In vitro activity: ML402 is a potent and selective activator of TREK-1/-2 and an agonist for OPRM1-OPRD1 (OPRM1: opioid receptor mu 1; OPRD1: delta opioid receptor) heterdimerization. ML402 selectively activates OPRM1-OPRD1 heterodimerization which may have potential use in the treatment of pain and alleviate unwanted effects associated with opiate use.

Kinase Assay: Xenopus oocyte two-electrode voltage-clamp measurements show that ML335 and ML402 activate K2P2.1 and K2P10.1 but not K2P4.1 (14.3±2.7 μM, K2P2.1-ML335; 13.7±7.0 μM, K2P2.1-ML402; 5.2±0.5 μM, K2P10.1-ML335; and 5.9±1.6 μM, K2P10.1-ML402). Swapping the Lys271 equivalent between K2P2.1 and K2P4.1 results in a clear phenotype reversal for ML335 and M402 activation. ML335 and ML402 activate K2P2.1 in HEK293 cells similar to their effects in Xenopus oocytes (5.2±0.8 μM and 5.9±1.6 μM for ML335 and ML402, respectively (n≥3));

Primary uHTS assay to identify agonists of OPRD1-OPRM1 heterodimerization : the purpose of this assay is to identify compounds that activate heterodimer formation between the OPRM1 and OPRD1 opioid receptors, resulting in membrane recruitment of beta-arrestin. The assay monitors GPCR-beta-arrestin proximity using low affinity fragment complementation of beta-galactosidase (beta-gal). This assay employs U2OS cells which express OPRD1, OPRM1 fused to a beta-gal peptide fragment (enzyme donor), and beta-arrestin fused to the complementary beta-gal fragment (enzyme acceptor). Cells are incubated with test compound, followed by measurement of well luminescence. As designed, compounds that induce formation of OPRD1 homodimers or OPRM1-OPRD1 heterodimers will cause beta-arrestin recruitment, resulting in reconstitution of the beta-gal holoenzyme. The reconstituted holoenzyme can then catalyze the hydrolysis of a substrate which yields a chemiluminescent signal, resulting in increased well luminescence. Deltorphin B is used as the high control for agonists, and wells containing cells treated with DMSO are used as the low control. Compounds were tested in singlicate or triplicate at a final nominal concentration of 9.3 μM, or in triplicate using a 10-point, 1:3 dilution series, starting at a nominal test concentration of 93 μM.

Cell Assay: Mouse K2P2.1, human K2P4.1, and mutants are expressed from a previously described pIRES2-EGFP vector in HEK293T cells (ATTC). 70% confluent cells are transfected (in 35-mm diameter wells) with LipofectAMINE 2000 for 6 h, and plated onto coverslips coated with Matrigel. Effects of ML335, ML402 and arachidonic acid on K2P2.1 current at 0 mV are measured by whole-cell patch-clamp experiments 24 h after transfection. Acquisition and analysis are performed using pCLAMP9 and an Axopatch 200B amplifier. Pipette resistance ranges from 1 to 1.5 MΩ. Pipette solution contains the following: 145 mM KCl, 3 mM MgCl2, 5 mM EGTA and 20 mM HEPES (pH 7.2 with KOH). Bath solution contains the following: 145 mM NaCl, 5 mM KCl, 1 mM CaCl2, 3 mM MgCl2 and 20 mM HEPES (pH 7.4 with NaOH). K2P2.1 currents are elicited by a 1 s ramp from -100 to +50 mV from a -80 mV holding potential. After stabilization of the basal current, ML335 and ML402 are perfused at 200 mL per hour until potentiation is stably reached.