Discovery and chemical optimisation of a Potent, Bi-cyclic (Bicycle®) Antimicrobial Inhibitor of Escherichia coli PBP3

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 ( Ec PBP3) 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. ### Competing Interest Statement The authors declare the following competing financial interest(s): C.E.R., C.K., H.N., K.v.R., M.J.S., N.L., N.B., S.J.S, T.T.M., and P.B. are shareholders and/or share option holders in Bicycle Therapeutics plc, the parent company of BicycleTx Ltd. * caMHB : cation-adjusted Mueller Hinton broth Cbg : cyclobutylglycine EcPBP3 : E. coli PBP3 EDC : 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride FP : fluorescence polarisation HArg : homoarginine HPhe : homophenylalanine MHB : Mueller Hinton broth MIC : minimal inhibitory concentration MOPS : 3-morpholin-4-ylpropane-1-sulfonic acid NB : no binding NHS : N-hydroxy succinimide NT : not tested SPR : surface plasmon resonance TATA : 1,3,5-triacryoyl-1,3,5-triazinane tBuGly : tert-butyl-leucine 2FPhe : 2-fluoro-phenylalanine 2MePhe : 2-methyl-phenylalanine 2Pal : 2-pyridylalanine 3FPhe : 3-fluoro-phenylalanine 3QuinAla : 3-Quinoyl-L-alanine 4MeOTrp : 4-methoxytryptophan 4MePhe : 4-methyl-phenylalanine 4Pal : 4-pyridylalanine 5FPhe : pentafluoro-phenylalanine 5MeOTrp : 5-methoxytryptophan 6ClTrp : 6-chlorotryptophan 6FlTrp : 6-fluorotryptophan.