Fig. 1. Screening for small-molecule activators and inhibitors of the CPD of TcdB.

(A) Superposition of the crystal structures of TcdB (red) and TcdA (yellow) CPD domains with an IP₆ allosteric activator bound. (B) Structure of the activity-based probe TAMRA–AWP-19 used for the high-throughput screen (HTS). The fluorophore is shaded in red, and the acyloxymethyl ketone electrophile specific for cysteine proteases is shown in blue. (C) Schematic of the fluorescence polarization (fluopol)–activity-based probe screen workflow. Compounds were added to the purified CPD domain from TcdB followed by addition of TAMRA–AWP-19 probe. The unbound probe produced low fluorescence polarization [millipolarization (mP)], which increased upon binding to the activated CPD. This change in millipolarization was measured on a plate reader and was used to assess the ability of a compound to either directly stimulate or inhibit (upon IP₆ addition) CPD activation.