BRAF is among the most frequently mutated oncogenes in human cancers. Multiple small molecule BRAF kinase inhibitors have been approved for treating melanoma carrying BRAF-V600 mutations. However, the benefits of BRAF kinase inhibitors are generally short-lived. Small molecule-mediated targeted protein degradation has recently emerged as a novel pharmaceutical strategy to remove disease proteins through hijacking the cellular ubiquitin proteasome system (UPS). In this study, we developed thalidomide-based heterobifunctional compounds that induced selective degradation of BRAF-V600E, but not the wild-type BRAF. Downregulation of BRAF-V600E suppressed the MEK/ERK kinase cascade in melanoma cells and impaired cell growth in culture. Abolishing the interaction between degraders and cereblon or blocking the UPS significantly impaired the activities of these degraders, validating a mechanistic role of UPS in mediating targeted degradation of BRAF-V600E. These findings highlight a new approach to modulate the functions of oncogenic BRAF mutants and provide a framework to treat BRAF-dependent human cancers.
Highly potent human glucagon receptor (hGluR) antagonists have been prepared employing both medicinal chemistry and targeted libraries based on modification of the core (proximal) dimethoxyphenyl group, the benzyl ether linkage, as well as the (distal) benzylic aryl group of the lead 2, 3-cyano-4-hydroxybenzoic acid (3,5-dimethoxy-4-isopropylbenzyloxybenzylidene)hydrazide. Electron-rich proximal aryl moieties such as mono- and dimethoxy benzenes, naphthalenes, and indoles were found to be active. The SAR was found to be quite insensitive regarding the linkage to the distal aryl group, since long and short as well as polar and apolar linkers gave highly potent compounds. The presence of a distal aryl group was not crucial for obtaining high binding affinity to the hGluR. In many cases, however, the affinity could be further optimized with substituted distal aryl groups. Representative compounds have been tested for in vitro metabolism, and structure-metabolism relationships are described. These efforts lead to the discovery of 74, NNC 25-2504, 3-cyano-4-hydroxybenzoic acid [1-(2,3,5,6-tetramethylbenzyl)-1H-indol-4-ylmethylene]hydrazide, with low in vitro metabolic turnover. 74 was a highly potent noncompetitive antagonist of the human glucagon receptor (IC(50) = 2.3 nM, K(B) = 760 pM) and of the isolated rat receptor (IC(50) = 430 pM, K(B) = 380 pM). Glucagon-stimulated glucose production from isolated primary rat hepatocytes was inhibited competitively by 74 (K(i) = 14 nM). This compound was orally available in dogs (F(po) = 15%) and was active in a glucagon-challenged rat model of hyperglucagonemia and hyperglycemia.
A weak human glucagon receptor antagonist with an IC50 of 7 microM was initially found by screening of libraries originally targeted to mimic the binding of the glucagon-like peptide (GLP-1) hormone to its receptor. Optimization of this hit for binding affinity for the glucagon receptor led to ligands with affinity in the nanomolar range. In addition to receptor binding, optimization efforts were made to stabilize the molecules against fast metabolic turnover. A potent antagonist of the human human glucagon receptor was obtained that had 17% oral availability in rats with a plasma half-life of 90 min. The major metabolites of this lead were identified and used to further optimize this series with respect to pharmacokinetic properties. This final optimization led to a potent glucagon antagonist that was orally available in rats and dogs and was efficacious in lowering blood glucose levels in a diabetic animal model.
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