Year
2013
Degree Name
Doctor of Philosophy
Department
School of Chemistry
Recommended Citation
Yin, Zhou, Fragment-based inhibitor design targeting the E. coli sliding clamp, Doctor of Philosophy thesis, School of Chemistry, University of Wollongong, 2013. https://ro.uow.edu.au/theses/4100
Abstract
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.
FoR codes (2008)
0304 MEDICINAL AND BIOMOLECULAR CHEMISTRY, 0307 THEORETICAL AND COMPUTATIONAL CHEMISTRY, 0601 BIOCHEMISTRY AND CELL BIOLOGY, 1115 PHARMACOLOGY AND PHARMACEUTICAL SCIENCES
Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.