Protein-Catalyzed Capture Agents Agnew, Heather D; Coppock, Matthew B; Idso, Matthew N ...
Chemical reviews,
09/2019, Volume:
119, Issue:
17
Journal Article
Peer reviewed
Open access
Protein-catalyzed capture agents (PCCs) are synthetic and modular peptide-based affinity agents that are developed through the use of single-generation in situ click chemistry screens against large ...peptide libraries. In such screens, the target protein, or a synthetic epitope fragment of that protein, provides a template for selectively promoting the noncopper catalyzed azide–alkyne dipolar cycloaddition click reaction between either a library peptide and a known ligand or a library peptide and the synthetic epitope. The development of epitope-targeted PCCs was motivated by the desire to fully generalize pioneering work from the Sharpless and Finn groups in which in situ click screens were used to develop potent, divalent enzymatic inhibitors. In fact, a large degree of generality has now been achieved. Various PCCs have demonstrated utility for selective protein detection, as allosteric or direct inhibitors, as modulators of protein folding, and as tools for in vivo tumor imaging. We provide a historical context for PCCs and place them within the broader scope of biological and synthetic aptamers. The development of PCCs is presented as (i) Generation I PCCs, which are branched ligands engineered through an iterative, nonepitope-targeted process, and (ii) Generation II PCCs, which are typically developed from macrocyclic peptide libraries and are precisely epitope-targeted. We provide statistical comparisons of Generation II PCCs relative to monoclonal antibodies in which the protein target is the same. Finally, we discuss current challenges and future opportunities of PCCs.
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The protein catalyzed capture agent (PCC) method is a powerful combinatorial screening strategy for discovering synthetic macrocyclic peptide ligands, called PCCs, to designated protein epitopes. The ...foundational concept of the PCC method is the use of in situ click chemistry to survey large combinatorial libraries of peptides for ligands to designated biological targets. State-of-the-art PCC screens integrate synthetic libraries of constrained macrocyclic peptides with epitope-specific targeting strategies to identify high-affinity (<100 nM) binders de novo. Automated instrumentation can accelerate PCC discovery to a rapid 2-week timeframe. Here, we describe methods to perform combinatorial screens that yield epitope-targeted PCCs.
Antibiotic resistant infections are projected to cause over 10 million deaths by 2050, yet the development of new antibiotics has slowed. This points to an urgent need for methodologies for the rapid ...development of antibiotics against emerging drug resistant pathogens. We report on a generalizable combined computational and synthetic approach, called antibody-recruiting protein-catalyzed capture agents (AR-PCCs), to address this challenge. We applied the combinatorial protein catalyzed capture agent (PCC) technology to identify macrocyclic peptide ligands against highly conserved surface protein epitopes of carbapenem-resistant
Klebsiella pneumoniae
, an opportunistic Gram-negative pathogen with drug resistant strains. Multi-omic data combined with bioinformatic analyses identified epitopes of the highly expressed MrkA surface protein of
K. pneumoniae
for targeting in PCC screens. The top-performing ligand exhibited high-affinity (EC
50
∼50 nM) to full-length MrkA, and selectively bound to MrkA-expressing
K. pneumoniae
, but not to other pathogenic bacterial species. AR-PCCs that bear a hapten moiety promoted antibody recruitment to
K. pneumoniae
, leading to enhanced phagocytosis and phagocytic killing by macrophages. The rapid development of this highly targeted antibiotic implies that the integrated computational and synthetic toolkit described here can be used for the accelerated production of antibiotics against drug resistant bacteria.
Antibody-recruiting protein-catalyzed capture agent (AR-PCCs) are a new class of all-synthetic and highly targeted antibiotics that recruit endogenous immune responses to eliminate drug-resistant microbes.
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Chemically synthesized, small peptides that bind with high affinity and specificity to CD8-expressing (CD8+) tumor-infiltrating T cells, yet retain the desirable characteristics of small molecules, ...hold valuable potential for diagnostic molecular imaging of immune response. Here, we report the development of
F-labeled peptides targeting human CD8α with nanomolar affinity via the strain-promoted sydnone-alkyne cycloaddition with 4-
Ffluorophenyl sydnone. The
F-sydnone is produced in one step, in high radiochemical yield, and the peptide labeling proceeds rapidly. A hydrophilic chemical linker results in a tracer with favorable pharmacokinetic properties and improved image contrast, as demonstrated by
PET imaging studies.
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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.
Cooperative binding: Macrocyclic peptide ligands were developed against distinct and discontinuous epitopes of two interleukin‐17 isoforms. These ligands achieved differential detection of the targeted isoform. Knowledge of the targeted epitopes informed a potentially general strategy for conjoining two ligands by a chemical linker to yield a cooperative biligand with picomolar binding affinity.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
We report on a method to improve in vitro diagnostic assays that detect immune response, with specific application to HIV-1. The inherent polyclonal diversity of the humoral immune response was ...addressed by using sequential in situ click chemistry to develop a cocktail of peptide-based capture agents, the components of which were raised against different, representative anti-HIV antibodies that bind to a conserved epitope of the HIV-1 envelope protein gp41. The cocktail was used to detect anti-HIV-1 antibodies from a panel of sera collected from HIV-positive patients, with improved signal-to-noise ratio relative to the gold standard commercial recombinant protein antigen. The capture agents were stable when stored as a powder for two months at temperatures close to 60(o)C.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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 (T1/2) 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.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
α-Synuclein Tertiary Contact Dynamics Lee, Jennifer C; Lai, Bert T; Kozak, John J ...
The journal of physical chemistry. B,
03/2007, Volume:
111, Issue:
8
Journal Article
Peer reviewed
Open access
Tertiary contact formation rates in α-synuclein, an intrinsically disordered polypeptide implicated in Parkinson's disease, have been determined from measurements of diffusion-limited ...electron-transfer kinetics between triplet-excited tryptophan:3-nitrotyrosine pairs separated by 10, 12, 55, and 90 residues. Calculations based on a Markovian lattice model developed to describe intrachain diffusion dynamics for a disordered polypeptide give contact quenching rates for various loop sizes ranging from 6 to 48 that are in reasonable agreement with experimentally determined values for small loops (10−20 residues). Contrary to expectations, measured contact rates in α-synuclein do not continue to decrease as the loop size increases (≥35 residues), and substantial deviations from calculated rates are found for the pairs W4−Y94, Y39−W94, and W4−Y136. The contact rates for these large loops indicate much shorter average donor−acceptor separations than expected for a random polymer.
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Metallic nanowires composed of nickel and gold as well as bimetallic nickel−gold nanowires were fabricated via templated electrodeposition in nanoporous alumina membranes. Gold surfaces were ...functionalized with alkanethiols with terminal hexa(ethylene glycol) groups (EG6), while nickel surfaces were functionalized with palmitic acid, a 16-carbon fatty acid. When exposed to a fluorescently tagged protein, hydrophobic nickel wires exhibited bright fluorescence while EG6-terminated gold wires did not, indicating that the protein did not adhere to the EG6-functionalized nanowires. Nickel−gold nanowires presenting distinct segments of alkyl and EG6 surfaces were also exposed to the fluorescent protein. Intense fluorescence was only observed on the nickel segment of these wires, demonstrating that proteins selectively adsorbed to one portion of these multicomponent nanostructures.
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10.
Deamidation of α‐synuclein Robinson, Noah E.; Robinson, Matthew L.; Schulze, Stephanie E. S. ...
Protein science,
August 2009, Volume:
18, Issue:
8
Journal Article
Peer reviewed
Open access
The rates of deamidation of α‐synuclein and single Asn residues in 13 Asn‐sequence mutants have been measured for 5 × 10−5M protein in both the absence and presence of 10−2M sodium dodecyl sulfate ...(SDS). In the course of these experiments, 370 quantitative protein deamidation measurements were performed and 37 deamidation rates were determined by ion cyclotron resonance Fourier transform mass spectrometry, using an improved whole protein isotopic envelope method and a mass defect method with both enzymatic and collision‐induced fragmentation. The measured deamidation index of α‐synuclein was found to be 0.23 for an overall deamidation half‐time of 23 days, without or with SDS micelles, owing primarily to the deamidation of Asn(103) and Asn(122). Deamidation rates of 15 Asn residues in the wild‐type and mutant proteins were found to be primary sequence controlled without SDS. However, the presence of SDS micelles slowed the deamidation rates of nine N‐terminal region Asn residues, caused by the known three‐dimensional structures induced through protein binding to SDS micelles.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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