ABSTRACT␣4␤2 nicotinic acetylcholine receptors (nAChRs) are recognized as the principal nicotine binding site in brain. Recombinant ␣4␤2 nAChR demonstrate biphasic concentration-response relationships with low-and high-EC 50 components. This study shows that untranslated regions (UTR) can influence expression of high-sensitivity subforms of ␣4␤2 and ␣3␤2 nAChR. Oocytes injected with ␣4 and ␤2 RNA lacking UTR expressed biphasic concentration-response relationships for acetylcholine with high-sensitivity EC 50 values of 0.5 to 2.5 M (14 -24% of the population) and low-sensitivity EC 50 values of 110 to 180 M (76 -86%). In contrast, message with UTR expressed exclusively the high-sensitivity ␣4␤2 nAChR subform with an acetylcholine EC 50 value of 2.2 M. Additional studies revealed pharmacological differences between highand low-sensitivity ␣4␤2 subforms. Whereas the antagonists dihydro-␤-erythroidine (IC 50 of 3-6 nM) and methyllycaconitine (IC 50 of 40 -135 nM) were not selective between high-and low-sensitivity ␣4␤2, chlorisondamine, mecamylamine, and dtubocurarine were, respectively, 100-, 8-, and 5-fold selective for the ␣4␤2 subform with low sensitivity to acetylcholine. Conversely, agonists that selectively activated the high-sensitivity ␣4␤2 subform with respect to efficacy as well as potency were identified. Furthermore, two of these agonists were shown to activate mouse brain ␣4␤2 as well as the ferret high-sensitivity ␣4␤2 expressed in Xenopus laevis oocytes. With the use of UTR-containing RNA, exclusive expression of a novel highsensitivity ␣3␤2 nAChR was also achieved. These studies 1) provide further evidence for the existence of multiple subforms of ␣4␤2 nAChR, 2) extend that to ␣3␤2 nAChR, 3) demonstrate UTR influence on ␤2-containing nAChR properties, and 4) reveal compounds that interact with ␣4␤2 in a subformselective manner.Nicotinic acetylcholine receptors (nAChRs) are a diverse group of ligand-gated ion channels found in brain and spinal cord; autonomic, enteric, and sensory nervous systems; skeletal muscle; cochlea; and several non-neuronal cell types

The focus of the present study is the molecular and functional characterization of four splice variants of the human Nav1.3 alpha subunit. These subtypes arise due to the use of alternative splice donor sites of exon 12, which encodes a region of the alpha subunit that resides in the intracellular loop between domains I and II. This region contains several important phosphorylation sites that modulate Na+ channel kinetics in related sodium channels, i.e. Nav1.2. While three of the four Nav1.3 isoforms, 12v1, 12v3 and 12v4 have been previously identified in human, 12v2 has only been reported in rat. Herein, we evaluate the distribution of these splice variants in human tissues and the functional characterization of each of these subtypes. We demonstrate by reverse transcriptase-polymerase chain reaction (RT-PCR) that each subtype is expressed in the spinal cord, thalamus, amygdala, cerebellum, adult and fetal whole brain and heart. To investigate the functional properties of these different splice variants, each alpha subunit isoform was cloned by RT-PCR from human fetal brain and expressed in Xenopus oocytes. Each isoform exhibited functional voltage-dependent Na+ channels with similar sensitivities to tetrodotoxin (TTX) and comparable current amplitudes. Subtle shifts in the V 1/2 of activation and inactivation (2-3 mV) were observed among the four isoforms, although the functional significance of these differences remains unclear. This study has demonstrated that all four human splice variants of the Nav1.3 channel alpha subunit are widely expressed and generate functional TTX-sensitive Na+ channels that likely modulate cellular excitability.

IL-36 cytokines are pro-inflammatory members of the IL-1 family that are upregulated in inflammatory disorders. Specifically, IL-36γ is highly expressed in active psoriatic lesions and can drive pro-inflammatory processes in 3D human skin equivalents supporting a role for this target in skin inflammation. Small molecule antagonists of interleukins have been historically challenging to generate. Nevertheless, we performed a small molecule high-throughput screen to identify IL-36 antagonists using a novel TR-FRET binding assay. Several compounds, including 2-oxypyrimidine containing structural analogs of the marketed endothelin receptor A antagonist Ambrisentan, were identified as hits from the screen. A-552 was identified as a the most potent antagonist of human IL-36γ, but not the closely related family member IL-36α, was capable of attenuating IL-36γ induced responses in mouse and human disease models. Additionally, x-ray crystallography studies identified key amino acid residues in the binding pocket present in human IL-36γ that are absent in human IL-36α. A-552 represents a first-in-class small molecule antagonist of IL-36 signaling that could be used as a chemical tool to further investigate the role of this pathway in inflammatory skin diseases such as psoriasis.

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A HTS campaign identified compound 1, an excellent hit-like molecule to initiate medicinal chemistry efforts to optimize a dual ROCK1 and ROCK2 inhibitor. Substitution (2-Cl, 2-NH2, 2-F, 3-F) of the pyridine hinge binding motif or replacement with pyrimidine afforded compounds with a clean CYP inhibition profile. Cocrystal structures of an early lead compound were obtained in PKA, ROCK1, and ROCK2. This provided critical structural information for medicinal chemistry to drive compound design. The structural data indicated the preferred configuration at the central benzylic carbon would be (R), and application of this information to compound design resulted in compound 16. This compound was shown to be a potent and selective dual ROCK inhibitor in both enzyme and cell assays and efficacious in the retinal nerve fiber layer model after oral dosing. This tool compound has been made available through the AbbVie Compound Toolbox. Finally, the cocrystal structures also identified that aspartic acid residues 176 and 218 in ROCK2, which are glutamic acids in PKA, could be targeted as residues to drive both potency and kinome selectivity. Introduction of a piperidin-3-ylmethanamine group to the compound series resulted in compound 58, a potent and selective dual ROCK inhibitor with excellent predicted drug-like properties.