Novel Ubc13 Inhibitors Figure 3. In vitro activities of compounds Ia and IIa. (A) Inhibition of the Ubc13-Uev1 interaction by compounds Ia and IIa, determined by GST- pull-down assays. Matrix-bound GST-Ubc13, preincubated with the test or control compounds at a final concentration of 100 nM, were allowed to interact with recombinant Uev1. Fraction E corresponds to the eluted fraction of Uev1. Fraction B corresponds to the Uev1 that remaind bound to GST-Ubc13, and that are eluted with reduced glutathione. All fractions were assayed by Western blotting with anti-Ubc13 and anti-Uev antibodies. (B) Surface plasmon resonance Ubc13-Uev1 binding competition assay. Ubc13 was pre-incubated with varying concentrations of compounds Ia or IIa and injected into a CM5 chip on which Uev1 had been immobilized. The IC50 for binding inhibition was 10 pM for compound Ia, and 1.1 mM for compound IIa. (C) Surface plasmon resonance sensorgrams for the interaction of UBC13 with immobilized compound Ia (top) or IIa (bottom). Lysine derivatives of either compound were immobilized onto CM chips, and Ubc13 was run in the mobile phase at the indicated concentrations. From the signals, dissociation constants for Ubc13 were calculated, (4.4610212 M for compound Ia and 4.6861027 M for compound IIa). (D) Effect of compound Ia on in vitro wild-type ubiquitin free chain elongation dependent on Ubc13 and Uev1. Purified recombinant proteins (E1, Ubc13, Uev1) were used to synthesize free ubiquitin chains for the indicated times, in the absence (Control) or presence of compound Ia (1 mM). Ubiquitin chains were detected by Western blotting with anti-ubiquitin. (E) Graphic representation of the kinetic experiment shown in (D), with quantitation of the accumulated levels of triubiquitin (Ub3) free chains. (F) Effect of compound Ia on in vitro chain elongation of K63- or K48-only ubiquitin mutants. The experiment was performed as in (D), except that mutant ubiquitins were substituted for the wild-type form. doi:10.1371/journal.pone.0011403.g003 PLoS ONE | www.plosone.org 5 June 2010 | Volume 5 | Issue 6 | e11403