Synthesis and biological evaluation of indole-2-carboxamides bearing photoactivatable functionalities as novel allosteric modulators for the cannabinoid CB1 receptor

Abstract

5-Chloro-3-ethyl-N-(4-(piperidin-1-yl)phenethyl)-1H–indole-2-carboxamide (ORG27569, 1) is a prototypical allosteric modulator for the cannabinoid CB1 receptor. Based on this indole-2-carboxamide scaffold, we designed and synthesized novel CB1 allosteric modulators that possess photoactivatable functionalities, which include benzophenone, phenyl azide, aliphatic azide and phenyltrifluoromethyldiazrine. To assess their allosteric effects, the dissociation constant (KB) and allosteric binding cooperativity factor (α) were determined and compared to their parent compounds. Within this series, benzophenone-containing compounds 26 and 27, phenylazide-containing compound 28, and the aliphatic azide containing compound 36b showed allosteric binding parameters (KB and α) comparable to their parent compound 1, 7, 8, and 9, respectively. We further assessed these modulators for their impact on G-protein coupling activity. Interestingly, these compounds exhibited negative allosteric modulator properties in a manner similar to their parent compounds, which antagonize agonist-induced G-protein coupling. These novel CB1 allosteric modulators, possessing photoactivatable functionalities, provide valuable tools for future photo-affinity labeling and mapping the CB1 allosteric binding site(s).

Assessing Allosteric Modulation of CB1 at the Receptor and Cellular Levels

“…Strikingly, the reduced expression of β-arrestin 1 nearly abolished ORG27569-induced ERK1/2 phosphorylation (but not that by CP55940), whereas cotransfection with β-arrestin 2 siRNA did not alter patterns of ERK1/2 phosphorylation (induced by either ligand) compared to those observed by control siRNA transfection. Analogues of ORG27569 are typically comparable (Ahn et al, 2012; Ahn, Mahmoud, Samala, et al, 2013; Ahn, Mahmoud, Shim, et al, 2013; Khurana et al, 2014; Mahmoud et al, 2013; Qiao et al, 2016), but other results have been observed (Gamage, Anderson, & Abood, 2016). …”

“…For example, Ross and colleagues used equilibrium binding and kinetic binding assays to evaluate ORG27569 and related compounds and found that they elicit a conformational change that increases agonist affinity for the orthosteric site, although probe dependence was also observed (Baillie et al, 2013; Khajehali et al, 2015; Price et al, 2005). It should be noted that some functional assays show that ORG27569 and several derivatives do not induce G i protein coupling with CB 1 (Ahn et al, 2012; Ahn, Mahmoud, Samala, et al, 2013; Baillie et al, 2013; Khurana et al, 2014; Mahmoud et al, 2013; Price et al, 2005; Qiao et al, 2016) unlike many orthosteric agonists that do (Howlett & Fleming, 1984), but these compounds do induce effects consistent with coupling to the β-arrestin isoforms 1 and 2 (Ahn et al, 2012; Ahn, Mahmoud, Samala, et al, 2013; Ahn, Mahmoud, Shim, et al, 2013; Khurana et al, 2014). …”

“…G protein binding assays determine the extent to which a PAM induces CB 1 coupling to the G i protein. To evaluate the extent of G protein coupling, as a result of the impact of an allosteric modulator, stimulation of 35 S-guanine 5′-3- O -(thio)triphosphate (GTPγS) binding (Ahn et al, 2012; Ahn, Mahmoud, Samala, et al, 2013; Baillie et al, 2013; Khurana et al, 2014; Mahmoud et al, 2013; Price et al, 2005; Qiao et al, 2016) or the level of cAMP accumulation is monitored (Baillie et al, 2013; Cawston et al, 2013; Khajehali et al, 2015; Laprairie, Kulkarni, et al, 2017) using cells or inhibition of electrically stimulated contractions in a vas deferens assay from mice is evaluated (Laprairie, Kulkarni, et al, 2017; Price et al, 2005). This has been carried out for ORG27569, PSNCBAM-1, and several analogues of these that function as PAMs with respect to agonist binding, yet most show inhibition of the extent of G protein coupling and some specifically G i coupling (Ahn et al, 2012; Ahn, Mahmoud, Samala, et al, 2013; Baillie et al, 2013; Khurana et al, 2014, 2017; Mahmoud et al, 2013; Qiao et al, 2016).…”

“…Since binding assays and G protein coupling assays can give apparently conflicting results (Ahn et al, 2012; Ahn, Mahmoud, Samala, et al, 2013; Khurana et al, 2014, 2017; Mahmoud et al, 2013; Price et al, 2005; Qiao et al, 2016), it is helpful to evaluate allosteric modulators by other means to determine if CB 1 functions as if it is activated or inactivated by the modulator. For example, following prolonged activation, GPCRs typically become desensitized and become internalized in cells (Appleyard, Patterson, Jin, & Chavkin, 1997; Jin et al, 1999; Kovoor, Nappey, Kieffer, & Chavkin, 1997; Krupnick & Benovic, 1998; Zhang et al, 1997).…”

Modulation of CB1 cannabinoid receptor by allosteric ligands: Pharmacology and therapeutic opportunities

“…molecular dynamics simulations) integrating crystal structure information with site-directed mutagenesis which may shed new light on the allosteric binding sites on the CB 1 receptor. Alternatively, photoactivatable analogues of indole-2-carboxamides such as ORG27569 with functionalities such as benzophenone, aliphatic or phenyl azide and phenyltrifluoromethyldiazrine have also been reported which may provide novel tools for mapping the allosteric binding site(s) on the CB 1 receptor (Qiao et al, 2016). …”