Aldehyde Oxidase: Reaction Mechanism and Prediction of Site of Metabolism

Montefiori, Marco; Jørgensen, Flemming Steen; Olsen, Lars

doi:10.1021/acsomega.7b00658

Cited by 37 publications

(50 citation statements)

References 34 publications

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“…The functional significance of these enzymes in MECs is not clear. However, they have been reported to play a role in the clearance of drugs containing aldehydes and N-containing heterocyclic fragments in liver 69 .…”

Section: Scientific Reports |mentioning

confidence: 99%

In-depth proteome analysis of more than 12,500 proteins in buffalo mammary epithelial cell line identifies protein signatures for active proliferation and lactation

Jaswal

Anand

Kumar

et al. 2020

Sci Rep

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The mature mammary gland is made up of a network of ducts that terminates in alveoli. The innermost layer of alveoli is surrounded by the differentiated mammary epithelial cells (MECs), which are responsible for milk synthesis and secretion during lactation. However, the MECs are in a state of active proliferation during pregnancy, when they give rise to network like structures in the mammary gland. Buffalo (Bubalus bubalis) constitute a major source of milk for human consumption, and the MECs are the major precursor cells which are mainly responsible for their lactation potential. The proteome of MECs defines their functional state and suggests their role in various cellular activities such as proliferation and lactation. To date, the proteome profile of MECs from buffalo origin is not available. In the present study, we have profiled in-depth proteome of in vitro cultured buffalo MECs (BuMECs) during active proliferation using high throughput tandem mass spectrometry (MS). MS analysis identified a total of 8330, 5970, 5289, 4818 proteins in four sub-cellular fractions (SCFs) that included cytosolic (SCF-I), membranous and membranous organelle's (SCF-II), nuclear (SCF-III), and cytoskeletal (SCF-IV). However, 792 proteins were identified in the conditioned media, which represented the secretome. Altogether, combined analysis of all the five fractions (SCFs-I to IV, and secretome) revealed a total of 12,609 non-redundant proteins. The KEGG analysis suggested that these proteins were associated with 325 molecular pathways. Some of the highly enriched molecular pathways observed were metabolic, MAPK, PI3-AKT, insulin, estrogen, and cGMP-PKG signalling pathway. The newly identified proteins in this study are reported to be involved in NOTCH signalling, transport and secretion processes.The mammary gland, a unique organ of mammals, is a derivative of ventral skin 1 and has evolved to provide nutrition and immune protection to the offspring. Though the anatomical and physiological features of a mammary gland are diverse, the basic structure is similar in all species. The mature mammary gland is constituted by a network of tubular-alveolar structures, with their lumen lined by a layer of polarized secretory mammary epithelial cells (MECs). The MECs are dynamic components of the mammary gland, which undergo repeated rounds of proliferation and differentiation during different developmental stages such as pregnancy, and lactation 2 . The quantity and functional differentiation of MECs in the udder determine the milk-producing ability in livestock. Bovine (ruminants) are physiologically distinct from other mammals such as humans, mice and rats. The present study on in-depth proteome analysis of buffalo (Bubalus bubalis) MECs (BuMECs) advances our knowledge on the protein machinery operating in mammary gland development and lactogenesis in buffalo, which can be extrapolated in other mammalian species as well. Among the domestic livestock species, cattle (Bos taurus) and

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Section: Scientific Reports |mentioning

confidence: 99%

In-depth proteome analysis of more than 12,500 proteins in buffalo mammary epithelial cell line identifies protein signatures for active proliferation and lactation

Jaswal

Anand

Kumar

et al. 2020

Sci Rep

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“…Several well-known structure modification strategies exist to guide the medicinal chemist through CYP-mediated instabilities [5,6]. However, strategies to attenuate non-CYP-mediated liabilities are sparsely reported in the literature [7][8][9][10]. A few in-house projects at the National Center for Advancing Translational Sciences (NCATS) were recently discovered to have cytosol-mediated metabolic liabilities.…”

Section: Introductionmentioning

confidence: 99%

Predicting liver cytosol stability of small molecules

et al. 2020

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Over the last few decades, chemists have become skilled at designing compounds that avoid cytochrome P (CYP) 450 mediated metabolism. Typical screening assays are performed in liver microsomal fractions and it is possible to overlook the contribution of cytosolic enzymes until much later in the drug discovery process. Few data exist on cytosolic enzyme-mediated metabolism and no reliable tools are available to chemists to help design away from such liabilities. In this study, we screened 1450 compounds for liver cytosol-mediated metabolic stability and extracted transformation rules that might help medicinal chemists in optimizing compounds with these liabilities. In vitro half-life data were collected by performing in-house experiments in mouse (CD-1 male) and human (mixed gender) cytosol fractions. Matched molecular pairs analysis was performed in conjunction with qualitative-structure activity relationship modeling to identify chemical structure transformations affecting cytosolic stability. The transformation rules were prospectively validated on the test set. In addition, selected rules were validated on a diverse chemical library and the resulting pairs were experimentally tested to confirm whether the identified transformations could be generalized. The validation results, comprising nearly 250 library compounds and corresponding half-life data, are made publicly available. The datasets were also used to generate in silico classification models, based on different molecular descriptors and machine learning methods, to predict cytosol-mediated liabilities. To the best of our knowledge, this is the first systematic in silico effort to address cytosolic enzyme-mediated liabilities.

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“…Aldehyde oxidase (AO) is a cytosolic molybdenum-containing enzyme that very efficiently oxidizes a range of N-containing heterocyclic aromatic rings and aldehydes (Montefiori et al, 2017). The limited knowledge about AO activity, abundance, and translation from in vitro to in vivo has led to poor prediction of in vivo clearance (CL) and consequently to clinical failure of some AO substrates (Fan et al, 2016;Jensen et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

A Laboratory-Specific Scaling Factor to Predict the In Vivo Human Clearance of Aldehyde Oxidase Substrates

et al. 2020

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Aldehyde oxidase (AO) efficiently metabolizes a range of compounds with N-containing heterocyclic aromatic rings and/or aldehydes. The limited knowledge of AO activity and abundance (in vitro and in vivo) has led to poor prediction of in vivo systemic clearance (CL) using in vitro-to-in vivo extrapolation (IVIVE) approaches, which for drugs in development can lead to their discontinuation. We aimed to identify appropriate scaling factors (SF) to predict AO CL of future New Chemical Entities (NCE). The metabolism of six AO substrates was measured in human liver cytosol (HLC) and S9 fractions. Measured blood-to-plasma ratios and free fractions (in the in vitro system and in plasma) were used to develop physiologically-based pharmacokinetic (PBPK) models for each compound. The impact of extrahepatic metabolism was explored, and the intrinsic clearance required to recover in vivo profiles was estimated and compared to in vitro measurements. Using HLC data and assuming only hepatic metabolism, a systematic underprediction of clearance was observed (average fold under-prediction was 3.8). Adding extrahepatic metabolism improved the accuracy of the results (average fold error of 1.9). A workflow for predicting metabolism of an NCE by AO is proposed, an empirical (lab-specific) SF of 3 on the predicted CL IV allows a reasonable prediction of the available clinical data. Alternatively, considering also extrahepatic metabolism, a SF of 6.5 applied on the intrinsic clearance (CL int,AO) could be used. Future research should focus on the impact of the in vitro study designs and the contribution of extrahepatic metabolism to AO-mediated clearance to understand the mechanisms behind the systematic under-prediction. Significance Statement This works describes the development of scaling factors to allow in vitro-in vivo extrapolation of the clearance of compounds by aldehyde oxidase metabolism in humans. In addition, PBPK models were developed for each of the AO substrate compounds investigated.

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Aldehyde Oxidase: Reaction Mechanism and Prediction of Site of Metabolism

Cited by 37 publications

References 34 publications

In-depth proteome analysis of more than 12,500 proteins in buffalo mammary epithelial cell line identifies protein signatures for active proliferation and lactation

In-depth proteome analysis of more than 12,500 proteins in buffalo mammary epithelial cell line identifies protein signatures for active proliferation and lactation

Predicting liver cytosol stability of small molecules

A Laboratory-Specific Scaling Factor to Predict the In Vivo Human Clearance of Aldehyde Oxidase Substrates

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