Validation of Trifluoromethylphenyl Diazirine Cholesterol Analogues As Cholesterol Mimetics and Photolabeling Reagents

Abstract

Aliphatic diazirine analogues of cholesterol have been used previously to elaborate the cholesterol proteome and identify cholesterol binding sites on proteins. Cholesterol analogues containing the trifluoromethylphenyl diazirine (TPD) group have not been reported. Both classes of diazirines have been prepared for neurosteroid photolabeling studies and their combined use provided information that was not obtainable with either diazirine class alone. Hence, we prepared cholesterol TPD analogues and used them along with previously reported aliphatic diazirine analogues as photoaffinity labeling reagents to obtain additional information on the cholesterol binding sites of the pentameric Gloeobacter ligand-gated ion channel (GLIC). We first validated the TPD analogues as cholesterol substitutes and compared their actions with those of previously reported aliphatic diazirines in cell culture assays. All the probes bound to the same cholesterol binding site on GLIC but with differences in photolabeling efficiencies and residues identified. Photolabeling of mammalian (HEK) cell membranes demonstrated differences in the pattern of proteins labeled by the two classes of probes. Collectively, these date indicate that cholesterol photoaffinity labeling reagents containing an aliphatic diazirine or TPD group provide complementary information and will both be useful tools in future studies of cholesterol biology.

A bifunctional imidazolium-based cholesterol analog for the tracking of cellular cholesterol distributions and cholesterol–protein interactions

“…While photoactivable lipid analogs (including multifunctional ones) in general have been widely used for the investigation and manipulation of lipid biology, only comparably few examples have been reported on the development and application of corresponding cholesterol-based analogs. 11,13,14 Here, X-CHIM's structure differs from comparable, previously reported compounds in which the diazirine (and click functionality) is integrated in the membrane-embedded part of the molecule, thus potentially affecting crosslinking scope as well as biological behavior such as membrane integration. 14 To first evaluate basic functional properties of the synthesized X-CHIM we performed test-clicking/-crosslinking experiments under simplified conditions either in solution only (MeOH/H 2 O 70/30 + 0.1% formic acid) or in preformed DPPC liposomes in solution (Fig.…”

“…11,13,14 Here, X-CHIM's structure differs from comparable, previously reported compounds in which the diazirine (and click functionality) is integrated in the membrane-embedded part of the molecule, thus potentially affecting crosslinking scope as well as biological behavior such as membrane integration. 14 To first evaluate basic functional properties of the synthesized X-CHIM we performed test-clicking/-crosslinking experiments under simplified conditions either in solution only (MeOH/H 2 O 70/30 + 0.1% formic acid) or in preformed DPPC liposomes in solution (Fig. 2B).…”

Druggable Lipid Binding Sites in Pentameric Ligand-Gated Ion Channels and Transient Receptor Potential Channels

“…There is, however, some controversy whether PS inhibition of the GABA A R is mediated by this inner leaflet site, since this anionic neurosteroid is a rapid and effective inhibitor when applied extracellularly (Germann et al, 2019). This site may also be a cholesterol binding site based on a recent photolabeling study (Krishnan et al, 2021). Cryo-EM structures of the GABA A R and GlyR also show phospholipid densities in this site.…”

“…Moreover, lipid densities from cryo-EM and crystallography studies may not provide the resolution to definitively distinguish one lipid species from another. For these reasons, photo-affinity labeling, combined with residue-level identification of labeled sites by mass spectrometry, offers a complementary approach for identifying lipid binding sites, and has been utilized to identify cholesterol and neurosteroid binding sites in membrane proteins (Budelier et al, 2017a,b;Cheng et al, 2018;Krishnan et al, 2021). A recent study also showed that photo-affinity labeling can be used to identify binding sites for the polyunsaturated fatty acid, DHA, in a pLGIC, and to assess state-dependent binding of DHA at these sites (Dietzen et al, 2021).…”

“…The same strategy should be feasible for other lipids, such as sphingolipids (Dadsena et al, 2019), phospholipids and lysophospholipids (Xia and Peng, 2013;Peng et al, 2014), endocannabinoids (Dixon et al, 2012), or fatty acid metabolites for residue-level identification of binding sites. Developing new reagents with optimal photochemistry, especially the use of trifluoromethylphenyl diazirine (TPD) photoreactive groups, may be essential for this effort (Cheng et al, 2019;Dietzen et al, 2021;Krishnan et al, 2021). Once a site(s) is identified, competition photolabeling experiments with any lipid or ligand can be used to determine relative binding affinities at individual sites as well as state-dependent changes in binding affinity, as has extensively applied for anesthetic binding sites in the GABA A R (Li et al, 2010;Chiara et al, 2013;Jayakar et al, 2014Jayakar et al, , 2019.…”