Chemical mechanisms for photoaffinity labeling of receptors

Cavalla, David; Neff, Norton H.

doi:10.1016/0006-2952(85)90001-2

Cited by 39 publications

(11 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…37 Membranes from human CB2 receptors (hCB2) expressed in HEK293 38 were incubated with the azido ligands ( 20 and 17 ) in concentrations of 10-fold their K i values for 30 min at 37 °C in a water bath with gentle agitation and then exposed to UV (254 nm) for 1 min to activate the ligand. 39 Unbound excess ligand was washed out twice with 1% BSA in TME. This was followed by an additional washing to remove residual BSA, and the membranes were isolated by centrifugation (Beckman Coulter, JA 20, 17 000 rpm, 10 min, 25 °C).…”

Section: Methodsmentioning

confidence: 99%

3′-Functionalized Adamantyl Cannabinoid Receptor Probes

et al. 2015

View full text Add to dashboard Cite

The aliphatic side chain plays a pivotal role in determining the cannabinergic potency of tricyclic classical cannabinoids, and we have previously shown that this chain could be substituted successfully by adamantyl or other polycyclic groups. In an effort to explore the pharmacophoric features of these conformationally fixed groups, we have synthesized a series of analogues in which the C3 position is substituted directly with an adamantyl group bearing functionality at one of the tertiary carbon atoms. These substituents included the electrophilic isothiocyanate and photoactivatable azido groups, both of which are capable of covalent attachment with the target protein. Our results show that substitution at the 3′-adamantyl position can lead to ligands with improved affinities and CB1/CB2 selectivities. Our work has also led to the development of two successful covalent probes with high affinities for both cannabinoid receptors, namely, the electrophilic isothiocyanate AM994 and the photoactivatable aliphatic azido AM993 analogues.

show abstract

Section: Methodsmentioning

confidence: 99%

3′-Functionalized Adamantyl Cannabinoid Receptor Probes

et al. 2015

View full text Add to dashboard Cite

show abstract

“…Covalent ligands can generally be divided into two categories, namely, photoactivatable and electrophilic ligands. Photoactivatable ligands carry a chemically inert group which upon irradiation with a suitable light source is converted to a highly reactive species—usually a carbene or nitrene—capable of forming covalent bonds with amino acid residues situated in their immediate vicinity (39,40). Ligands carrying an aromatic azido group have found many applications as photolabeling reagents.…”

Section: Covalent Probesmentioning

confidence: 99%

“…Ligands carrying an aromatic azido group have found many applications as photolabeling reagents. Irradiation of these compounds after they are allowed to bind to their respective binding site(s), results in the formation of a highly reactive nitrene that is capable of insertion into single and multiple bonds (39,40). Thus, the ligand is first allowed to interact selectively but noncovalently with its binding site and subsequently activated to develop a covalent attachment with an amino acid residue at or near the binding site.…”

Section: Covalent Probesmentioning

confidence: 99%

Ligand based structural studies of the CB1 cannabinoid receptor

Picone

Fournier

Makriyannis

2002

The Journal of Peptide Research

View full text Add to dashboard Cite

The structural characterization of G-protein coupled receptors (GPCRs) is quite important as these proteins represent a vast number of therapeutic targets involved in drug discovery. However, solving the three-dimensional structure of GPCR has been a significant obstacle in structural biology. A variety of reasons, including their large molecular weight, intricate interhelical packing, as well as their membrane-associated topology, has hindered efforts aimed at their purification. In the absence of pure protein, available in the native conformation, classical methods of structural analysis such as X-ray crystallography and nuclear magnetic resonance spectroscopy cannot be utilized successfully. Alternative methods must therefore be explored to facilitate the structural features involved in drug-receptor interactions. The methods described herein detail the use of covalent probes, or affinity labels, capable of binding covalently to a target GPCR at its binding site(s). Our approach involves the incorporation of a number of reactive moieties in different regions of the ligand molecule each of which is expected to react with different amino acid residues. Information obtained from such work coupled with computer modeling and validated by the use of site-directed mutagenesis of GPCRs allows for three-dimensional mapping of the receptor binding site. It also sheds light on the different possible binding motifs for the various classes of agonists and antagonists and identifies amino acid residues involved with GPCR activation or inactivation.

show abstract

“…An advantage of photoactivated ligands is that the highly reactive species generated in situ is able to form a covalent bond with any amino acid nearby, compared with electrophilic covalent ligands that are limited to reaction with only one or two nucleophilic amino acid side chains (Sumranjit and Chung, 2013). The highly reactive species generated can covalently bind to the receptors through insertion into bonds (e.g., C-H) in the peptide backbone and amino acid side chains (Cavalla and Neff, 1985). Desirable properties of a photoactivated ligand include high receptor affinity, activatable at a wavelength that does not cause damage to the target receptor or biosystem, the generation of a reactive group with a short lifetime, indiscriminate or finely-tuned formation of a covalent bond with any nearby amino acid, and formation of a stable irreversible adduct (Vodovozova, 2007).…”

Section: B Applications Of Covalent Toolsmentioning

confidence: 99%

Chemical Tools for Studying Lipid-Binding Class A G Protein–Coupled Receptors

et al. 2017

View full text Add to dashboard Cite

Cannabinoid, free fatty acid, lysophosphatidic acid, sphingosine 1-phosphate, prostanoid, leukotriene, bile acid, and platelet-activating factor receptor families are class A G protein-coupled receptors with endogenous lipid ligands. Pharmacological tools are crucial for studying these receptors and addressing the many unanswered questions surrounding expression of these receptors in normal and diseased tissues. An inherent challenge for developing tools for these lipid receptors is balancing the often lipophilic requirements of the receptor-binding pharmacophore with favorable physicochemical properties to optimize highly specific binding. In this study, we review the radioligands, fluorescent ligands, covalent ligands, and antibodies that have been used to study these lipid-binding receptors. For each tool type, the characteristics and design rationale along with in vitro and in vivo applications are detailed.

show abstract

Chemical mechanisms for photoaffinity labeling of receptors

Cited by 39 publications

References 44 publications

3′-Functionalized Adamantyl Cannabinoid Receptor Probes

3′-Functionalized Adamantyl Cannabinoid Receptor Probes

Ligand based structural studies of the CB1 cannabinoid receptor

Chemical Tools for Studying Lipid-Binding Class A G Protein–Coupled Receptors

Contact Info

Product

Resources

About