Fragment-based inhibitor design targeting the E. coli sliding clamp

The emergence of antibiotic-resistant bacteria necessitates the discovery of new classes of antibiotics acting through novel mechanisms. The bacterial sliding clamp, also known as the DNA polymerase III β subunit, engages in multiple protein-protein interactions by recognizing conserved penta- or hexapeptide motifs in various binding partners during DNA replication and repair. The central role of the sliding clamp in DNA replication and repair make it an attractive antibiotic target. Protein-protein interactions based on linear motif (LM) recognition play roles in many cell regulatory processes. The E. coli sliding clamp uses a common surface pocket composed of two sub-sites (I and II) to interact with LMs in multiple binding partners. A structural and thermodynamic dissection of sliding clamp-LM recognition has been performed, providing support for a sequential binding model. According to the model, a hydrophobic C-terminal LM dipeptide sub-motif acts as an anchor to establish initial contacts within subsite I and this is followed by formation of a stabilizing hydrogenbonding network between the flanking LM residues and subsite II. Differential solvation/desolvation effects during positioning of the sub-motifs are proposed to act as drivers of sequential binding. This model provides general insights into linear motif recognition and should guide the design of small-molecule inhibitors of the E. coli sliding clamp. Following the study of the LM recognition, fragment-based screening against the E. coli sliding clamp using X-ray crystallography, was carried out. Screening 352 fragment compounds produced hits bound at one of the two LM-binding subsites (subsites I). These fragment compounds were estimated to have millimolar binding affinity to the E. coli sliding clamp. A number of small-molecule inhibitors were designed based on the fragment hits, aided by molecular docking, and were found to bind to subsite I and inhibit LM-binding by the E. coli sliding clamp. They showed increased affinity at the micromolar level. From these, compounds with a tetrahydrocarbazole scaffold were chosen for lead development. A lead compound with tetrahydrocarbazole scaffold was designed to target both subsite I and subsite II. This work yielded a range of inhibitors with low-micromolar affinity with the potential to be a new class of antibiotics. This work reveals a sequential binding model of LM recognition by the E. coli sliding clamp. Fragment-based inhibitor design was carried out with the aim to find novel antibiotics with novel mechanism(s) of action. The inhibitor design improved the affinity from millimolar to low-micromolar level, providing a tetrahydrocarbazole scaffold with the potential for further optimization.