ag-490 and degrasyn

AG490 is a tyrosine kinase inhibitor with activity against Jak2 and apoptotic activity in specific leukemias. Due to its weak kinase inhibitory activity and poor pharmacology, we conducted a cell-based screen for derivatives with improved Jak2 inhibition and activity in animals. Two hits emerged from an initial small chemical library screen, and more detailed structure-activity relationship studies led to the development of WP1130 with 50-fold greater activity in suppressing Jak2-dependent cytokine signaling than AG490. However, WP1130 did not directly suppress Jak2 kinase activity, but mediated Jak2 ubiquitination resulting in its trafficking through HDAC6 to perinuclear aggresomes without cytokine stimulation or SOCS-1 induction. Jak2 primarily contained K63-linked ubiquitin polymers, and mutation of this lysine blocked Jak2 ubiquitination and mobilization in WP1130-treated cells. Further analysis demonstrated that WP1130, but not AG490, acts as a deubiquitinating enzyme (DUB) inhibitor, possibly through a Michael addition reaction. We conclude that chemical modification of AG490 resulted in development of a DUB inhibitor with activity against a DUB capable of modulating Jak2 ubiquitination, trafficking and signal transduction.

Topics: Amino Acid Substitution; Cell Line, Tumor; Cell Proliferation; Cyanoacrylates; Drug Evaluation, Preclinical; Endopeptidases; Enzyme Assays; Histone Deacetylase 6; Histone Deacetylases; HSP90 Heat-Shock Proteins; Humans; Inhibitory Concentration 50; Interleukin-6; Janus Kinase 2; Mutant Proteins; Nitriles; Phosphorylation; Protease Inhibitors; Proteasome Inhibitors; Protein Transport; Proteolysis; Pyridines; Signal Transduction; STAT3 Transcription Factor; STAT5 Transcription Factor; Tyrphostins; Ubiquitin; Ubiquitination