With additional hits identified from the iterative screen, we sought to obtain further structural and biophysical information to better understand how these reactive fragments bind to BCL6. A total of eight SF hits were resynthesised and purified: the four from the original screen (2f-i), and four from the iterative screen (2p, 2q, 2s, 2t). To confirm that these eight hits targeted the same binding site as previously reported binders, GSK137 (biochemical pIC50 = 8) was used as a known inhibitor for a displacement experiment. The SFs (100 µM) were incubated for 24 h with BCL6 in the presence of GSK137 (100 µM) or DMSO as a control, and the samples were analysed by intact protein LC-MS. The resultant mass spectra showed that covalent modification was attenuated by 78-96% for all eight SFs, indicating that all hits were competing for the same site (Fig. ). We subsequently analysed protein-fragment interactions using differential scanning fluorimetry (DSF) to explore the impact of these binding events on protein stability. The azetidinyl/piperidinyl fragments exhibited negligible thermal shifts (see Fig. &), however, the meta-substituted benzamide complexes were highly thermally stabilised (ΔTm ~7 °C) relative to unmodified BCL6, which implied the presence of intermolecular interactions from ligand binding (Fig. ). This level of stabilisation was comparable to that observed for the potent, non-covalent inhibitor GSK137 (ΔTm ~12 °C, see Fig. ). Kinetic analyses of the four hits from the original screen (2f-i) revealed that for the meta-substituted benzamides, the reversible affinities of these fragments were relatively weak, with KI = 56 and 80 µM for 2f and 2g, respectively (see Fig. ). Therefore, it was interesting to observe a thermal shift similar to an inhibitor with biochemical pIC50 = 8; this illustrates the impact of covalent modification on protein-ligand binding.