Peptide epitope tags offer a valuable means for detection and manipulation of protein targets for which high quality detection reagents are not available. Most commonly used epitope tags are bound by conventional, full-size antibodies (Abs). The complex architecture of Abs complicates their application in protein engineering and intracellular applications. To address these shortcomings, single domain antibodies (nanobodies, Nbs) that recognize short peptide epitopes have become increasingly prized. Here we characterize the interaction between a Nb (Nb6E) and a 14-mer peptide epitope. We identify residues in the peptide epitope essential for high affinity binding. Using this information in combination with computational modeling we propose a mode of interaction between Nb6E and this epitope. We apply this nanobody-epitope pair to augment the potency of a ligand at an engineered adenosine A2A receptor. This characterization of the nanobody-epitope pair opens the door to diverse applications including mechanistic studies of G protein-coupled receptor function.
Linking of fragments in neighboring binding sites is one of the optimization strategies in fragment-based drug discovery, where additive or even more substantial bioactivity improvements can be realized. However, such efforts present a considerable challenge when one fragment binds covalently to the target protein, as small modifications can influence the correct positioning of the covalent warhead toward the targeted nucleophilic residue. Here, we present a case study of fragment linking that yielded single-digit micromolar, covalent inhibitors of the SARS-CoV-2 main protease, starting from fragments that were inactive in the biochemical assay. Using structural information from a recent, high-throughput crystallographic fragment screen, we show that the success of fragment linking in the design of targeted covalent inhibitors is heavily impacted by several factors, including the warhead type, the labeling chemistry, and even subtle changes in the designed linker. Notably, we observe that induced fit effects might override the original fragment orientations in the linked molecule, highlighting the need for reliable structure verification, especially in consecutive rounds of fragment elaboration.
Levente Kollár, Levente M. Mihalovits, Dávid Bajusz, DamijanKnez, József Simon, Blake H. Balcomb, Daren Fearon, Stanislav Gobec, György M. K...
