Discovery and chemical optimisation of a Potent, Bi-cyclic (Bicycle^®) Antimicrobial Inhibitor ofEscherichia coliPBP3

Abstract

Penicillin binding proteins (PBPs) are well validated antimicrobial targets, but the prevalence of β-lactamase driven resistance and, more rarely, target-based mutations, necessitates new classes of PBP-targeting drugs. Here we describe the discovery and optimisation of novel, bicyclic peptide (Bicycle®) inhibitors of E. coli PBP3 (EcPBP3) using a proprietary phage display platform, and their conjugation to linear antimicrobial peptides to confer outer membrane permeation. These molecules exhibited high-affinity binding to E. coli PBP3 and a viable spectrum of killing activity against clinically relevant species of the Enterobacterales. X-ray crystallography was used to explore the mode of binding to PBP3, enabling increased target affinity and improvement of in vitro stability. These compounds bind to the transpeptidase active site cleft of PBP3 and represent a novel non-β-lactam chemical class of high affinity, non-covalent penicillin binding protein inhibitors.

Investigating Penetration and Antimicrobial Activity of Vector-Bicycle Conjugates

“…To synthesize MAP-Bicycle conjugates, different variants of a previously tested MAP (DRAMP18563 in Wagstaff et al , 2020) were generated with a C-terminal 3-azido- l -alanine (CAzal) group. A previously identified Bicycle molecule, which inhibits its periplasmic target (described previously as peptide 2 ) was also generated with a C-terminal Lys­(pentynoyl)-CONH ( AB1 ) . Copper­(I)-catalyzed azide–alkyne cycloaddition (CuAAC) was used to “click” the vector variant, with the Bicycle molecule generating the MAP-Bicycle conjugates (Table ).…”

“…In this study, we conjugated a previously identified MAP to a Bicycle molecule selected against a periplasmic target and generated variations based on amino acid changes of the vector (Table ). The MAP-Bicycle conjugate shows enhanced antimicrobial activity compared to either the standalone vector or unconjugated Bicycle molecule, suggesting that MAP conjugation enhances OM penetration and delivery of the Bicycle molecule to its periplasmic target (Table , Figure A).…”

“…12,19 In this study, we set out to explore which physicochemical factors of AV1 drive OM penetration when conjugated to an E. coli PBP3-targeting Bicyclic peptide. 9 By varying amino acids within the "vector" component of a MAP-Bicycle conjugate, we show that variants with higher positive charge and hydrophobicity enhanced antibacterial activity. Interestingly, altering the amphipathicity did not affect the antimicrobial activity of the conjugate but impaired the ability of the "vector" to adopt a secondary structure.…”

“…It is well established that MAPs often contain a high number of positively charged and hydrophobic residues, while others assume a secondary structure when in contact with a membrane, such as the OM. , In this study, we set out to explore which physicochemical factors of AV1 drive OM penetration when conjugated to an E. coli PBP3-targeting Bicyclic peptide . By varying amino acids within the “vector” component of a MAP-Bicycle conjugate, we show that variants with higher positive charge and hydrophobicity enhanced antibacterial activity.…”

“…The increasing difficulty in treating Gram-negative multidrug resistant pathogens using current antibiotics means there is a pressing need to develop new classes of antibiotics. , Constrained peptides have shown therapeutic potential as a novel modality in many disease areas, including as antimicrobials where they structurally resemble many naturally occurring ligands. , Naturally occurring bicyclic compounds with various biological activities, such as inhibition of eukaryotic cell division and antitumor activity, have been previously isolated. , Bicycle Ⓡ molecules are a specific class of constrained peptides where a central small molecule scaffold is covalently reacted to cysteines within a linear peptide sequence. This provides structural constraint and creates a bicyclic structure. ,, Enormously diverse libraries of these Bicycle molecules can rapidly be screened, using modified bacteriophage display, to identify high-affinity binders against a variety of prokaryotic targets. These can be rapidly optimized using simple peptide and medicinal chemistry approaches to build in desired drug-like properties, which makes Bicycle molecules a potentially promising new group of constrained peptides for antibiotic discovery .…”