1.The 19S Deubiquitinase inhibitor b-AP15 is enriched in cells and elicits rapid commitment to cell death.
Wang X1, Stafford W, Mazurkiewicz M, Fryknäs M, Brjnic S, Zhang X, Gullbo J, Larsson R, Arnér ES, D'Arcy P, Linder S. Mol Pharmacol. 2014 Jun;85(6):932-45. doi: 10.1124/mol.113.091322. Epub 2014 Apr 8.
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.
2.Inhibitory effect of b-AP15 on the 20S proteasome.
Huang L1, Jung K2, Chen CH3. Biomolecules. 2014 Oct 14;4(4):931-9. doi: 10.3390/biom4040931.
The 26S proteasome is a cellular proteolytic complex containing 19S regulatory particles and the 20S core proteasome. It was reported that the small molecule b-AP15 targets the proteasome by inhibiting deubiquitination of the 19S regulatory particles of the proteasome complex. An investigation of b-AP15 on the 20S proteasome core suggested that this compound can also inhibit the 20S proteasome with a potency equivalent to that found to inhibit the 19S regulatory particles.
3.Proapoptotic effects of the novel proteasome inhibitor b-AP15 on multiple myeloma cells and natural killer cells.
Feng X1, Holmlund T1, Zheng C2, Fadeel B3. Exp Hematol. 2014 Mar;42(3):172-82. doi: 10.1016/j.exphem.2013.11.010. Epub 2013 Nov 27.
The small molecule b-AP15 is a novel inhibitor of proteasome deubiquitination. Recent studies have shown that b-AP15 displays antitumor activity in several preclinical, solid tumor models. In this study, we show that b-AP15 triggers time- and dose-dependent apoptosis of the human multiple myeloma (MM) cell lines RPMI8226 and U266, as determined by phosphatidylserine exposure. Apoptosis was dependent on caspase activation and was partially dependent on cathepsin D. Furthermore, b-AP15 triggered processing of pro-caspase-3 and cleavage of poly (ADP-ribose) polymerase in MM cells. b-AP15 also induced caspase-independent apoptosis in primary human natural killer cells. We also demonstrate that b-AP15 induces activation of the mitochondrial apoptosis pathway in MM cells, with activation of the proapoptotic protein Bax and a pronounced loss of the mitochondrial transmembrane potential. The latter events, however, appeared largely independent of caspase activation.
4.Synthesis and evaluation of derivatives of the proteasome deubiquitinase inhibitor b-AP15.
Wang X1, D'Arcy P1, Caulfield TR2, Paulus A3, Chitta K3, Mohanty C4, Gullbo J5, Chanan-Khan A3, Linder S1,4. Chem Biol Drug Des. 2015 Nov;86(5):1036-48. doi: 10.1111/cbdd.12571. Epub 2015 May 27.
The ubiquitin-proteasome system (UPS) is increasingly recognized as a therapeutic target for the development of anticancer therapies. The success of the 20S proteasome core particle (20S CP) inhibitor bortezomib in the clinical management of multiple myeloma has raised the possibility of identifying other UPS components for therapeutic intervention. We previously identified the small molecule b-AP15 as an inhibitor of 19S proteasome deubiquitinase (DUB) activity. Building upon our previous data, we performed a structure-activity relationship (SAR) study on b-AP15 and identified VLX1570 as an analog with promising properties, including enhanced potency and improved solubility in aqueous solution. In silico modeling was consistent with interaction of VLX1570 with key cysteine residues located at the active sites of the proteasome DUBs USP14 and UCHL5. VLX1570 was found to inhibit proteasome deubiquitinase activity in vitro in a manner consistent with competitive inhibition.