Collections of peptides, peptide agents, and methods of use thereof
Inventors
MCGEE, John Hanney • Thomson, Ty Matthew • Wahl, Sebastian Christof Theodor • Verdine, Gregory L. • Rezaei Araghi, Raheleh • Zhang, Yue-Mei • Mulvihill, Mark Joseph
Assignees
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Publication Number
US-12398178-B2
Publication Date
2025-08-26
Expiration Date
Abstract
The present disclosure provides powerful technologies for the development, production, characterization, and/or use of stapled peptide compositions. Among other things, the present disclosure provides strategies for defining amino acid sequences particularly amenable or useful for stapling, as well as technologies, reagents, and systems for developing, producing, characterizing, and/or using stapled peptides having such amino acid sequences.
Core Innovation
The invention provides a collection of stapled peptides, each independently comprising ADPAXXXCXXAAXXCXXX (SEQ ID NO: 3), where each X is independently an amino acid residue. Two cysteine residues are covalently linked with one another via a linker having the structure —S-Ls2-S—, where each S is independently a sulfur atom of one of the two cysteine residues.
The disclosure characterizes staple chemistry for stapled peptides, including hydrocarbon staple and non-hydrocarbon staple embodiments, carbamate staples, amino staples, staple stereochemistry at peptide-staple connection points, permissible staple heteroatoms, and the effect of olefin E/Z configuration. Staple length is defined using (i,i+m) relationships with m in the range of 3–12, together with staple-length characterization using chain-atom ranges.
The collection further includes discrete identifiers associated with individual stapled peptides, enabling independent identification of the amino acid sequence. The identifier comprises a nucleic acid sequence that encodes the amino acid sequence of the stapled peptide or a portion thereof, and the claimed amino acid sequence is expressed by a phage system that comprises the identifier, with each stapled peptide fused to a phage protein.
Claims Coverage
The consolidated claim coverage centers on one independent claim for a collection of stapled peptides defined by a specific sequence pattern and a cysteine-to-cysteine covalent linker. Dependent claim refinements add discrete identifiers, including nucleic acid encoding, and phage-related association, including covalent attachment to a phage particle and fusion to a phage protein.
Sequence pattern of stapled peptides
A collection of stapled peptides, each independently comprising ADPAXXXCXXAAXXCXXX (SEQ ID NO: 3), wherein each X is independently an amino acid residue.
Cysteine-to-cysteine covalent linker with defined structure
The two cysteine residues are covalently linked with one another via a linker having the structure —S-Ls2-S—, where each S is independently a sulfur atom of one of the two cysteine residues.
Discrete identifier for independent sequence identification
Each stapled peptide in the collection is discretely associated with an identifier so its amino acid sequence can be independently identified.
Nucleic-acid identifier encoding the stapled-peptide sequence
The identifier comprises a nucleic acid sequence that encodes the amino acid sequence of the stapled peptide or a portion thereof.
Phage system expressing the amino acid sequence associated with the identifier
The claimed amino acid sequence is expressed by a phage system that comprises the identifier.
Covalent attachment of each stapled peptide to a phage particle
Each stapled peptide is covalently bound to a phage particle.
Fusion of each stapled peptide to a phage protein
Each stapled peptide in the collection is fused to a phage protein.
Overall, the claim coverage is a collection of stapled peptides defined by ADPAXXXCXXAAXXCXXX (SEQ ID NO: 3) and a cysteine-to-cysteine linker of the form —S-Ls2-S—. The dependent refinements add identifier-based independent sequence identification and phage-system association, including nucleic-acid encoding, covalent attachment to a phage particle, and fusion to a phage protein.
Stated Advantages
The document describes highly diverse stapled peptide libraries for target binding discovery and characterization.
The peptide-associated identifiers enable sequencing-based genotype decoding of target-binding peptides using high-throughput sequencing.
Quantifies sequence enrichment using spiked-in internal standards added post-screening prior to sequencing.
Documented Applications
Target binding discovery and characterization.
Phage display systems with barcode-enabled identification.
Sequencing-based genotype decoding of target-binding peptides using high-throughput sequencing.
Assessment of stapling efficiency for beta-catenin peptide binders/non-binders (including (i,i+4) and (i,i+7) formats) using LC-MS integration and time-point comparisons.
Binding assessment of stapled peptides using a fluorescence polarization (FP) assay.
Exploration of stapled peptides in phage display/phagemid contexts using sequence identifiers and next-generation sequencing-based sequence family clustering.
Measuring sequence enrichment in peptide/phage display libraries using high-throughput sequencing of encoding nucleic acids with spiked-in internal standards.
Exemplary targets such as beta-catenin and Cbl-b.
Assay use of purified/assayed binders.
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