Novel Ubc13 Inhibitors Figure 1. Peptoid combinatorial libraries as sources of structures that antagonize protein-protein interactions. (A) General structure of the combinatorial N-alkyl glycine library. (B) Yeast two-hybrid screening for peptoid pools that inhibit the Ubc13-Uev1 interaction. As a control interaction, the p53-large T interaction was assayed with the same pools in parallel assays. The b-galactosidase activity was normalized to the values for the control interaction, and to the values for the Ubc13-Uev1 interaction in the presence of solvent only (without peptoids). (C) Structures of the individual peptoids inferred as the most likely active inhibitors of the Ubc13-Uev1 interaction. doi:10.1371/journal.pone.0011403.g001 to virtual screening to select the molecules with the best fit to Ubc13. For each ligand, the energetically most favourable orientations for interaction with the Ubc13 surface were computed and ranked with CDOCK [39] (Tables S1 and S2). The ligands with the most favourable interactions corresponded to conformers of cyclic families I and II, both derived from peptoid N37-37-9C. Figures 2A and 2B show Connolly surface representations of the computed interactions of these two compounds with Ubc13. These two structures differ mainly in their cyclical components, with either seven (family I) or six (family II) atom rings, that occupy the center of the hydrophobic pocket on Ubc13, with the side chains adapting with favorable interaction energies to the three grooves that stem from the pocket (Fig. 2A and B). These models suggest that family I compounds have more favorable interaction energies on Ubc13 than family II compound (Tables S1 and S2). In vitro activities of Ubc13-Uev1 antagonists Two cyclic compounds were synthesized on the basis of the structures selected from the virtual screening, and designated hereafter Ia (family I) and IIa (family II) (Fig. 2C and 2D). Both compounds interfered with the Ubc13-Uev1 interaction at micromolar concentrations on yeast two-hybrid assays (Fig. S1). In competition assays with recombinant proteins, compound Ia inhibited the Ubc13-Uev1 interaction at nanomolar concentra- tions, and compound IIa at micromolar concentrations (Fig. 3A). These actitivies were specific to these two compounds, since an unrelated control cyclic compound with a similar ring structure (of the family I type) did not detectably interfere with the Ubc13-Uev1 interaction at the same concentrations (Fig. 3A). This activity was quantitated by surface plasmon resonance (SPR). With this technique, the dissociation constant for the Ubc13-Uev1 interaction was 1.061029 M, indicating a high- affinity binding of the heterodimer, with values close to those reported by isothermal titration calorimetry [40] that are expected to require high affinity binding by any potential competitor. SPR determinations yielded a IC50 of 1.0610211 M for compound Ia, and of 1.161026 M for compound IIa (Fig. 3B), indicating a significantly more effective inhibition of the Ubc13-Uev1 interaction by compound Ia. They also indicated that the binding of the two active compounds on Ubc13 must occur at high affinities, in order to successfully compete with the high affinity Ubc13-Uev1 interaction. To determine the binding efficiency of these compounds to Ubc13, lysine-conjugated derivatives (Fig. S2) were immobilized on SPR sensor chips, and Ubc13 subsequently applied in the mobile phase. These assays yielded dissociation constants for Ubc13 of 4.4610212 M for compound Ia and of 4.6861027 M for compound IIa (Figure 3C). This low dissociation constant for compound Ia reinforces the conclusion that it specifically occupies with high affinity the Ubc13 interface normally used to interact with Uev1, and that this is the likely mechanism by which it antagonizes this interaction. PLoS ONE | www.plosone.org 3 June 2010 | Volume 5 | Issue 6 | e11403