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Aliphatic Azides as Selective Cysteine Labeling Reagents for Integral Membrane Proteins
Dennis Szymanski1, Malvina Papanastasiou1, Lakshmipathi Pandarinathan1
1Center for Drug Discovery , Northeastern University , 116 Mugar Hall, 360 Huntington Avenue , Boston , Massachusetts 02115 , United States.
Journal of Medicinal Chemistry
|November 17, 2018
Summary
Cannabinoid receptor inverse agonists with aliphatic azides covalently label proteins. These probes, upon UV activation, form imines that selectively attach to cysteine residues, revealing a precise labeling mechanism.
Area of Science:
- Medicinal Chemistry
- Biochemistry
- Pharmacology
Background:
- Cannabinoid receptors are key targets for G-protein-coupled receptor (GPCR) drug development.
- Aliphatic azido ligands offer a route for covalent labeling of these receptors upon ultraviolet activation.
Purpose of the Study:
- To elucidate the mechanism of covalent attachment of AM1335 and its deuterated analog AM1335(d10) to model substrates.
- To investigate the selectivity of these covalent probes for protein labeling.
Main Methods:
- Utilized human cannabinoid 2 receptor (hCB2R) TMH6 peptide and N-acyl-protected cysteine (NAC) as model substrates.
- Analyzed photochemical reaction products using tandem electrospray ionization mass spectrometry and deuterium exchange mass spectrometry.
Main Results:
- Identified a reaction pathway involving nitrene rearrangement to an imine intermediate.
- Demonstrated preferential labeling of protein cysteine residues by the aliphatic azido ligands.
- AM1335 and AM1335(d10) showed selective covalent attachment to cysteine.
Conclusions:
- The mechanism involves UV-induced nitrene formation, rearrangement to an imine, and subsequent reaction with cysteine sulfhydryl groups.
- Aliphatic azido covalent probes exhibit greater selectivity than previously assumed.
- These findings support the use of such probes for targeted protein labeling in biological systems.
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