G protein-receptor kinases 5/6 are the key regulators of G protein-coupled receptor 35-arrestin interactions.

Human G protein-coupled receptor 35 is regulated by agonist-mediated phosphorylation of a set of five phospho-acceptor amino acids within its C-terminal tail. Alteration of both Ser and Ser to alanine in the GPR35a isoform greatly reduces the ability of receptor agonists to promote interactions with arrestin adapter proteins. Here, we have integrated the use of cell lines genome edited to lack expression of combinations of G protein receptor kinases (GRKs), selective small molecule inhibitors of subsets of these kinases, and antisera able to specifically identify either human GPR35a or mouse GPR35 only when Ser and Ser (orce; the equivalent residues in mouse GPR35) have become phosphorylated to demonstrate that GRK5 and GRK6 cause agonist-dependent phosphorylation of these residues.

G protein-coupled receptor kinase 6 (GRK6) regulates insulin processing and secretion via effects on proinsulin conversion to insulin.

Recent studies identified a missense mutation in the gene coding for G protein-coupled receptor kinase 6 (GRK6) that segregates with type 2 diabetes (T2D). To better understand how GRK6 might be involved in T2D, we used pharmacological inhibition and genetic knockdown in the mouse β-cell line, MIN6, to determine whether GRK6 regulates insulin dynamics. We show inhibition of GRK5 and GRK6 increased insulin secretion but reduced insulin processing while GRK6 knockdown revealed these same processing defects with reduced levels of cellular insulin.

Design, Synthesis, and Characterization of 4-Aminoquinazolines as Potent Inhibitors of the G Protein-Coupled Receptor Kinase 6 (GRK6) for the Treatment of Multiple Myeloma.

Both previous and additional genetic knockdown studies reported herein implicate G protein-coupled receptor kinase 6 (GRK6) as a critical kinase required for the survival of multiple myeloma (MM) cells. Therefore, we sought to develop a small molecule GRK6 inhibitor as an MM therapeutic. From a focused library of known kinase inhibitors, we identified two hits with moderate biochemical potencies against GRK6.

Identification of RSK and TTK as Modulators of Blood Vessel Morphogenesis Using an Embryonic Stem Cell-Based Vascular Differentiation Assay.

Blood vessels are formed through vasculogenesis, followed by remodeling of the endothelial network through angiogenesis. Many events that occur during embryonic vascular development are recapitulated during adult neoangiogenesis, which is critical to tumor growth and metastasis. Current antiangiogenic tumor therapies, based largely on targeting the vascular endothelial growth factor pathway, show limited clinical benefits, thus necessitating the discovery of alternative targets.

Recent progress on MAP kinase pathway inhibitors.

The RAS-RAF-MEK-ERK, or ERK signaling pathway propagates signals through an intracellular signal transduction cascade. Since approximately one third of human cancers are impacted by mutations in the ERK signaling pathway, intensive efforts to develop drugs targeting members of this cascade are ongoing. While efforts to develop drugs aimed at inhibiting RAS are still at an early stage, substantial progress in discovering clinical drugs targeting RAF, MEK, and ERK have been made.

Dabrafenib; preclinical characterization, increased efficacy when combined with trametinib, while BRAF/MEK tool combination reduced skin lesions.

Mitogen-Activated Protein Kinase (MAPK) pathway activation has been implicated in many types of human cancer. BRAF mutations that constitutively activate MAPK signalling and bypass the need for upstream stimuli occur with high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma. In this report we characterize the novel, potent, and selective BRAF inhibitor, dabrafenib (GSK2118436).

Discovery of Dabrafenib: A Selective Inhibitor of Raf Kinases with Antitumor Activity against B-Raf-Driven Tumors.

Hyperactive signaling of the MAP kinase pathway resulting from the constitutively active B-Raf(V600E) mutated enzyme has been observed in a number of human tumors, including melanomas. Herein we report the discovery and biological evaluation of GSK2118436, a selective inhibitor of Raf kinases with potent in vitro activity in oncogenic B-Raf-driven melanoma and colorectal carcinoma cells and robust in vivo antitumor and pharmacodynamic activity in mouse models of B-Raf(V600E) human melanoma. GSK2118436 was identified as a development candidate, and early clinical results have shown significant activity in patients with B-Raf mutant melanoma.

Development of potent B-RafV600E inhibitors containing an arylsulfonamide headgroup.

A potent series of inhibitors against the B-Raf(V600E) kinase have been developed that show excellent activity in cellular assays and good oral bioavailability in rats. The key structural features of the series are an arylsulfonamide headgroup, a thiazole core, and a fluorine ortho to the sulfonamide nitrogen.

Synthesis and evaluation of aniline headgroups for alkynyl thienopyrimidine dual EGFR/ErbB-2 kinase inhibitors.

Aniline 'headgroups' were synthesized and incorporated into an alkynyl thienopyrimidine series of EGFR and ErbB-2 inhibitors. Potent inhibition of enzyme activity and cellular proliferation was observed. In certain instances, protein binding was reduced and oral exposure was found to be somewhat improved relative to compounds containing the reference aniline.

Thienopyrimidine-based dual EGFR/ErbB-2 inhibitors.

Two new series of potent and selective dual EGFR/ErbB-2 kinase inhibitors derived from novel thienopyrimidine cores have been identified. Isomeric thienopyrimidine cores were evaluated as isosteres for a 4-anilinoquinazoline core and several analogs containing the thieno[3,2-d]pyrimidine core showed anti-proliferative activity with IC(50) values less than 1 microM against human tumor cells in vitro.

Discovery and optimization of imidazo[1,2-a]pyridine inhibitors of insulin-like growth factor-1 receptor (IGF-1R).

The optimization of imidazo[1,2-a]pyridine inhibitors as potent and selective inhibitors of IGF-1R is presented. Further optimization of oral exposure in mice is also discussed. Detailed selectivity, in vitro activity, and in vivo PK profiles of an optimized compound is also highlighted.

Synthesis and stereochemical effects of pyrrolidinyl-acetylenic thieno[3,2-d]pyrimidines as EGFR and ErbB-2 inhibitors.

A novel class of pyrrolidinyl-acetyleneic thieno[3,2-d]pyrimidines has been identified which potently inhibit the EGFR and ErbB-2 receptor tyrosine kinases. Synthetic modifications of the pyrrolidine carbamate moiety result in a range of effects on enzyme and cellular potency. In addition, the impact of the absolute stereochemical configuration on cellular potency and oral mouse pharmacokinetics is described.

Dual EGFR/ErbB-2 inhibitors from novel pyrrolidinyl-acetylenic thieno[3,2-d]pyrimidines.

A novel class of substituted pyrrolidinyl-acetylenic thieno[3,2-d]pyrimidines has been identified that are potent and selective inhibitors of both EGFR/ErbB-2 receptor tyrosine kinases. The inhibitors are found to display a range of enzyme and cellular potency and also to display a varying level of covalent modification of the kinase targets. Selected molecules, including compound 15h, were found to be potent in enzymatic and cellular assays while also demonstrating exposure in the mouse from an oral dose.

6-Ethynylthieno[3,2-d]- and 6-ethynylthieno[2,3-d]pyrimidin-4-anilines as tunable covalent modifiers of ErbB kinases.

Analysis of the x-ray crystal structure of mono-substituted acetylenic thienopyrimidine 6 complexed with the ErbB family enzyme ErbB-4 revealed a covalent bond between the terminal carbon of the acetylene moiety and the sulfhydryl group of Cys-803 at the solvent interface. The identification of this covalent adduct suggested that acetylenic thienopyrimidine 6 and related analogs might also be capable of forming an analogous covalent adduct with EGFR, which has a conserved cysteine (797) near the ATP binding pocket. To test this hypothesis, we treated a truncated, catalytically competent form of EGFR (678-1020) with a structurally related propargylic amine (8).

Synthesis and evaluation of potent and selective beta3 adrenergic receptor agonists containing heterobiaryl carboxylic acids.

The design, synthesis, and SAR of a novel series of heterobiaryl phenethanolamine beta3 adrenergic receptor agonists are described. The furan analogue 49 was shown to elicit a significant dose-dependent lowering of plasma glucose in a rodent model of type 2 diabetes.

Biarylaniline phenethanolamines as potent and selective beta3 adrenergic receptor agonists.

The synthesis of a series of phenethanolamine aniline agonists that contain an aniline ring on the right-hand side of the molecule substituted at the meta position with a benzoic acid or a pyridyl carboxylate is described. Several of the analogues (e.g.

Alkynyl pyrimidines as dual EGFR/ErbB2 kinase inhibitors.

Anilinoalkynylpyrimidines were prepared and evaluated as dual EGFR/ErbB2 kinase inhibitors. A preference was found for substituted phenyl and heteroaromatic rings attached to the alkyne. In addition, the presence of a potential hydrogen bond donor appended to this ring was favored.

Synthesis and evaluation of potent and selective beta(3) adrenergic receptor agonists containing acylsulfonamide, sulfonylsulfonamide, and sulfonylurea carboxylic acid isosteres.

Starting from phenethanolamine aniline leads 3a and 3b, we have identified a series of functionally potent and selective beta(3) adrenergic receptor (AR) agonists containing acylsulfonamide, sulfonylsulfonamide, or sulfonylurea groups within the aniline phenethanolamine series. In beta(3), beta(2), and beta(1) AR cAMP functional assays, 3a and other right-hand side (RHS) carboxylate analogues were found to be full agonists that were modestly selective against beta(1) or beta(2) ARs, while analogues lacking RHS acid functionality were active at beta(3) AR but not selective. Replacement of the carboxylate with acylthiazole and acylmethylsulfone gave potent, but only modestly selective, compounds.