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

ABSTRACT: 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 ?M. 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.

SUBMITTER: Sodhi JK

PROVIDER: S-EPMC4429680 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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

Sodhi Jasleen K JK Wong Susan S Kirkpatrick Donald S DS Liu Lichuan L Khojasteh S Cyrus SC Hop Cornelis E C A CE Barr John T JT Jones Jeffrey P JP Halladay Jason S JS

Drug metabolism and disposition: the biological fate of chemicals 20150406 6

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-tetrahydrobenz ...[more]

PMID: 25845827

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Project description:Butyrylcholinesterase (BChE)-cocaine binding and the fundamental pathway for BChE-catalyzed hydrolysis of cocaine have been studied by molecular modeling, molecular dynamics (MD) simulations, and ab initio calculations. Modeling and simulations indicate that the structures of the prereactive BChE/substrate complexes for (-)-cocaine and (+)-cocaine are all similar to that of the corresponding prereactive BChE/butyrylcholine (BCh) complex. The overall binding of BChE with (-)-cocaine and (+)-cocaine is also similar to that proposed with butyrylthiocholine and succinyldithiocholine, i.e., (-)- or (+)-cocaine first slides down the substrate-binding gorge to bind to Trp-82 and stands vertically in the gorge between Asp-70 and Trp-82 (nonprereactive complex) and then rotates to a position in the catalytic site within a favorable distance for nucleophilic attack and hydrolysis by Ser-198 (prereactive complex). In the prereactive complex, cocaine lies horizontally at the bottom of the gorge. The fundamental catalytic hydrolysis pathway, consisting of acylation and deacylation stages similar to those for ester hydrolysis by other serine hydrolases, was proposed on the basis of the simulated prereactive complex and confirmed theoretically by ab initio reaction coordinate calculations. Both the acylation and deacylation follow a double-proton-transfer mechanism. The calculated energetic results show that within the chemical reaction process the highest energy barrier and Gibbs free energy barrier are all associated with the first step of deacylation. The calculated ratio of the rate constant (k(cat)) for the catalytic hydrolysis to that (k(0)) for the spontaneous hydrolysis is approximately 9.0 x 10(7). The estimated k(cat)/k(0) value of approximately 9.0 x 10(7) is in excellent agreement with the experimentally derived k(cat)/k(0) value of approximately 7.2 x 10(7) for (+)-cocaine, whereas it is approximately 2000 times larger than the experimentally derived k(cat)/k(0) value of approximately 4.4 x 10(4) for (-)-cocaine. All of the results suggest that the rate-determining step of the BChE-catalyzed hydrolysis of (+)-cocaine is the first step of deacylation, whereas for (-)-cocaine the change from the nonprereactive complex to the prereactive complex is rate-determining and has a Gibbs free energy barrier higher than that for the first step of deacylation by approximately 4 kcal/mol. A further analysis of the structural changes from the nonprereactive complex to the prereactive complex reveals specific amino acid residues hindering the structural changes, providing initial clues for the rational design of BChE mutants with improved catalytic activity for (-)-cocaine.

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

Publications

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

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