM), were used to investigate this apparent compound-dependent disconnect between CYP3A4 protein alkylation and activity loss. The compound's effect on 1) enzymatic activity, 2) carbon monoxide (CO) binding capacity, 3) intact heme content, and 4) protein conformation were measured. Results showed that PM had a large time-dependent loss of enzyme activity, whereas PIA did not. The differential effect on enzymatic activity between PM and PIA was mirrored in the CO binding data. Despite disruption of CO binding, neither compound affected the heme concentrations, inferring there was no destruction or alkylation of the heme. Lastly, differential scanning fluorescence showed PM-treated CYP3A4 caused a shift in the onset temperature required to induce protein aggregation, which was not observed for CYP3A4 treated with PIA. In conclusion, alkylation of CYP3A4 apoprotein can have a variable impact on catalytic activity, CO binding, and protein conformation that may be compound-dependent. These results highlight the need for careful interpretation of experimental results aimed at characterizing the nature of P450 enzyme inactivation. SIGNIFICANCE STATEMENT: Understanding the mechanism of CYP3A4 time-dependent inhibition is critical to drug discovery. In this study, we use two cysteine-targeting electrophiles to probe how subtle variation in inhibitor structure may impact the mechanism of CYP3A4 time-dependent inhibition and confound interpretation of traditional diagnostic experiments. Ultimately, this simplified system was used to reveal insights into CYP3A4 biochemical behavior. The insights may have implications that aid in understanding the susceptibility of CYP enzymes to the effects of electrophilic intermediates generated via bioactivation.

Fraction Unbound for Liver Microsome and Hepatocyte Incubations for All Major Species Can Be Approximated Using a Single-Species Surrogate.

It is well recognized that nonspecific binding of a drug within an in vitro assay (f u) can have a large impact on in vitro to in vivo correlations of intrinsic clearance. Typically, this value is determined experimentally across multiple species in the drug-discovery stage. Herein we examine the feasibility of using a single species (rat) as a surrogate for other species using a panel of small molecules representing highly diverse structures and physiochemical classes. The study demonstrated that 86% and 92% of the tested compounds measured in the mouse, dog, monkey, and human were within 2-fold of rat values for f u in microsomes and hepatocytes, respectively. One compound, amiodarone, exhibited unique species-dependent binding where the f u was approximately 10-fold higher in human microsomes and 20-fold higher in human hepatocytes compared with the average of the other species tested. Overall, these data indicate that using a single species (rat) f u as a surrogate for other major species, including humans, is a means to increase the throughput of measuring nonspecific binding in vitro.

A novel reaction mediated by human aldehyde oxidase: amide hydrolysis of GDC-0834.

GDC-0834, a Bruton's tyrosine kinase inhibitor investigated as a potential treatment of rheumatoid arthritis, was previously reported to be extensively metabolized by amide hydrolysis such that no measurable levels of this compound were detected in human circulation after oral administration. In vitro studies in human liver cytosol determined that GDC-0834 (R)-N-(3-(6-(4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenylamino)-4-methyl-5-oxo- 4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b] thiophene-2-carboxamide) was rapidly hydrolyzed with a CLint of 0.511 ml/min per milligram of protein. Aldehyde oxidase (AO) and carboxylesterase (CES) were putatively identified as the enzymes responsible after cytosolic fractionation and mass spectrometry-proteomics analysis of the enzymatically active fractions. Results were confirmed by a series of kinetic experiments with inhibitors of AO, CES, and xanthine oxidase (XO), which implicated AO and CES, but not XO, as mediating GDC-0834 amide hydrolysis. Further supporting the interaction between GDC-0834 and AO, GDC-0834 was shown to be a potent reversible inhibitor of six known AO substrates with IC50 values ranging from 0.86 to 1.87 µM. Additionally, in silico modeling studies suggest that GDC-0834 is capable of binding in the active site of AO with the amide bond of GDC-0834 near the molybdenum cofactor (MoCo), orientated in such a way to enable potential nucleophilic attack on the carbonyl of the amide bond by the hydroxyl of MoCo. Together, the in vitro and in silico results suggest the involvement of AO in the amide hydrolysis of GDC-0834.

Inhibition of human aldehyde oxidase activity by diet-derived constituents: structural influence, enzyme-ligand interactions, and clinical relevance.

The mechanistic understanding of interactions between diet-derived substances and conventional medications in humans is nascent. Most investigations have examined cytochrome P450-mediated interactions. Interactions mediated by other phase I enzymes are understudied. Aldehyde oxidase (AO) is a phase I hydroxylase that is gaining recognition in drug design and development programs. Taken together, a panel of structurally diverse phytoconstituents (n = 24) was screened for inhibitors of the AO-mediated oxidation of the probe substrate O(6)-benzylguanine. Based on the estimated IC50 (<100 µM), 17 constituents were advanced for Ki determination. Three constituents were described best by a competitive inhibition model, whereas 14 constituents were described best by a mixed-mode model. The latter model consists of two Ki terms, Kis and Kii, which ranged from 0.26-73 and 0.80-120 µM, respectively. Molecular modeling was used to glean mechanistic insight into AO inhibition. Docking studies indicated that the tested constituents bound within the AO active site and elucidated key enzyme-inhibitor interactions. Quantitative structure-activity relationship modeling identified three structural descriptors that correlated with inhibition potency (r(2) = 0.85), providing a framework for developing in silico models to predict the AO inhibitory activity of a xenobiotic based solely on chemical structure. Finally, a simple static model was used to assess potential clinically relevant AO-mediated dietary substance-drug interactions. Epicatechin gallate and epigallocatechin gallate, prominent constituents in green tea, were predicted to have moderate to high risk. Further characterization of this uncharted type of interaction is warranted, including dynamic modeling and, potentially, clinical evaluation.

Determination of the Structure and Catalytic Mechanism of Sorghum bicolor Caffeic Acid O-Methyltransferase and the Structural Impact of Three brown midrib12 Mutations.

Using S-adenosyl-methionine as the methyl donor, caffeic acid O-methyltransferase from sorghum (Sorghum bicolor; SbCOMT) methylates the 5-hydroxyl group of its preferred substrate, 5-hydroxyconiferaldehyde. In order to determine the mechanism of SbCOMT and understand the observed reduction in the lignin syringyl-to-guaiacyl ratio of three brown midrib12 mutants that carry COMT gene missense mutations, we determined the apo-form and S-adenosyl-methionine binary complex SbCOMT crystal structures and established the ternary complex structure with 5-hydroxyconiferaldehyde by molecular modeling. These structures revealed many features shared with monocot ryegrass (Lolium perenne) and dicot alfalfa (Medicago sativa) COMTs. SbCOMT steady-state kinetic and calorimetric data suggest a random bi-bi mechanism. Based on our structural, kinetic, and thermodynamic results, we propose that the observed reactivity hierarchy among 4,5-dihydroxy-3-methoxycinnamyl (and 3,4-dihydroxycinnamyl) aldehyde, alcohol, and acid substrates arises from the ability of the aldehyde to stabilize the anionic intermediate that results from deprotonation of the 5-hydroxyl group by histidine-267. Additionally, despite the presence of other phenylpropanoid substrates in vivo, sinapaldehyde is the preferential product, as demonstrated by its low Km for 5-hydroxyconiferaldehyde. Unlike its acid and alcohol substrates, the aldehydes exhibit product inhibition, and we propose that this is due to nonproductive binding of the S-cis-form of the aldehydes inhibiting productive binding of the S-trans-form. The S-cis-aldehydes most likely act only as inhibitors, because the high rotational energy barrier around the 2-propenyl bond prevents S-trans-conversion, unlike alcohol substrates, whose low 2-propenyl bond rotational energy barrier enables rapid S-cis/S-trans-interconversion.

Enzyme kinetics, inhibition, and regioselectivity of aldehyde oxidase.

The aldehyde oxidase (AO) enzyme family plays an increasing role in drug development. However, a number of compounds that are AO substrates have failed in the clinic because the clearance or toxicity is underestimated by preclinical species. Human AO is much more active than rodent AO, and dogs do not have functional AO. While AOs normally make non-reactive metabolites such as lactams, the metabolic products often have much lower solubility that can lead to renal failure. While an endogenous substrate for the oxidation reaction is not known, electron acceptors for the reductive part of the reaction include oxygen and nitrites. Reduction of oxygen leads to the reactive oxygen species (ROS) superoxide radical anion, and hydrogen peroxide. Reduction of nitrite leads to the formation of nitric oxide with potential pharmacological implications. To date, no clinically important drug-drug interactions (DDIs) have been observed for AOs. However, the inhibition kinetics are complex, and multiple probe substrates should be used when assessing the potential for DDIs. Finally, AO appears to be amenable to computational predictions of both regioselectivity and rates of reaction, which holds promise for virtual screening.

Case study 1. Practical considerations with experimental design and interpretation.

At some point, anyone with knowledge of drug metabolism and enzyme kinetics started out knowing little about these topics. This chapter was specifically written with the novice in mind. Regardless of the enzyme one is working with or the goal of the experiment itself, there are fundamental components and concepts of every experiment using drug metabolism enzymes. The following case studies provide practical tips, techniques, and answers to questions that may arise in the course of conducting such experiments. Issues ranging from assay design and development to data interpretation are addressed. The goal of this section is to act as a starting point to provide the reader with key questions and guidance while attempting his/her own work.

Why do most human liver cytosol preparations lack xanthine oxidase activity?

When investigating the potential for xanthine oxidase (XO)-mediated metabolism of a new chemical entity in vitro, selective chemical inhibition experiments are typically used. Most commonly, these inhibition experiments are performed using the inhibitor allopurinol (AP) and commercially prepared human liver cytosol (HLC) as the enzyme source. For reasons detailed herein, it is also a common practice to perfuse livers with solutions containing AP prior to liver harvest. The exposure to AP in HLC preparations could obviously pose a problem for measuring in vitro XO activity. To investigate this potential problem, an HPLC-MS/MS assay was developed to determine whether AP and its primary metabolite, oxypurinol, are retained within the cytosol for livers that were treated with AP during liver harvest. Differences in enzymatic activity for XO and aldehyde oxidase (AO) in human cytosol that can be ascribed to AP exposure were also evaluated. The results confirmed the presence of residual AP (some) and oxypurinol (all) human liver cytosol preparations that had been perfused with an AP-containing solution. In every case where oxypurinol was detected, XO activity was not observed. In contrast, the presence of AP and oxypurinol did not appear to have an impact on AO activity. Pooled HLC that was purchased from a commercial source also contained residual oxypurinol and did not show any XO activity. In the future, it is recommended that each HLC batch is screened for oxypurinol and/or XO activity prior to testing for XO-mediated metabolism of a new chemical entity.

Absolute quantification of aldehyde oxidase protein in human liver using liquid chromatography-tandem mass spectrometry.

The function of the enzyme human aldehyde oxidase (AOX1) is uncertain; however, recent studies have implicated significant biochemical involvement in humans. AOX1 has also rapidly become an important drug-metabolizing enzyme. Until now, quantitation of AOX1 in complex matrices such as tissue has not been achieved. Herein, we developed and employed a trypsin digest and subsequent liquid chromatography-tandem mass spectrometry analysis to determine absolute amounts of AOX1 in human liver. E. coli expressed human purified AOX1 was used to validate the linearity, sensitivity, and selectivity of the method. Overall, the method is highly efficient and sensitive for determination of AOX1 in cytosolic liver fractions. Using this method, we observed substantial batch-to-batch variation in AOX1 content (21-40 pmol AOX1/mg total protein) between various pooled human liver cytosol preparations. We also observed interbatch variation in Vmax (3.3-4.9 nmol min(-1) mg(-1)) and a modest correlation between enzyme concentration and activity. In addition, we measured a large difference in kcat/Km, between purified (kcat/Km of 1.4) and human liver cytosol (kcat/Km of 15-20) indicating cytosol to be 11-14 times more efficient in the turnover of DACA than the E. coli expressed purified enzyme. Finally, we discussed the future impact of this method for the development of drug metabolism models and understanding the biochemical role of this enzyme.

Evaluation of rhesus monkey and guinea pig hepatic cytosol fractions as models for human aldehyde oxidase.

Aldehyde oxidase (AOX) is a cytosolic enzyme expressed across a wide range of species, including guinea pig and rhesus monkey. These species are believed to be the best preclinical models for studying human AOX-mediated metabolism. We compared AOX activity in rhesus monkeys, guinea pigs, and humans using phthalazine and N-[2-(dimethylamino)ethyl]acridone-4-carboxamide (DACA) as substrates and raloxifene as an inhibitor. Michaelis-Menten kinetics was observed for phthalazine oxidation in rhesus monkey, guinea pig, and human liver cytosol, whereas substrate inhibition was seen with DACA oxidase activity in all three livers. Raloxifene inhibited phthalazine and DACA oxidase activity uncompetitively in guinea pig, whereas mixed-mode inhibition was seen in rhesus monkey. Our analysis of the primary sequence alignment of rhesus monkey, guinea pig, and human aldehyde oxidase isoform 1 (AOX1) along with homology modeling has led to the identification of several amino acid residue differences within the active site and substrate entrance channel of AOX1. We speculate that some of these residues might be responsible for the differences observed in activity. Overall, our data indicate that rhesus monkeys and guinea pigs would overestimate intrinsic clearance in humans and would be unsuitable to use as animal models. Our study also showed that AOX metabolism in species is substrate-dependent and no single animal model can be reliably used to predict every drug response in humans.

Potential role of cardiac calsequestrin in the lethal arrhythmic effects of cocaine.

BACKGROUND: Cocaine-related deaths are continuously rising and its overdose is often associated with lethal cardiotoxic effects. METHODS AND RESULTS: Our approach, employing isothermal titration calorimetry (ITC) and light scattering in parallel, has confirmed the significant affinity of human cardiac calsequestrin (CASQ2) for cocaine. Calsequestrin (CASQ) is a major Ca(2+)-storage protein within the sarcoplasmic reticulum (SR) of both cardiac and skeletal muscles. CASQ acts as a Ca(2+) buffer and Ca(2+)-channel regulator through its unique Ca(2+)-dependent oligomerization. Equilibrium dialysis and atomic absorption spectroscopy experiments illustrated the perturbational effect of cocaine on CASQ2 polymerization, resulting in substantial reduction of its Ca(2+)-binding capacity. We also confirmed the accumulation of cocaine in rat heart tissue and the substantial effects cocaine has on cultured C2C12 cells. The same experiments were performed with methamphetamine as a control, which displayed neither affinity for CASQ2 nor any significant effects on its function. Since cocaine did not have any direct effect on the Ca(2+)-release channel judging from our single channel recordings, these studies provide new insights into how cocaine may interfere with the normal E-C coupling mechanism with lethal arrhythmogenic consequences. CONCLUSION: We propose that cocaine accumulates in SR through its affinity for CASQ2 and affects both SR Ca(2+) storage and release by altering the normal CASQ2 Ca(2+)-dependent polymerization. By this mechanism, cocaine use could produce serious cardiac problems, especially in people who have genetically-impaired CASQ2, defects in other E-C coupling components, or compromised cocaine metabolism and clearance.

Evidence for substrate-dependent inhibition profiles for human liver aldehyde oxidase.

The goal of this study was to provide a reasonable assessment of how probe substrate selection may impact the results of in vitro aldehyde oxidase (AO) inhibition experiments. Here, we used a previously studied set of seven known AO inhibitors to probe the inhibition profile of a pharmacologically relevant substrate N-[(2-dimethylamino)ethyl]acridine-4-carboxamide (DACA). DACA oxidation in human liver cytosol was characterized with a measured V(max) of 2.3 ± 0.08 nmol product · min(-1) · mg(-1) and a K(m) of 6.3 ± 0.8 µM. The K(ii) and K(is) values describing the inhibition of DACA oxidation by the panel of seven inhibitors were tabulated and compared with previous findings with phthalazine as the substrate. In every case, the inhibition profile shifted to a much less uncompetitive mode of inhibition for DACA relative to phthalazine. With the exception of one inhibitor, raloxifene, this change in inhibition profile seems to be a result of a decrease in the uncompetitive mode of inhibition (an affected K(ii) value), whereas the competitive mode (K(is)) seems to be relatively consistent between substrates. Raloxifene was found to inhibit competitively when using DACA as a probe, and a previous report showed that raloxifene inhibited uncompetitively with other substrates. The relevance of these data to the mechanistic understanding of aldehyde oxidase inhibition and potential implications on drug-drug interactions is discussed. Overall, it appears that the choice in substrate may be critical when conducting mechanistic inhibition or in vitro drug-drug interactions prediction studies with AO.

Inhibition of human liver aldehyde oxidase: implications for potential drug-drug interactions.

During the course of our research efforts to understand the kinetics of human aldehyde oxidase as a xenobiotic-clearing enzyme, we investigated the effect of eight different inhibitors on the oxidation of the probe substrate phthalazine. Saturation kinetic parameters for phthalazine oxidation in human liver cytosol were found to be the following: K(m) = 8.0 ± 0.4 µM and V(max) = 4.3 ± 0.1 nmol · min(-1) · mg protein(-1). Inhibitory potency of the inhibitors tested ranged from 0.1 to 5 µM. Of the eight different inhibitor compounds tested, seven were observed to inhibit through a mixed mode and one through a strictly competitive mode. A ratio of the K(ii) and K(is) values was used to assess the relative competitiveness of each inhibitor. For the mixed inhibitors, the mode of inhibition varied from mostly uncompetitive to predominantly competitive (K(ii)/K(is) values ranging from 0.1 to 15). The implications for potential drug-drug interactions and inhibition mechanism are discussed. We found two inhibitors, clozapine and chlorpromazine, that have a moderate predicted risk of drug-drug interactions based on the K(i) value relative to the inhibitor concentration in human plasma, having a calculated [I]/K(i) value of 0.4 and 0.8, respectively.

A retrospective look at the carcinogenic potency of vinyl chloride

Failure to find any cases of vinyl chloride-induced cancer aoutside the heavily exposed reactor cleaner group calls into question the human relevance of predictive methods for carcinogenic potency. Potency estimates by six conventional routes are reviewed with special attention to the implication of each route for vinyl choride. New metabolic and mechanistic data are used to suggest a basis for the failure of "classical" risk assessment methods to provide accurate predictions for humans in this instance.(AU)