Design, synthesis and evaluation of peptides and peptidomimetics inhibiting the bacterial DsbA-DsbB interaction

Abstract

The rapid development and dissemination of antibiotic resistance in pathogenic bacteria requires new strategies and new structural scaffolds of antimicrobial compounds to be investigated. A developing trend targets virulence factors rather than pathways essential for survival in an effort to neutralize pathogens while minimizing the risk of resistance. The thesis focuses on the design of new molecules from peptides to peptidomimetics aimed at disrupting the bacterial periplasmic oxidative system, a new antivirulence target.This thesis initially describes in chapter I the recent emergence of peptidomimetics in antimicrobial treatments aimed at well-known mechanisms as well as novel targets.Peptidomimetics may be particularly efficient for disrupting protein-protein interactions and have been successful for instance in preventing post-translational modifications or the formation of pili machinery. The thesis also highlights the mechanism and context of the interaction between DsbA and DsbB, both primary factors in the periplasmic oxidative system of gram-negative bacteria and the target proteins of this PhD.Developing inhibitors requires a solid screening pipeline of biophysical assays to measure binding parameters such as affinity, thermodynamics and kinetics. Chapter II highlights the building and optimization of such a pipeline by evaluating differential scanning fluorimetry, isothermal titration calorimetry, surface plasmon resonance, substrate oxidation assay and crystallography in order to characterize the designed peptide scaffolds.Chapter III focuses on the structure-activity relationship studies of 31 manually synthesized peptide sequences screened through this pipeline. Based on peptide length scouting, alanine scanning and rational substitution of specific residues, a final heptameric peptide was found to bind Escherichia coli DsbA with a Kd of 2.9 ± 0.3 µM. Moreover, the data suggest a probable disulfide bond formation between peptide and DsbA essential to the binding interface, while cysteine-free peptides present very weak affinity towards EcDsbA. This chapter generated a manuscript submitted to the Journal of Medicinal Chemistry.iiiIn a slightly different strategy, Chapter IV evaluates this best heptamer peptide sequence against a newly characterized Proteus Mirabilis DsbA, a structurally related protein (59% similarity with E. coli DsbA) showing promise for co-crystallization. With the peptide showing the same affinity against wild type E. coli and P. mirabilis DsbA, a highresolution (1.6 Å) co-crystal structure of the heptamer peptide bound to a cysteine mutant of P. mirabilis DsbA was solved. This structure shows the peptide binding to the P.mirabilis DsbA active site in a similar fashion to the native E. coli DsbA/DsbB interaction and differently from E. coli DsbA substrates. In-depth analysis of the high-resolution structure identifies two binding anchors, a H-bond-rich region and a hydrophobic pocket, which can be optimized for tighter affinities. This chapter generated a manuscript su...