Year

2015

Degree Name

Doctor of Philosophy

Department

School of Chemistry

Abstract

The rise of “superbugs” – antibiotic resistant infection-causing bacteria - poses a catastrophic threat to public health, and prompts the investigation of new drug targets. In this work, the inhibition of specific proteins in bacterial DNA replication machinery – a rich source of new targets - was investigated.

The single stranded DNA-binding protein, SSB, is an interaction hub that engages in vital interactions with several partner proteins through a flexible C-terminal peptide motif (DDDIPF; SSB-Ct). Based on available crystal structures of SSB-Ct with partner proteins, molecular dynamics simulations were used to identify mobile elements of SSB-Ct and important interactions in SSB-Ct binding-pockets.

Fragment-based screening using various methods was undertaken, targeting the SSBbinding partner DnaG, the replicative primase from E. coli. Initially two fragment libraries (by Zenobia Therapeutics and the Monash Institute of Pharmaceutical Science) containing ~1600 fragments were screened against DnaG primase using SPR competition and STD-NMR assays. The binding confirmation and binding site location of identified fragments was confirmed by ligand- and protein-based NMR techniques. Common features were identified in some fragment hits: carboxylic group containing aromatic compounds.

In silico fragment-to-hit optimisation led to the identification of phenoxy-phenyl tetrazoles; ZINC72447025 and CDS001350 with 1.3 mM and 1.2 mM binding affinities. It was hypothesized that these compounds could inhibit other proteins that bind to SSB-Ct. The tetrazole derivatives and other fragment hits were shown binding to other SSB-Ct binding partners such as E. coli χ subunit of Pol III, PriA, RNAse HI and A. baumannii χ.

This work demonstrates the potential for inhibitions SSB/binding partner interactions, and the possibility of inhibiting multiple SSB-binding proteins with similar ligands.

Share

COinS