title: 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 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.
authors.affiliation: Departments of Drug Metabolism and Pharmacokinetics (J.K.S., S.W., S.C.K., C.E.C.A.H., J.S.H.), Clinical Pharmacology (L.L.), and Protein Chemistry (D.S.K.), Genentech, Inc., South San Francisco, California; and Department of Chemistry, Washington State University, Pullman, Washington (J.T.B., J.P.J.) jasleens@gene.com.
authors.forename: Jasleen K
authors.initials: JK
authors.lastname: Sodhi
authors.affiliation: Departments of Drug Metabolism and Pharmacokinetics (J.K.S., S.W., S.C.K., C.E.C.A.H., J.S.H.), Clinical Pharmacology (L.L.), and Protein Chemistry (D.S.K.), Genentech, Inc., South San Francisco, California; and Department of Chemistry, Washington State University, Pullman, Washington (J.T.B., J.P.J.).
authors.forename: Susan
authors.initials: S
authors.lastname: Wong
authors.affiliation: Departments of Drug Metabolism and Pharmacokinetics (J.K.S., S.W., S.C.K., C.E.C.A.H., J.S.H.), Clinical Pharmacology (L.L.), and Protein Chemistry (D.S.K.), Genentech, Inc., South San Francisco, California; and Department of Chemistry, Washington State University, Pullman, Washington (J.T.B., J.P.J.).
authors.forename: Donald S
authors.initials: DS
authors.lastname: Kirkpatrick
authors.affiliation: Departments of Drug Metabolism and Pharmacokinetics (J.K.S., S.W., S.C.K., C.E.C.A.H., J.S.H.), Clinical Pharmacology (L.L.), and Protein Chemistry (D.S.K.), Genentech, Inc., South San Francisco, California; and Department of Chemistry, Washington State University, Pullman, Washington (J.T.B., J.P.J.).
authors.forename: Lichuan
authors.initials: L
authors.lastname: Liu
authors.affiliation: Departments of Drug Metabolism and Pharmacokinetics (J.K.S., S.W., S.C.K., C.E.C.A.H., J.S.H.), Clinical Pharmacology (L.L.), and Protein Chemistry (D.S.K.), Genentech, Inc., South San Francisco, California; and Department of Chemistry, Washington State University, Pullman, Washington (J.T.B., J.P.J.).
authors.forename: S Cyrus
authors.initials: SC
authors.lastname: Khojasteh
authors.affiliation: Departments of Drug Metabolism and Pharmacokinetics (J.K.S., S.W., S.C.K., C.E.C.A.H., J.S.H.), Clinical Pharmacology (L.L.), and Protein Chemistry (D.S.K.), Genentech, Inc., South San Francisco, California; and Department of Chemistry, Washington State University, Pullman, Washington (J.T.B., J.P.J.).
authors.forename: Cornelis E C A
authors.initials: CE
authors.lastname: Hop
authors.affiliation: Departments of Drug Metabolism and Pharmacokinetics (J.K.S., S.W., S.C.K., C.E.C.A.H., J.S.H.), Clinical Pharmacology (L.L.), and Protein Chemistry (D.S.K.), Genentech, Inc., South San Francisco, California; and Department of Chemistry, Washington State University, Pullman, Washington (J.T.B., J.P.J.).
authors.forename: John T
authors.initials: JT
authors.lastname: Barr
authors.affiliation: Departments of Drug Metabolism and Pharmacokinetics (J.K.S., S.W., S.C.K., C.E.C.A.H., J.S.H.), Clinical Pharmacology (L.L.), and Protein Chemistry (D.S.K.), Genentech, Inc., South San Francisco, California; and Department of Chemistry, Washington State University, Pullman, Washington (J.T.B., J.P.J.).
authors.forename: Jeffrey P
authors.initials: JP
authors.lastname: Jones
authors.affiliation: Departments of Drug Metabolism and Pharmacokinetics (J.K.S., S.W., S.C.K., C.E.C.A.H., J.S.H.), Clinical Pharmacology (L.L.), and Protein Chemistry (D.S.K.), Genentech, Inc., South San Francisco, California; and Department of Chemistry, Washington State University, Pullman, Washington (J.T.B., J.P.J.).
authors.forename: Jason S
authors.initials: JS
authors.lastname: Halladay
mesh_terms: D000077329:Agammaglobulinaemia Tyrosine Kinase; D042931:Aldehyde Oxidase / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000097:blood / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D055162:Biocatalysis; D020134:Catalytic Domain; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D004355:Drug Stability; D015507:Drugs, Investigational / Q000032:analysis / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D020869:Gene Expression Profiling; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D062105:Molecular Docking Simulation; D047428:Protein Kinase Inhibitors / Q000097:blood / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D011744:Pyrimidinones / Q000097:blood / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D013379:Substrate Specificity; D013876:Thiophenes / Q000097:blood / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D015507:Drugs, Investigational; C560621: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; D047428:Protein Kinase Inhibitors; D011744:Pyrimidinones; D013876:Thiophenes; C526436:AOX1 protein, human; D042931:Aldehyde Oxidase; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase
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