Chemical probes to potently and selectively inhibit endocannabinoid cellular reuptake

Abstract

The extracellular effects of the endocannabinoids anandamide and 2-arachidonoyl glycerol are terminated by enzymatic hydrolysis after crossing cellular membranes by facilitated diffusion. The lack of potent and selective inhibitors for endocannabinoid transport has prevented the molecular characterization of this process, thus hindering its biochemical investigation and pharmacological exploitation. Here, we report the design, chemical synthesis, and biological profiling of natural product-derived -substituted 2,4-dodecadienamides as a selective endocannabinoid uptake inhibitor. The highly potent (IC = 10 nM) inhibitor -(3,4-dimethoxyphenyl)ethyl amide (WOBE437) exerted pronounced cannabinoid receptor-dependent anxiolytic, antiinflammatory, and analgesic effects in mice by increasing endocannabinoid levels. A tailored WOBE437-derived diazirine-containing photoaffinity probe (RX-055) irreversibly blocked membrane transport of both endocannabinoids, providing mechanistic insights into this complex process. Moreover, RX-055 exerted site-specific anxiolytic effects on in situ photoactivation in the brain. This study describes suitable inhibitors to target endocannabinoid membrane trafficking and uncovers an alternative endocannabinoid pharmacology.

Novel analogs of PSNCBAM-1 as allosteric modulators of cannabinoid CB1 receptor

“…39, 40 Finally the functional activity of the new compounds was determined by SRE assay using HEK293 cells transfected with CB1R. 41 …”

“…39,40 Briefly, FAAH and MAGL activity assays were performed using U937 cell homogenate (100 μg) which were diluted in 200 μL of Tris–HCl 10 mM, EDTA 1 mM, pH 8 containing 0.1% fatty acid-free BSA. Compounds were added at the screening concentration of 10 μM and incubated for 30 min at 37 °C.…”

The Application of Fluorine‐Containing Reagents in Structural Proteomics

“…Instead of directly modulating the pharmacophore, a linker can be used, and the modified drug is challenged to undergo direct attachment at the pharmacophore through the CF 3 ‐diazirine group (Figure d). Chicca et al . synthesized probe 8 by replacing a methyl ether with an amide‐derived TPD and showed that the use of a linker can maintain efficacy.…”

“…Instead of directly modulating the pharmacophore,al inker can be used, and the modified drug is challenged to undergo direct attachment at the pharmacophore through the CF 3 -diazirine group (Figure 1d). Chicca et al [10] synthesized probe 8 by replacing amethyl ether with an amide-derived TPD and showed that the use of al inker can maintain efficacy.The design of TPD-based probes needs to be evaluated on ac ase-by-case basis.T PDs with aromatic rings may be analogues for pharmacophores that also contain aromatic rings to which aC F 3 -diazirine group can be attached. Even when the CF 3 -diazirine group is directly incorporated, structure-reactivity relationships (SARs) should be carefully considered.…”

Fluorierte Reagenzien in der Strukturproteomik

“…Anstatt den Pharmakophor direkt zu modifizieren, kann ein Linker verwendet werden, wenn ein modifizierter Wirkstoff dazu gezwungen wird, direkt mittels der CF 3 -Diazirin-Funktion an den Pharmakophor zu binden (Abbildung 1d). Chicca und Mitarbeiter synthetisierten Reagenz 8, [10] in dem eine Methyletherfunktion durch ein Amid-derivatisiertes TPD ersetzt wurde,w obei die Effizienz trotz des Linkers erhalten blieb.A lles in allem muss das Design eines TPD-basierten Reagenzes von Fall zu Fall evaluiert werden. TPDs mit aromatischen Ringen kçnnen als Analoga für Pharmakophore wirken, in denen eine CF 3 -Diazirin-Gruppe an das Aren gebunden wird.…”