Phage-encoded combinatorial chemical libraries based on bicyclic peptides

Abstract

Here we describe a phage strategy for the selection of ligands based on bicyclic or linear peptides attached covalently to an organic core. We designed peptide repertoires with three reactive cysteine residues, each spaced apart by several random amino acid residues, and we fused the repertoires to the phage gene-3-protein. Conjugation with tris-(bromomethyl)benzene via the reactive cysteines generated repertoires of peptide conjugates with two peptide loops anchored to a mesitylene core. Iterative affinity selections yielded several enzyme inhibitors; after further mutagenesis and selection, we were able to chemically synthesize a lead inhibitor (PK15; Ki =1.5 nM) specific to human plasma kallikrein that efficiently interrupted the intrinsic coagulation pathway in human plasma tested ex vivo. This approach offers a powerful means of generating and selecting bicyclic macrocycles (or if cleaved, linear derivatives thereof) as ligands poised at the interface of small-molecule drugs and biologics.

Peptide and small molecule inhibitors of HECT-type ubiquitin ligases

“…The bicycle method makes use of a phage library displaying peptides containing three Cys residues. The phages are treated under mild conditions with Tris-(bromomethyl)benzene, which reacts with all three cysteines, forming two peptide loops of six amino acids linked to the benzene ring (14,16). We screened this library for phages that bound to His-tagged Smurf2 HECT domain immobilized on Ni-NTA beads (Methods).…”

Multiple Toxin Production in the Cyanobacterium Microcystis: Isolation of the Toxic Protease Inhibitor Cyanopeptolin 1020

“…The enzyme inhibition assays were carried out in using modified literature procedures. 19 The kinetic fluorescence measurements were realized with transparent microplates (Nunc) using a fluorescence microplate reader (Tecan infinite M1000) set at  em = 355 nm,  ex = 480 nm and measuring a datapoint every 30 seconds for 1 h. To obtain the initial reaction rate, linear regression was performed with Microsoft Excel. The enzymes were dissolved in a buffer containing 50 mM Tris-HCl pH 7.4, 100 mM NaCl, 10 mM MgCl 2 , 1 mM CaCl 2 , 0.1% BSA and 0.01% Triton X-100.…”

A Handbook for DNA‐Encoded Chemistry

“…To date, display techniques employing such principles as phage display [7,8], ribosome display [9,10], yeast display [11], covalent display [12], mRNA display [13,14], and other conceptually analogous methodologies [15,16] profoundly impact the way novel drugs are discovered, yielding new and more efficacious classes of therapeutic agents (e.g., antibody drug conjugates) [17,18]. DNA-encoded library technology aims to extend the realm and the potential of these display approaches to the en masse interrogation of small synthetic organic molecules.…”