RT Journal Article SR Electronic T1 The 19S Deubiquitinase Inhibitor b-AP15 is Enriched in Cells and Elicits Rapid Commitment to Cell Death JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP mol.113.091322 DO 10.1124/mol.113.091322 A1 Xin Wang A1 William Stafford A1 Magdalena Mazurkiewicz A1 Marten Fryknas A1 Slavica Brjnic A1 Xiaonan Zhang A1 Joachim Gullbo A1 Rolf Larsson A1 Elias Arner A1 Padraig D'Arcy A1 Stig Linder YR 2014 UL http://molpharm.aspetjournals.org/content/early/2014/04/08/mol.113.091322.abstract AB b-AP15 is a small molecule inhibitor of the USP14/UCHL5 deubiquitinases of the 19S proteasome which shows anti-tumor activity in a number of tumor models, including multiple myeloma. b-AP15 contains an α, β-unsaturated carbonyl unit which is likely to react with intracellular nucleophiles such as cysteine thiolates by Michael addition. We found that binding of b-AP15 to USP14 is partially reversible, and that inhibition of proteasome function is reversible in cells. Despite reversible binding, tumor cells are rapidly committed to apoptosis/cell death after exposure to b-AP15. We show that b-AP15 is rapidly taken up from the medium and enriched in cells. Enrichment provides an explanation to the stronger potency of the compound in cellular assays compared to in vitro biochemical assays. Cellular uptake was impaired by 30 min pre-treatment of cells with low concentrations of N-ethylmaleimide (10 μM), suggesting that enrichment was thiol dependent. We report that in addition to inhibition of deubiquitinases, b-AP15 inhibits the selenoprotein thioredoxin reductase (TrxR). Whereas proteasome inhibition was closely associated with cell death induction, inhibition of TrxR was not. TrxR inhibition is, however, likely to contribute to triggering of oxidative stress observed with b-AP15. Furthermore, we present structure-activity, in vivo pharmacokinetic and hepatocyte metabolism data for b-AP15. We conclude that the strong enrichment of b-AP15 in cells and a rapid commitment to apoptosis/cell death are factors that are likely to contribute to the strong anti-tumor activity of this compound.