The 19S Deubiquitinase inhibitor b-AP15 is enriched in cells and elicits rapid commitment to cell death.
b-AP15 [(3E,5E)-3,5-bis[(4-nitrophenyl)methylidene]-1-(prop-2-enoyl)piperidin-4-one] is a small molecule inhibitor of the ubiquitin specific peptidase (USP) 14/ubiquitin carboxyl-terminal hydrolase (UCH) L5 deubiquitinases of the 19S proteasome that shows antitumor activity in a number of tumor models, including multiple myeloma. b-AP15 contains an α,β-unsaturated carbonyl unit that 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 of the stronger potency of the compound in cellular assays compared with in vitro biochemical assays. Cellular uptake was impaired by 30-minute pretreatment 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 likely contribute to the strong antitumor activity of this compound.
Department of Oncology and Pathology, Cancer Center Karolinska (X.W., M.M., S.B., X.Z., P.D., S.L.), and Division of Biochemistry, Department of Medical Biochemistry and Biophysics (W.S., E.S.J.A.), Karolinska Institute, Stockholm, Sweden; and Division of Clinical Pharmacology, Department of Medical Sciences, Uppsala University, Uppsala, Sweden (M.F., J.G., R.L., S.L.).
Xin
X
Wang
William
W
Stafford
Magdalena
M
Mazurkiewicz
Mårten
M
Fryknäs
Slavica
S
Brjnic
Xiaonan
X
Zhang
Joachim
J
Gullbo
Rolf
R
Larsson
Elias S J
ES
Arnér
Padraig
P
D'Arcy
Stig
S
Linder
D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D018384:Oxidative Stress / Q000187:drug effects; D010881:Piperidones / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects; D064570:Ubiquitin-Specific Proteases / Q000037:antagonists & inhibitors*
C569633:3,5-bis((4-nitrophenyl)methylidene)-1-prop-2-enoylpiperidin-4-one; D004791:Enzyme Inhibitors; D010881:Piperidones; D064570:Ubiquitin-Specific Proteases; D046988:Proteasome Endopeptidase Complex