Ultra-large chemical libraries for the discovery of high-affinity peptide binders

Abstract

High-diversity genetically-encoded combinatorial libraries (10 8 −10 13 members) are a rich source of peptide-based binding molecules, identified by affinity selection. Synthetic libraries can access broader chemical space, but typically examine only~10 6 compounds by screening. Here we show that in-solution affinity selection can be interfaced with nano-liquid chromatography-tandem mass spectrometry peptide sequencing to identify binders from fully randomized synthetic libraries of 10 8 members-a 100-fold gain in diversity over standard practice. To validate this approach, we show that binders to a monoclonal antibody are identified in proportion to library diversity, as diversity is increased from 10 6-10 8. These results are then applied to the discovery of p53-like binders to MDM2, and to a family of 3-19 nM-affinity, α/β-peptide-based binders to 14-3-3. An X-ray structure of one of these binders in complex with 14-3-3σ is determined, illustrating the role of β-amino acids in facilitating a key binding contact.

De Novo Discovery of High Affinity Peptide Binders for the SARS-CoV-2 Spike Protein

“…Taken together, we have shown the use of our affinity selection -mass spectrometry platform 27 for the rapid discovery of peptides binding to the SARS-CoV-2-spike-RBD. From 800 million synthetic peptides screened, we identified three sequences with a shared residue motif and nanomolar affinity.…”

“…SARS-CoV-2-spike-RBD was biotinylated and immobilized on streptavidin-coated magnetic beads. Following an AS-MS protocol recently established by our group, 27 we screened four peptide libraries with ~200 million members each against the immobilized spike RBD. The peptide library design was X12K, where X = any canonical amino acid except Ile and Cys.…”

“…Here, we report the discovery of synthetic peptides (13 residues) with nanomolar affinity for the SARS-CoV-2-spike-RBD. We leveraged a combinatorial affinity selection-mass spectrometry (AS-MS) platform 27 for the rapid identification of sequences that display high affinity toward RBD with selectivity over human proteins. After synthesis of the identified peptides, validation of their selective binding activity was observed using biolayer interferometry (BLI) and a magnetic bead pulldown assay.…”

Targeting the SARS-CoV-2-spike protein: from antibodies to miniproteins and peptides

“…In the Pentelute laboratory, we used a recently established high throughput affinity selectionmass spectrometry (AS -MS) platform 38,39 to identify SARS-CoV-2-spike-RBD binders from synthetic peptide libraries (Fig. 4).…”

Erythrocyte-targeted immunomodulatory antigens enabled by in vivo selection of D-peptides

“…When administered as an antigen-CTP conjugate, these molecules have anti-inflammatory effects. This in vivo selection technique marries the benefits of customizable synthetic peptide libraries to a high fidelity 'in solution' selection 17,18 . We envision that this platform can be used to discover synthetic CTPs that recognize targets beyond erythrocytes, including specific cells, tissues, and organs of therapeutic interest, including tumors.…”