Design of Aminobenzothiazole Inhibitors of Rho Kinases 1 and 2 by Using Protein Kinase†A as a Structure Surrogate.

We describe the design, synthesis, and structure-activity relationships (SARs) of a series of 2-aminobenzothiazole inhibitors of Rho kinases (ROCKs) 1 and 2, which were optimized to low nanomolar potencies by use of protein kinase A (PKA) as a structure surrogate to guide compound design. A subset of these molecules also showed robust activity in a cell-based myosin phosphatase assay and in a mechanical hyperalgesia in vivo pain model.

Small Molecule IL-36γ Antagonist as a Novel Therapeutic Approach for Plaque Psoriasis.

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.

Identification of Selective Dual ROCK1 and ROCK2 Inhibitors Using Structure-Based Drug Design.

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.

Structure-activity studies and analgesic efficacy of N-(3-pyridinyl)-bridged bicyclic diamines, exceptionally potent agonists at nicotinic acetylcholine receptors.

A series of exceptionally potent agonists at neuronal nicotinic acetylcholine receptors (nAChRs) has been investigated. Several N-(3-pyridinyl) derivatives of bridged bicyclic diamines exhibit double-digit-picomolar binding affinities for the alpha 4 beta 2 subtype, placing them with epibatidine among the most potent nAChR ligands described to date. Structure-activity studies have revealed that substitutions, particularly hydrophilic groups in the pyridine 5-position, differentially modulate the agonist activity at ganglionic vs central nAChR subtypes, so that improved subtype selectivity can be demonstrated in vitro. Analgesic efficacy has been achieved across a broad range of pain states, including rodent models of acute thermal nociception, persistent pain, and neuropathic allodynia. Unfortunately, the hydrophilic pyridine substituents that were shown to enhance agonist selectivity for central nAChRs in vitro tend to limit CNS penetration in vivo, so that analgesic efficacy with an improved therapeutic window was not realized with those compounds.

High- and low-sensitivity subforms of alpha4beta2 and alpha3beta2 nAChRs.

Previous studies in other laboratories have shown that alpha4beta2 nicotinic acetylcholine receptor (nAChR) exhibits a biphasic concentration-response relationship for ACh with low and high EC50 components, and that the low EC50 component can be augmented by decreasing the alpha4:beta2 message ratio or incubating overnight in nicotine or at low temperature (Zwart and Vijverberg, 1998; Covernton and Connolly, 2000; Buisson and Bertrand, 2001; Nelson et al., 2003; Zhou et al., 2003). In the process of cloning ferret nAChR subunits, we found alpha4 and beta2 messages with long untranslated regions (UTRs), as well as those with no UTRs. Combinations of these messages revealed that the presence of UTRs influenced the ability to exclusively express high-sensitivity subforms of alpha4beta2 and alpha3beta2 nAChRs. Injection of oocytes with alpha4 and beta2 RNAs lacking UTRs (1:1 ratio) led to expression of a biphasic concentration-response relationship for ACh with EC50 values of 0.5 (high sensitivity) and 114 microM(low sensitivity). Decreasing the alpha4:beta2 message ratio to as much as 1:120 increased the high-sensitivity component slightly, but the ACh concentration response remained biphasic. In contrast, injection of messages with UTRs (1:1 ratio) led to expression of a monophasic concentration response to ACh and a high-sensitivity EC50 value of 2.3 microM, as shown in Fig. 1.

Untranslated region-dependent exclusive expression of high-sensitivity subforms of alpha4beta2 and alpha3beta2 nicotinic acetylcholine receptors.

alpha4beta2 nicotinic acetylcholine receptors (nAChRs) are recognized as the principal nicotine binding site in brain. Recombinant alpha4beta2 nAChR demonstrate biphasic concentration-response relationships with low- and high-EC50 components. This study shows that untranslated regions (UTR) can influence expression of high-sensitivity subforms of alpha4beta2 and alpha3beta2 nAChR. Oocytes injected with alpha4 and beta2 RNA lacking UTR expressed biphasic concentration-response relationships for acetylcholine with high-sensitivity EC50 values of 0.5 to 2.5 microM (14-24% of the population) and low-sensitivity EC50 values of 110 to 180 microM (76-86%). In contrast, message with UTR expressed exclusively the high-sensitivity alpha4beta2 nAChR subform with an acetylcholine EC50 value of 2.2 microM. Additional studies revealed pharmacological differences between high- and low-sensitivity alpha4beta2 subforms. Whereas the antagonists dihydro-beta-erythroidine (IC50 of 3-6 nM) and methyllycaconitine (IC50 of 40-135 nM) were not selective between high- and low-sensitivity alpha4beta2, chlorisondamine, mecamylamine, and d-tubocurarine were, respectively, 100-, 8-, and 5-fold selective for the alpha4beta2 subform with low sensitivity to acetylcholine. Conversely, agonists that selectively activated the high-sensitivity alpha4beta2 subform with respect to efficacy as well as potency were identified. Furthermore, two of these agonists were shown to activate mouse brain alpha4beta2 as well as the ferret high-sensitivity alpha4beta2 expressed in Xenopus laevis oocytes. With the use of UTR-containing RNA, exclusive expression of a novel high-sensitivity alpha3beta2 nAChR was also achieved. These studies 1) provide further evidence for the existence of multiple subforms of alpha4beta2 nAChR, 2) extend that to alpha3beta2 nAChR, 3) demonstrate UTR influence on beta2-containing nAChR properties, and 4) reveal compounds that interact with alpha4beta2 in a subform-selective manner.