In vitro activity: ML335 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. ML335 selectively activates OPRM1-OPRD1 heterodimerization which may have potential use in the treatment of pain and alleviate unwanted effects associated with opiate use. The Scripps Research Institute Molecular Screening Center (SRIMSC) discovered ML335 as an agonist for OPRM1-OPRD1 heterdimerization with an EC50 of 403 nM, and selectivities vs. OPRM1, OPRD1, and HTR5A of 37, 2.7, and>99, respectively. Heteromerization of OPRM1 with OPRD1 leads to the modulation of receptor binding and signaling properties. It has further been shown that the selective activation of the OPRM1-OPRD1 heteromer by a combination of OPRM1 agonist with OPRD1 antagonist can be blocked by antibodies that selectively recognize the heteromer.

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.