Affinity Enhancement of Protein Ligands by Reversible Covalent Modification of Neighboring Lysine Residues

Abstract

The discovery of protein ligands, capable of forming a reversible covalent bond with amino acid residues on a protein target of interest, may represent a general strategy for the discovery of potent small‐molecule inhibitors. We analyzed the ability of different aromatic aldehydes to form imines by reaction with lysine using 1H NMR techniques. 2‐Hydroxybenzaldehyde derivatives were found to efficiently form imines in the millimolar concentration range. These benzaldehyde derivatives could increase the binding affinity of protein ligands towards the cognate protein target. Affinity maturation was achieved not only by displaying ligand and aldehyde moieties on two complementary locked nucleic acid strands but also by incorporating the binding fragments in a single small‐molecule ligand. The affinity gain was only observed when lysine residues were accessible in the immediate surroundings of the ligand‐binding site and could be abrogated by quenching with a molar excess of hydroxylamine.

RGD Cyclopeptide Equipped with a Lysine‐Engaging Salicylaldehyde Showing Enhanced Integrin Affinity and Cell Detachment Potency

“…Building on this information, we synthesized two derivatives of peptide 1 ( 1‐N‐SA and 1‐C‐SA in Scheme 1A) bearing the RGD integrin‐binding motif, a disulfide bond, and a SA tag, installed either at the peptide N (compound 1‐N‐SA ) or C (compound 1‐C‐SA ) termini through a triazole ring. Moreover, two additional peptides ( 1‐N‐BA and 1‐C‐BA , Scheme 1A) were prepared, where the SA tag at the peptide termini was substituted by benzaldehyde (BA), which does not form stable imines in water [10] . Finally, the cyclic pentapeptide 2 (Scheme 1A) was prepared as negative control, in which the Ser‐Gly‐Glu (SGE) tripeptide was installed in place of the integrin‐binding RGD sequence.…”

“…1‐N‐SA , 1‐N‐BA and 2‐N‐SA ) were rapidly prepared through microwave‐assisted solid phase peptide synthesis (SPPS, to give linear peptide 3 , bearing a N‐terminal azido group, Scheme 1B), followed by intramolecular disulfide bond formation in solution, using iodine as oxidant. Later on, the resulting cyclic peptide 4 was reacted with SA and BA tags 5 , [10] featuring a terminal alkyne group: copper‐catalyzed azide‐alkyne cycloaddition (CuAAC) allowed the peptide‐tag conjugation, leading to the isolation of final products after HPLC purification and lyophilization. Since the preparation of C‐modified peptides on resin is typically cumbersome, we devised the assembly of compounds 1‐C‐SA and 1‐C‐BA in solution.…”

Modulation of Amyloidogenic Protein Self-Assembly Using Tethered Small Molecules

“…The results presented here expand this covalent chemical tool approach, highlighting its power to facilitate analysis of the structure and role of specific oligomers in self-assembly pathways. We have demonstrated that covalently reinforced protein-ligand interactions [65][66][67] can be used to manipulate heterogeneous and dynamic PPIs, exemplified by those formed in the initial stages of amyloid formation, which are notoriously difficult to target specifically when using purely non-covalent approaches 68 . By assessing both the affinity of non-covalent interactions between a protein and covalent fragment, and the effect of these fragments on protein oligomerization, insights into the molecular basis of PPI modulation (i.e.…”

Reactivity and Selectivity Principles in Native Protein Bioconjugation

“…There have been a few recent contributions that add value to the knowledge in this perspective. The F K 1 could lock the non‐covalent ligands with proteins in another impressive contribution from Neri and coworkers [39] . Ottmann and coworkers argued that the imine generation could act as a molecular glue to hold the protein interactome [40] .…”