Epitope‐Targeted Macrocyclic Peptide Ligand with Picomolar Cooperative Binding to Interleukin‐17F

Abstract

The IL-17 cytokine family is associated with multiple immune and autoimmune diseases and comprises important diagnostic and therapeutic targets. This work reports the development of epitope-targeted ligands designed for differential detection of human IL-17F and its closest homologue IL-17A. Non-overlapping and unique epitopes on IL-17F and IL-17A were identified by comparative sequence analysis of the two proteins. Synthetic variants of these epitopes were utilized as targets for in situ click screens against a comprehensive library of synthetic peptide macrocycles with 5-mer variable regions. Single generation screens yielded selective binders for IL-17F and IL-17A with low cross-reactivity. Macrocyclic peptide binders against two distinct IL-17F epitopes were coupled using variable length chemical linkers to explore the physical chemistry of cooperative binding. The optimized linker length yielded a picomolar affinity binder, while retaining high selectivity. The presented method provides a rational approach towards targeting discontinuous epitopes, similar to what is naturally achieved by many B cell receptors.

Inhibition of heme sequestration of histidine‐rich protein 2 using multiple epitope‐targeted peptides

“…Two other motifs with high sequence homology (AHHAADAHHA and AHHAHHAAD) were also targeted, yielding consensus PCC sequences of Tyr‐Lys‐Tyr‐Tyr‐Arg (cyYKYYR, L2 ; EC 50 = 218nM) and Tyr‐(4F)Phe‐Tyr‐Arg‐Val (cyY 4F FYRV, L3 ; EC 50 = 540pM) (Figure C). We have previously demonstrated that the qualitative EC 50 values of PCC‐target binding, assayed using methods employed here, approximate (within a factor of 2) the quantitative binding constants (K d ) measured using fluorescence polarization . Thus, we stipulate that these EC 50 values approximate the K d of L1 , L2 , and L3 to HRP2.…”

“…We have previously demonstrated that the qualitative EC 50 values of PCC-target binding, assayed using methods employed here, approximate (within a factor of 2) the quantitative binding constants (K d ) measured using fluorescence polarization. 28 Thus, we stipulate that these EC 50 values approximate the K d of L1, L2, and L3 to HRP2.…”

Antibody-recruiting protein-catalyzed capture agents to combat antibiotic-resistant bacteria

“…60,61 Epitope-targeted PCC ligands against the MrkA protein PCC ligands against the four selected epitopes were identied from a combinatorial library of macrocyclic peptides by using the epitope-targeted PCC method. [42][43][44][45] This method exploits non-catalyzed click chemistry via an in situ click screen. For the screen, an alkyne-presenting, one-bead one-compound (OBOC) combinatorial library of approximately 1 M peptide macrocycles with a 5-residue variable region is screened against synthetic variants of the epitopes (SynEps).…”

“…Combinatorial in situ click screens were performed as described previously. 44 Briey, 500 mg (representing $1.4 copies of approximately 1 000 000 different compounds) of a combinatorial onebead one-compound libraries was incubated overnight with shaking in TBS buffer (25 mM Tris HCl, 150 mM NaCl, pH 7.6). A preclear to remove beads that bound SAv-AP was performed as follows.…”

“…1). To generate the AR-PCC ligand, we employed the recently reviewed, 41 all synthetic epitope-targeted PCC method, [42][43][44][45] coupled with a bioinformatics approach to identify epitopes for targeting. By targeting highly exposed epitopes of the type 3 Fimbrial Sha (MrkA) surface protein of K. pneumoniae, we developed a small macrocyclic peptide binder in a single generation screen.…”