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Mass spectral analysis of the multikinase inhibitor BZG and its metabolites and analysis of their binding to vascular endothelial growth factor receptor-2.

ABSTRACT: We previously showed that BZG is a novel multitarget kinase inhibitor, which inhibited hepatocellular carcinoma in vivo and in vitro. In the present study, we used ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) to characterize BZG and its metabolites generated in vivo. The probable metabolic mechanism was further confirmed by analysis of Phase I and Phase II metabolism in liver microsomes and with recombinant enzymes. In addition, the binding affinities of BZG metabolites to vascular endothelial growth factor receptor 2 (VEGFR2) were predicted using electronic high throughput screening (eHiTS). The results showed that BZG underwent phase I and phase II metabolism. We detected 11 BZG metabolites and identified hydroxylation, glucuronation, acetylation, sulfonation and degradation as the major metabolic processes in vivo and in vitro. Five of the eleven metabolites showed highly favorable eHiTS energy scores that were lower than sorafenib. Knowledge of the in vivo metabolic pathways of BZG and its binding affinities to VEGFR2 will be beneficial for further clinical development of BZG.

SUBMITTER: Lou Y

PROVIDER: S-EPMC5444716 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Mass spectral analysis of the multikinase inhibitor BZG and its metabolites and analysis of their binding to vascular endothelial growth factor receptor-2.

Lou Yan Y Qiu Wenqi W Wu Zhe Z Wang Qian Q Qiu Yunqing Y Zeng Su S

Oncotarget 20170501 18

We previously showed that BZG is a novel multitarget kinase inhibitor, which inhibited hepatocellular carcinoma in vivo and in vitro. In the present study, we used ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) to characterize BZG and its metabolites generated in vivo. The probable metabolic mechanism was further confirmed by analysis of Phase I and Phase II metabolism in liver microsomes and with recombinant enzymes. In addition, ...[more]

PMID: 28415783

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Project description:Vascular endothelial growth factor pathway inhibitors (VEGFi), used as anti-angiogenic drugs to treat cancer are associated with cardiovascular toxicities through unknown molecular mechanisms. Endothelial cell-derived microparticles (ECMPs) are biomarkers of endothelial injury and are also functionally active since they influence downstream target cell signalling and function. We questioned whether microparticle (MP) status is altered in cancer patients treated with VEGFi and whether they influence endothelial cell function associated with vascular dysfunction. Plasma MPs were isolated from cancer patients before and after treatment with VEGFi (pazopanib, sunitinib, or sorafenib). Human aortic endothelial cells (HAECs) were stimulated with isolated MPs (106 MPs/mL). Microparticle characterization was assessed by flow cytometry. Patients treated with VEGFi had significantly increased levels of plasma ECMP. Endothelial cells exposed to post-VEGFi treatment ECMPs induced an increase in pre-pro-ET-1 mRNA expression, corroborating the increase in endothelin-1 (ET-1) production in HAEC stimulated with vatalanib (VEGFi). Post-VEGFi treatment MPs increased generation of reactive oxygen species in HAEC, effects attenuated by ETA (BQ123) and ETB (BQ788) receptor blockers. VEGFi post-treatment MPs also increased phosphorylation of the inhibitory site of endothelial nitric oxide synthase (eNOS), decreased nitric oxide (NO), and increased ONOO- levels in HAEC, responses inhibited by ETB receptor blockade. Additionally, gene expression of proinflammatory mediators was increased in HAEC exposed to post-treatment MPs, effects inhibited by BQ123 and BQ788. Our findings define novel molecular mechanism involving interplay between microparticles, the ET-1 system and endothelial cell pro-inflammatory and redox signalling, which may be important in cardiovascular toxicity and hypertension associated with VEGFi anti-cancer treatment. New and noteworthy: our novel data identify MPs as biomarkers of VEGFi-induced endothelial injury and important mediators of ET-1-sensitive redox-regulated pro-inflammatory signalling in effector endothelial cells, processes that may contribute to cardiovascular toxicity in VEGFi-treated cancer patients.

Project description:AimsHemodynamic shear stress participates in maintaining vascular redox status. Elucidating flow-mediated endothelial metabolites enables us to discover metabolic biomarkers and therapeutic targets. We posited that flow-responsive vascular endothelial growth factor receptor (VEGFR)-protein kinase C isoform epsilon (PKC?)-6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) signaling modulates glycolytic metabolites for vascular repair.ResultsBidirectional oscillatory flow (oscillatory shear stress [OSS]: 0.1?±?3 dyne·cm-2 at 1?Hz) upregulated VEGFR-dependent PKC? expression to a greater degree than did unidirectional pulsatile flow (pulsatile shear stress [PSS]: 23?±?8 dyne·cm-2 at 1?Hz) in human aortic endothelial cells (p?<?0.05, n?=?3). PSS and OSS further upregulated PKC?-dependent PFKFB3 expression for glycolysis (p?<?0.05, n?=?4). Constitutively active PKC? increased, whereas dominant-negative PKC? reduced both basal and maximal extracellular acidification rates for glycolytic flux (p?<?0.01, n?=?4). Metabolomic analysis demonstrated an increase in PKC?-dependent glycolytic metabolite, dihydroxyacetone (DHA), but a decrease in gluconeogenic metabolite, aspartic acid (p?<?0.05 vs. control, n?=?6). In a New Zealand White rabbit model, both PKC? and PFKFB3 immunostaining was prominent in the PSS- and OSS-exposed aortic arch and descending aorta. In a transgenic Tg(flk-1:EGFP) zebrafish model, GATA-1a morpholino oligonucleotide injection (to reduce viscosity-dependent shear stress) impaired vascular regeneration after tail amputation (p?<?0.01, n?=?20), which was restored with PKC? messenger RNA (mRNA) rescue (p?<?0.05, n?=?5). As a corollary, siPKC? inhibited tube formation and vascular repair, which were restored by DHA treatment in our Matrigel and zebrafish models. Innovation and Conclusion: Flow-sensitive VEGFR-PKC?-PFKFB3 signaling increases the glycolytic metabolite, dihydroxyacetone, to promote vascular repair. Antioxid. Redox Signal. 28, 31-43.

Dataset Information

Mass spectral analysis of the multikinase inhibitor BZG and its metabolites and analysis of their binding to vascular endothelial growth factor receptor-2.

Publications

Mass spectral analysis of the multikinase inhibitor BZG and its metabolites and analysis of their binding to vascular endothelial growth factor receptor-2.

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