Protein Catalyzed Capture Agents with Tailored Performance for In Vitro and In Vivo Applications

We report on peptide-based ligands matured through the protein catalyzed capture (PCC) agent method to tailor molecular binders for in vitro sensing/diagnostics and in vivo pharmacokinetics parameters. A vascular endothelial growth factor (VEGF) binding peptide and a peptide against the protective antigen (PA) protein of Bacillus anthracis discovered through phage and bacterial display panning technologies, respectively, were modified with click handles and subjected to iterative in situ click chemistry screens using synthetic peptide libraries. Each azide-alkyne cycloaddition iteration, promoted by the respective target proteins, yielded improvements in metrics for the application of interest. The anti-VEGF PCC was explored as a stable in vivo imaging probe. It exhibited excellent stability against proteases and a mean elimination in vivo half-life (T_(1/2)) of 36 min. Intraperitoneal injection of the reagent results in slow clearance from the peritoneal cavity and kidney retention at extended times, while intravenous injection translates to rapid renal clearance. The ligand competed with the commercial antibody for binding to VEGF in vivo. The anti-PA ligand was developed for detection assays that perform in demanding physical environments. The matured anti-PA PCC exhibited no solution aggregation, no fragmentation when heated to 100°C, and  > 81% binding activity for PA after heating at 90°C for 1 h. We discuss the potential of the PCC agent screening process for the discovery and enrichment of next generation antibody alternatives

Protein Catalyzed Capture Agents with Tailored Performance for In Vitro and In Vivo Applications

We report on peptide-based ligands matured through the protein catalyzed capture (PCC) agent method to tailor molecular binders for in vitro sensing/diagnostics and in vivo pharmacokinetics parameters. A vascular endothelial growth factor (VEGF) binding peptide and a peptide against the protective antigen (PA) protein of Bacillus anthracis discovered through phage and bacterial display panning technologies, respectively, were modified with click handles and subjected to iterative in situ click chemistry screens using synthetic peptide libraries. Each azide-alkyne cycloaddition iteration, promoted by the respective target proteins, yielded improvements in metrics for the application of interest. The anti-VEGF PCC was explored as a stable in vivo imaging probe. It exhibited excellent stability against proteases and a mean elimination in vivo half-life (T_(1/2)) of 36 min. Intraperitoneal injection of the reagent results in slow clearance from the peritoneal cavity and kidney retention at extended times, while intravenous injection translates to rapid renal clearance. The ligand competed with the commercial antibody for binding to VEGF in vivo. The anti-PA ligand was developed for detection assays that perform in demanding physical environments. The matured anti-PA PCC exhibited no solution aggregation, no fragmentation when heated to 100°C, and  > 81% binding activity for PA after heating at 90°C for 1 h. We discuss the potential of the PCC agent screening process for the discovery and enrichment of next generation antibody alternatives

A General Synthetic Approach for Designing Epitope Targeted Macrocyclic Peptide Ligands

We describe a general synthetic strategy for developing high-affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify the best binder. We describe development of epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies

Unraveling the Roots of Selectivity of Peptide Affinity Reagents for Structurally Similar Ribosomal Inactivating Protein Derivatives

Peptide capture agents have become increasingly useful tools for a variety of sensing applications due to their ease of discovery, stability, and robustness. Despite the ability to rapidly discover candidates through biopanning bacterial display libraries and easily mature them to Protein Catalyzed Capture (PCC) agents with even higher affinity and selectivity, an ongoing challenge and critical selection criteria is that the peptide candidates and final reagent be selective enough to replace antibodies, the gold-standard across immunoassay platforms. Here, we have discovered peptide affinity reagents against abrax, a derivative of abrin with reduced toxicity. Using on-cell Fluorescence Activated Cell Sorting (FACS) assays, we show that the peptides are highly selective for abrax over RiVax, a similar derivative of ricin originally designed as a vaccine, with significant structural homology to abrax. We rank the newly discovered peptides for strongest affinity and analyze three observed consensus sequences with varying affinity and specificity. The strongest (Tier 1) consensus was FWDTWF, which is highly aromatic and hydrophobic. To better understand the observed selectivity, we use the XPairIt peptide–protein docking protocol to analyze binding location predictions of the individual Tier 1 peptides and consensus on abrax and RiVax. The binding location profiles on the two proteins are quite distinct, which we determine is due to differences in pocket size, pocket environment (including hydrophobicity and electronegativity), and steric hindrance. This study provides a model system to show that peptide capture candidates can be quite selective for a structurally similar protein system, even without further maturation, and offers an in silico method of analysis for understanding binding and down-selecting candidates

Biodiscovery of aluminum binding peptides

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Living Bacteria–Nanoparticle Hybrids Mediated through Surface-Displayed Peptides

In this study, we investigated the preparation of living bacteria–nanoparticle hybrids mediated by surface-displayed peptides. The assembly of metallic nanoparticles on living bacteria has been achieved under mild conditions utilizing metal–peptide interactions, whereas the viability of the bacterial cells was greatly preserved. Escherichia coli was engineered with inducible gene circuits to control the display of peptides with desired sequences. Several designed peptide sequences as well as known gold-binding peptides were expressed on the cell surface using enhanced circularly permuted outer membrane protein X (eCPX) scaffolds. Driven by metal–peptide affinity, “biofriendly” citrate-stabilized gold nanoparticles were self-assembled onto the surface of bacteria with displayed peptides, which required overcoming the repulsive force between negatively charged nanoparticles and negatively charged cells. The bacteria/Au nanoparticle hybrids were highly viable and maintained the ability to grow and divide, which is a crucial step toward the creation of living material systems. Further activity and preservation of the bacterial hybrid assembly was demonstrated. The method described herein enables the conjugation of bacterial surfaces with diverse metal-rich nanoparticles in an inducible, and therefore easily controlled, manner. The expressed peptide sequences can be easily modified to alter the binding affinity and specificity for a wide variety of materials to form on-demand, high-density living biohybrids