Using a Fragment‐Based Approach To Target Protein–Protein Interactions

Abstract

The ability to identify inhibitors of protein–protein interactions represents a major challenge in modern drug discovery and in the development of tools for chemical biology. In recent years, fragment-based approaches have emerged as a new methodology in drug discovery; however, few examples of small molecules that are active against chemotherapeutic targets have been published. Herein, we describe the fragment-based approach of targeting the interaction between the tumour suppressor BRCA2 and the recombination enzyme RAD51; it makes use of a screening pipeline of biophysical techniques that we expect to be more generally applicable to similar targets. Disruption of this interaction in vivo is hypothesised to give rise to cellular hypersensitivity to radiation and genotoxic drugs. We have used protein engineering to create a monomeric form of RAD51 by humanising a thermostable archaeal orthologue, RadA, and used this protein for fragment screening. The initial fragment hits were thoroughly validated biophysically by isothermal titration calorimetry (ITC) and NMR techniques and observed by X-ray crystallography to bind in a shallow surface pocket that is occupied in the native complex by the side chain of a phenylalanine from the conserved FxxA interaction motif found in BRCA2. This represents the first report of fragments or any small molecule binding at this protein–protein interaction site.

Identification of the β-Lactamase Inhibitor Protein-II (BLIP-II) Interface Residues Essential for Binding Affinity and Specificity for Class A β-Lactamases

“…However, the concept that hot spots provide the majority of the driving force for an interaction presents a more tractable problem (4,7,43). The alanine scanning approach taken here identified the hot spot residues for the interaction of BLIP-II with four diverse class A ␤-lactamases where the association rate constants (k on ) were only modestly affected in comparison with the dissociation rate constants (k off ).…”

Quinazolinone derivatives as inhibitors of homologous recombinase RAD51

“…However, RAD51 inhibition was minimally toxic to normal breast epithelial cell line MCF10A which has unperturbed access to both HR and NHEJ repair pathways (IC 50 ~ 48 µM, Table 1), suggesting a promising role in selectively inhibiting aggressive, metastatic breast cancer cells rather than normal cells. In conclusion, by altering the known RAD51 inhibitor B02, we have identified a new cinnamylquinazoline compound (17) that shows enhanced cytotoxicity via RAD51. 17 effectively inhibits both RAD51 foci formation, in response to DNA damage, and proliferation of TNBC cell lines.…”

“…The cleft was surrounded by hydrophobic residues (L104, M158, I160, A190, A192, L203, A207, L219). A second shallow indentation close to motif 3, accommodating the threonine side chain of the tetrapeptide in the crystal structure with a truncated RAD51 17 , is formed by hydrophobic residues (F166, P168, L171, V185, L186, V189). These features were used to design our compound library in this report.…”

“…Several years ago, we developed a homology model of human RAD51, based on a full-length homologue from Pyrococcus furiosus (PDB code: 1PZN 16 ), to aid compound design. B02 was docked into the model in several different putative binding sites, including the ATPase domain known to bind small fragments like tetrapeptide and bicyclic aromatics 17 .…”

Fragment-based drug discovery and protein–protein interactions

“…67 Attempts to generate stable, unpartnered RAD51 with an exposed "FXXA" binding region were unsuccessful. Therefore, the RAD51 ortholog, Pyrococcus furiosus RadA, which could be produced in a suitable form for fragment screening was used as a surrogate system.…”