Inactivation of Cytochrome P450 (P450) 3A4 but not P450 3A5 by OSI-930, a Thiophene-Containing Anticancer Drug

Lin, Hsia Lien; Zhang, Haoming; Medower, Christine; Hollenberg, Paul F.; Johnson, William W.

doi:10.1124/dmd.110.034074

Cited by 16 publications

(13 citation statements)

References 20 publications

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“…The crystal structure of the BMP (PDB code 4KEW) and the BMR homology model were fit to the 3D density volume using Chimera. The BMR homology model was constructed with Modeler 9v8 as described previously (39). BMP molecule is shown in magenta, and the FMN, FAD, and nucleotidebinding domains of the BMR are shown in green, blue, and cyan, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…Because the crystal structure of a full-length CYP102A1 is not available, we fit the crystal structure of the BMP domain (PDB code 4KEW, residues 3-455) and a BMR homology model (reside 478 -1048) instead. The BMR homology model was constructed on the template of rat POR (PDB code 1AMO) using Modeler 9v8 as described previously (39). This article cites 38 references, 13 of which can be accessed free at…”

Section: Em Sample Preparation Data Collection and 2d Processingmentioning

confidence: 99%

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The full-length cytochrome P450 enzyme CYP102A1 dimerizes at its reductase domains and has flexible heme domains for efficient catalysis

Zhang¹,

Yokom

Shen³

et al. 2018

Journal of Biological Chemistry

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The cytochrome P450 enzyme CYP102A1 from is a highly efficient hydroxylase of fatty acids, and there is a significant interest in using CYP102A1 for biotechnological applications. Here, we used size-exclusion chromatography-multiangle light scattering (SEC-MALS) analysis and negative-stain EM to investigate the molecular architecture of CYP102A1. The SEC-MALS analysis yielded a homogeneous peak with an average molecular mass of 235 ± 5 kDa, consistent with homodimeric CYP102A1. The negative-stain EM of dimeric CYP102A1 revealed four distinct lobes, representing the two heme and two reductase domains. Two of the lobes were in close contact, whereas the other two were often observed apart and at the ends of a U-shaped configuration. The overall dimension of the dimer was ∼130 Å. To determine the identity of the lobes, we FLAG-tagged the N or C terminus of CYP102A1 to visualize additional densities in EM and found that anti-FLAG Fab could bind only the N-tagged P450. Single-particle analysis of this anti-Flag Fab-CYP102A1 complex revealed additional density in the N-terminally tagged heme domains, indicating that the heme domains appear flexible, whereas the reductase domains remain tightly associated. The effects of truncation on CYP102A1 dimerization, identification of cross-linked sites by peptide mapping, and molecular modeling results all were consistent with the dimerization of the reductase domain. We conclude that functional CYP102A1 is a compact globular protein dimerized at its reductase domains, with its heme domains exhibiting multiple conformations that likely contribute to the highly efficient catalysis of CYP102A1.

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Section: Discussionmentioning

confidence: 99%

Section: Em Sample Preparation Data Collection and 2d Processingmentioning

confidence: 99%

The full-length cytochrome P450 enzyme CYP102A1 dimerizes at its reductase domains and has flexible heme domains for efficient catalysis

Zhang¹,

Yokom

Shen³

et al. 2018

Journal of Biological Chemistry

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“…Of the compounds that show promise but lack a full assessment are OSI-930 and azamulin. amino)-N-(4-(trifluoromethoxy)phenyl) thiophene-2-carboxamide] is an experimental anticancer agent which has been shown to inactivate CYP3A4 and not CYP3A5 (Lin et al, 2011). Studies therein demonstrated that OSI-930 was able to act as a time-dependent inhibitor of human CYP3A4 with a K I of 24 mM, a maximum inactivation rate (k inact ) of 0.04 minute…”

Section: Discussion Cytochrome P450smentioning

confidence: 99%

Reaction Phenotyping: Advances in the Experimental Strategies Used to Characterize the Contribution of Drug-Metabolizing Enzymes

2014

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During the process of drug discovery, the pharmaceutical industry is faced with numerous challenges. One challenge is the successful prediction of the major routes of human clearance of new medications. For compounds cleared by metabolism, accurate predictions help provide an early risk assessment of their potential to exhibit significant interpatient differences in pharmacokinetics via routes of metabolism catalyzed by functionally polymorphic enzymes and/or clinically significant metabolic drug-drug interactions. This review details the most recent and emerging in vitro strategies used by drug metabolism and pharmacokinetic scientists to better determine rates and routes of metabolic clearance and how to translate these parameters to estimate the amount these routes contribute to overall clearance, commonly referred to as fraction metabolized. The enzymes covered in this review include cytochrome P450s together with other enzymatic pathways whose involvement in metabolic clearance has become increasingly important as efforts to mitigate cytochrome P450 clearance are successful. Advances in the prediction of the fraction metabolized include newly developed methods to differentiate CYP3A4 from the polymorphic enzyme CYP3A5, scaling tools for UDP-glucuronosyltranferase, and estimation of fraction metabolized for substrates of aldehyde oxidase. IntroductionIn an era where combination drug therapy to treat several conditions simultaneously is common, drug companies emphasize the need for optimal absorption, distribution, metabolism, and excretion (ADME) properties, with the purpose of optimization of efficacy and minimization of the risk of adverse events. This includes a proper assignment and an extensive understanding of the routes of metabolism to aid in the prediction of human pharmacokinetics, and to help avoid a potential "object drug" scenario when coadministration is likely. The attributes of the coadministered drug and/or the patient has the potential to increase the probability of a drug-drug interaction (DDI), i.e., enzyme inhibitor, inducer, polymorphic genotype. Hence, the greater the percentage attributed to a single metabolic route, the greater the potential for a DDI and possible "black box warning" being issued as part of the drug package insert. Furthermore, the pharmacokinetic implications of a single polymorphic enzyme being responsible for a majority of the metabolism of a drug may have either an efficacy (extensive metabolizers) or toxicological (poor metabolizers) impact on exposure. To address these concerns, early drug discovery teams strive for balanced metabolism across multiple enzymes and clearance mechanisms (hepatic, renal, biliary). These discovery efforts center on in vitro reaction phenotyping to support enhanced chemical design.There is a general agreement among the major regulatory agencies that pharmaceutical companies should provide a characterization of the metabolic profile of a new chemical entity (NCE) and understand the enzymology of the major clearance mechanisms ...

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“…However, further exploration of raloxifene for reaction phenotyping purposes was not undertaken to demonstrate its potential practical utility. An experimental compound 3-((quinolin-4-ylmethyl)amino)-N-(4-(trifluoromethoxy)phenyl)thiophene-2-carboxamide (OSI-930) was also shown to inactivate CYP3A4 and not CYP3A5 (Lin et al, 2011). However, with all three of these agents, testing the putative selective inactivator was not done using HLM from CYP3A5 expressers and nonexpressers, and for any compound to be used as a selective tool for phenotyping purposes, this must be done.…”

Section: Discussionmentioning

confidence: 99%

Selective Mechanism-Based Inactivation of CYP3A4 by CYP3cide (PF-04981517) and Its Utility as an In Vitro Tool for Delineating the Relative Roles of CYP3A4 versus CYP3A5 in the Metabolism of Drugs

et al. 2012

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CYP3cide (PF-4981517; 1-methyl-3-[1-methyl-5-(4-methylphenyl)-1H-pyrazol-4-yl]-4-[(3S)-3-piperidin-1-ylpyrrolidin-1-yl]-1H-pyrazolo[3,4-d]pyrimidine) is a potent, efficient, and specific time-dependent inactivator of human CYP3A4. When investigating its inhibitory properties, an extreme metabolic inactivation efficiency (k inact /K I ) of 3300 to 3800 ml ⅐ min ؊1 ⅐ mol ؊1 was observed using human liver microsomes from donors of nonfunctioning CYP3A5 (CYP3A5 *3/*3). This observed efficiency equated to an apparent K I between 420 and 480 nM with a maximal inactivation rate (k inact ) equal to 1.6 min ؊1 . Similar results were achieved with testosterone, another CYP3A substrate, and other sources of the CYP3A4 enzyme. To further illustrate the abilities of CYP3cide, its partition ratio of inactivation was determined with recombinant CYP3A4. These studies produced a partition ratio approaching unity, thus underscoring the inactivation capacity of CYP3cide. WhenCYP3cide was tested at a concentration and preincubation time to completely inhibit CYP3A4 in a library of genotyped polymorphic CYP3A5 microsomes, the correlation of the remaining midazolam 1-hydroxylase activity to CYP3A5 abundance was significant (R 2 value equal to 0.51, p value of <0.0001). The work presented here supports these findings by fully characterizing the inhibitory properties and exploring CYP3cide's mechanism of action. To aid the researcher, multiple commercially available sources of CYP3cide were established, and a protocol was developed to quantitatively determine CYP3A4 contribution to the metabolism of an investigational compound. Through the establishment of this protocol and the evidence provided here, we believe that CYP3cide is a very useful tool for understanding the relative roles of CYP3A4 versus CYP3A5 and the impact of CYP3A5 genetic polymorphism on a compound's pharmacokinetics.

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Inactivation of Cytochrome P450 (P450) 3A4 but not P450 3A5 by OSI-930, a Thiophene-Containing Anticancer Drug

Cited by 16 publications

References 20 publications

The full-length cytochrome P450 enzyme CYP102A1 dimerizes at its reductase domains and has flexible heme domains for efficient catalysis

The full-length cytochrome P450 enzyme CYP102A1 dimerizes at its reductase domains and has flexible heme domains for efficient catalysis

Reaction Phenotyping: Advances in the Experimental Strategies Used to Characterize the Contribution of Drug-Metabolizing Enzymes

Selective Mechanism-Based Inactivation of CYP3A4 by CYP3cide (PF-04981517) and Its Utility as an In Vitro Tool for Delineating the Relative Roles of CYP3A4 versus CYP3A5 in the Metabolism of Drugs

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