Danial Yazdan, A. Michael Downey, Ana Gimeno, Caishun Li, Edward N. Schmidt, Jeffrey Y. K. Wong, Caleb Loo, Jaesoo Jung, Ryan Qiu, Ewa Lis, Lily Lindmeier, June Ereño-Orbea, Jesús Jiménez-Barbero, Matthew S. Macauley, Ratmir Derda

Journal of the American Chemical Society

DOI: 10.1021/jacs.6c14068

Abstract

Glycan-binding proteins (GBPs) are among the most difficult drug targets, limiting clinical progress against therapeutically important GBPs. We employed bicyclic genetically encoded libraries (BiGELs), produced by the chemical modification of phage-displayed peptide libraries with twofold symmetric linchpins, to discover inhibitors of therapeutically relevant Siglec-7:GD3 interactions. Next-generation sequencing (NGS) analysis of BiGEL panning against Siglec-7 yielded 815 candidates, of which 23 hits yielded K_D = 1–100 μM, as determined by surface plasmon resonance (SPR). Competitive enzyme-linked immunosorbent assays (ELISA) identified leads that disrupted the Siglec-7:GD3 interaction with IC₅₀ = 3-300 μM. Machine learning models trained on NGS datasets identified additional inhibitors with equivalent potencies. Alanine scans of 8c (SWCRPATVNC, IC₅₀ = 3.8 μM) and 12c (SFCHYPTHVC, IC₅₀ = 11 μM) identified residues crucial for activity. Ring reshaping studies of 8c highlighted the critical role of bicyclic topology, yielding analog 46e (SAAAAAWCRPATVNC, IC₅₀ = 9.5 μM), which was further evolved into 67e (STVTQHWCRPATVNC). The multivalent display of lead bicycles alongside ∼100 glycans in a Liquid Glycan Array (LiGA) enabled the comparison of binding to Siglec-7-expressing cells. LiGA assays confirmed the binding of the bicycles to Siglec-7 but revealed considerable nonspecific interactions with receptor-negative cells. Saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy revealed that 46e binds to Siglec-7 at a site distinct from the V-Ig domain, suggesting inhibition through an allosteric site. Together, these results demonstrate that BiGEL enables the discovery of bicyclic peptides for undruggable Siglec targets, but highlights future challenges in molecular discoveries that aim to identify small, noncarbohydrate inhibitors of GBPs.