Identification of alcohol‐binding site(s) in proteins using diazirine‐based photoaffinity labeling and mass spectrometry

Das, Joydip

doi:10.1111/cbdd.13403

Cited by 5 publications

(7 citation statements)

References 30 publications

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“…For example, to characterize the molecular targets of alcohols and to identify binding sites, diazirine-based azialcohol has been used as a photoaffinity labeling agent. It photoincorporates into alcohol-binding proteins, and the photoincorporation site can be elucidated by HRMS . Another example is the use of a nonradioactive photoaffinity labeled rapamycin analogue that was used to elucidate a binding site map of the FKBP12-Rapamycin-FRB interaction complex …”

Section: Photaffinity Labeling Of Tubulinmentioning

confidence: 99%

Utilization of Photoaffinity Labeling to Investigate Binding of Microtubule Stabilizing Agents to P-Glycoprotein and β-Tubulin

2022

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Photoaffinity labeling approaches have historically been used in pharmacology to identify molecular targets. This methodology has played a pivotal role in identifying drug-binding domains and searching for novel compounds that may interact at these domains. In this review we focus on studies of microtubule stabilizing agents of natural product origin, specifically taxol (paclitaxel). Taxol and other microtubule interacting agents bind to both P-glycoprotein (ABCB1), a drug efflux pump that reduces intracellular drug accumulation, and the tubulin/ microtubule system. Both binding relationships modulate drug efficacy and are of immense interest to basic and translational scientists, primarily because of their association with drug resistance for this class of molecules. We present this body of work and acknowledge its value as fundamental to understanding the mechanisms of taxol and elucidation of the taxol pharmacophore. Furthermore, we highlight the ability to multiplex photoaffinity approaches with other technologies to further enhance our understanding of pharmacologic interactions at an atomic level. Thus, photoaffinity approaches offer a relatively inexpensive and robust technique that will continue to play an important role in drug discovery for the foreseeable future.

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Section: Photaffinity Labeling Of Tubulinmentioning

confidence: 99%

Utilization of Photoaffinity Labeling to Investigate Binding of Microtubule Stabilizing Agents to P-Glycoprotein and β-Tubulin

2022

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“…Three stages may be described for this challenging task. First, drug-binding proteins can be identified using one of many strategies including affinity chromatography and photoaffinity labeling (Campos et al, 1996;Das, 2019;Muskens et al, 2019;Tulloch et al, 2017;Wang et al, 2012; Xiao and Li, 2020). Second, molecular effects of a drug can be documented in molecular studies after perturbing cells with drug concentrations that are not toxic (CEC 25 concentration, for example (Bachovchin et al, 2019)).…”

Section: Identification Of Physiological Targets Of Drugsmentioning

confidence: 99%

How Physiologic Targets Can Be Distinguished from Drug-Binding Proteins

Mensa-Wilmot

2021

Mol Pharmacol

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Some drugs in clinical trial owe their effectiveness to "off-target" activity. This and other observations raise a possibility that many studies to identify targets of drugs are incomplete. If "off-target" proteins are pharmacologically important it will be worthwhile to identify them early in the development process, for a better understanding of the molecular basis of drug action. Herein, we outline a multidisciplinary strategy for systematic identification of physiological targets of drugs in cells. A drugbinding protein whose genetic disruption yields very similar molecular effects as treatment of cells with the drug may be defined as a physiological target of the drug. For a drug developed with a "rational approach", it is desirable to verify experimentally that a protein used for hit optimization in vitro remains the sole polypeptide recognized by the drug in a cell.

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“…The highly reactive intermediates such as carbenes that are generated in the photoactivation process can indiscriminately label multiple sites and proteins within a cell. A modern more selective approach is detailed by Joydip Das (Das, 2018) to identify the binding sites of ethanol in a potential target protein Munc13-1, a presynaptic for inverse docking into the Astex data set : score-based (blue); receptor-average Z-score (orange); ligand average Z-score (green); and combined Z-score (red). The AUC value for each selection method is shown (from Kim et al, 2019) protein.…”

Section: F I G U R E 3 Peptide Sequencementioning

confidence: 99%

“…The highly reactive intermediates such as carbenes that are generated in the photoactivation process can indiscriminately label multiple sites and proteins within a cell. A modern more selective approach is detailed by Joydip Das (Das, ) to identify the binding sites of ethanol in a potential target protein Munc13‐1, a presynaptic protein. To do this, the author uses azi alcohols, derivatives of ethanol which contain a diaziridine moiety, for example, incubation of azi‐butanol with Munc 13‐1 followed by photochemical reaction and protein digestion gives a set of labelled peptides that can then be analysed by mass spectrometry and deconvoluted using the SEQUEST program to identify amino acids which have reacted with the generated carbene.…”

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confidence: 99%