Combination of biotransformation by P450 BM3 mutants with on-line post-column bioaffinity and mass spectrometric profiling as a novel strategy to diversify and characterize p38α kinase inhibitors

Rea, Vanina; Falck, David; Kool, Jeroen; Kanter, Frans J. J. de; Commandeur, Jan N. M.; Vermeulen, Nico; Niessen, W.M.A.; Honing, Maarten

doi:10.1039/c2md20283b

Cited by 14 publications

(23 citation statements)

References 29 publications

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“…Data on relative retention time (RRT) were used to assist in identifying the modification, e.g., to differentiate between hydroxylation and N ‐oxide formation . In the case of five BM3‐generated CPs of TAK‐715, the fourth step involved purification of the CPs by preparative LC and subsequent acquisition and interpretation of the 1 H NMR spectra, guided by the knowledge obtained from the MS n data . The data are summarized in Tables .…”

Section: Resultsmentioning

confidence: 99%

“…Electrochemistry adds (reduction) or removes (oxidation) electrons from the substrate on a surface electrode, which results in further reaction for example with the solvent . During incubation with (human) liver microsomes or with mutants of cytochrome P450 BM3, the enzymes present catalyse specific reactions such as hydroxylation. Oxidation with hydrogen peroxide occurs mainly via reactive oxygen species, whereas photochemical conversion is the result of the excitation of electrons in the substrate by intense visible light irradiation …”

Section: Methodsmentioning

confidence: 99%

“…For example, the use of electrochemistry in an on‐line combination with the HRS platform resulted in a number of related substances for p38α kinase inhibitors, including unstable and reactive conversion products (CPs) . Mixtures of related substances can also be generated enzymatically, e.g., biosynthetically using (human) microsomal incubations or by incubation with specific bacterial mutants (BM3) of cytochrome P450s . Alternatively, other (chemical) means such as photochemistry or oxidative reagents can be applied.…”

Section: Introductionmentioning

confidence: 99%

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Tandem mass spectrometry study of p38α kinase inhibitors and related substances

et al. 2013

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The p38 mitogen-activated protein kinase α (p38α) is an important drug target widely investigated for therapy of chronic inflammatory diseases. Its inhibitors are rather lipophilic and as such not very favourable lead compounds in drug discovery. Therefore, we explored various approaches to access new chemical space, create diversity, and generate lead libraries with improved solubility and reduced lipophilicity, based on known p38α inhibitors, e.g., BIRB796 and TAK-715. Compound modification strategies include incubation with human liver microsomes and bacterial cytochrome P450 mutants from Bacillus megaterium and treatment by electrochemical oxidation, H2O2, and intense light irradiation. The MS/MS fragmentation pathways of p38α inhibitors and their conversion products have been studied in an ion-trap-time-of-flight MS(n) instrument. Interpretation of accurate mass MS(n) data for four sets of related compounds revealed unexpected and peculiar fragmentation pathways that are discussed in detail. Emphasis is put on the usefulness of HRMS(n)-based structure elucidation in a screening setting and on peculiarities of the fragmentation with regard to the analytes and the MS instrument. In one example, an intramolecular rearrangement reaction accompanied by the loss of a bulky group is observed. For BIRB796, the double-charge precursor ion is used in MS(2), providing a wider range of fragment ions in our instrument. For TAK-715, a number of related compounds could be produced in a large-scale incubation with a Bacillus megaterium mutant, thus enabling comparison of the structure elucidation by (1)H NMR and MS(n). A surprisingly large number of homolytic cleavages are observed. Competition between two fragmentation pathways involving either the loss of CH3(•) or OH(•) radicals was observed for SB203580 and its conversion products.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Tandem mass spectrometry study of p38α kinase inhibitors and related substances

et al. 2013

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“…Also within our own lab, isolated CYP BM3 mutants have previously been successfully applied for biosynthesis of drug metabolites on a preparative scale [32][33][34][35][36]39,47,48]. During our in vitro experiment, bioconversion was monitored in time (see Fig.…”

Section: In Vitro Biotransformation Of Netmentioning

confidence: 99%

“…The 40 other previously described mutants have been based upon these four templates [28][29][30][31][32][33][34]. The 44 mutants have been shown to metabolize a wide range of substrates, including marketed drugs [28,30,35,36], steroids [29,32,34], ionones [33], kinase inhibitors [47], alkoxyresorufins [29,31], and endocrine disrupting chemicals [31,48], with varying catalytic activities while also displaying significant differences in the metabolic profiles generated. The 22 mutants that have not been described before were all created inhouse by site-directed mutagenesis using the appropriate mutant templates (see the Supporting Information, Table S2).…”

Section: Selection Of the Cyp Bm3 Mutant Librarymentioning

confidence: 99%