Potency assay (cells):
CellTiterGlo viability assay. A549/MM1S cells were treated with ABP A3 for 48h.
Target engagement assay (cells):
We also evaluated if ABP A3 inhibits closely related analogues SUMO E1, ISG15 E1, Ufm1 E1. To do so we treated A549 cells with ABP A3 and evaluated the amount of covalently labeled UBL proteins using click chemistry methods. Thus, A549 cells
were treated with each ABP (100 uM) for 1 hour, followed by cell lysis, conjugation to fluorescent dye via copper-mediated [3 + 2] cycloaddition reaction, and in-gel fluorescence scanning.
Under these conditions we did not observe SUMO labeling with ABP A3. Furthermore semi quantitative masspectrometry showed that ABP A3 potently labeled ubiquitin and Nedd8, but not SUMO1-3, Ufm1. Furthermore, western blotting was used to evaluate the effect of ABP A3 on the intracellular levels of UBL conjugates. Intracellular selectivity of ABP A3 on UBL conjugation. A549 cells were treated with different concentrations of ABP A3 or ABP1 for 16 hours, lysed, and immunoblotted using anti-Ub, Nedd8, SUMO1, SUMO2/3 and Ufm1 antibodies. Under these conditions ABP A3 inhibited the conjugation of Ub and Nedd8 to their intracellular substrates, and activated SUMO1-3 and Ufm1 conjugation. Activation of SUMO and Ufm1 pathways is due to the stress response.
Importantly, the length of the cell treatment with the probe affects the potency. For example, treatment of A549 cells with ABP A3 for 1 h leads to complete inhibition of Nedd8 conjugation at 6.3 uM of ABP A3, but ubiquitn conjugation is not changed. 16 h treatment of A549 cells with ABP A3 leads to inhibition of Nedd8 and Ub conjugation at ~3.2 uM. At 1 h treatment, complete inhibition of Ub conjugation requires ~25 uM of ABP A3.
In A549 cells, ~3 uM ABPA3 treatment for 16h caused an accumulation of p53, p21, induction of cleaved PARP (apoptosis) and activation of unfolded protein response (induction of CHOP, BiP, pPERK, and pelF2alpha). Furthermore, as expected ABP A3 did not cause an aggresome formation in A549 cells due to the inhibition of ubiquitin conjugation, while proteasome inhibitor MG132 did.