Towards mimicking short linear peptide motifs: identification of new mixed α,β-peptidomimetic ligands for SLAM-Associated Protein (SAP) by confocal on-bead screening.

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An array of chemical modifications have recently emerged, designed to improve the stability of natural peptides that inherently suffer from short in vivo half-lives, thereby preventing their use as therapeutics. The resultant peptidomimetics resemble native peptides; however, they contain synthetic elements (e.g. non-coded amino acids) which confer improved biophysical properties. An elegant approach towards the identification of peptidomimetics is through screening of large combinatorial chemical libraries incorporating both coded and non-coded amino acids (e.g. β amino acids). We apply here our recently developed Integrated Chemical Biophysics (ICB) platform, which combines microscale one-bead one-compound screening with fluorescence tagging of retrieved hit beads and subsequent affinity determination of hit compounds in homogenous solution, to the task of identifying novel mixed α, β peptidomimetic binders for the adaptor protein SLAM-associated protein (SAP), which acts as an intracellular adapter that transduces T and NK cell activation. An enhancement to the ICB process is introduced which enables ranking hit compounds from single-point measurements even if the library compound is <95% pure and without HPLC purification of single-bead-derived substance. Finally, a novel computational protocol enabling binding mode and SAR rationalisation of hit compounds is also described which we now utilise to inform future library design. Application of the full ICB process has allowed identification of a highly interesting motif, Ac-β(3)-Pro-α-pTyr, as a mimic for the -1 and -2 positions of the natural binding motif and provides a promising starting point for further optimization towards higher-affinity SAP inhibitors with enhanced metabolic stability.

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