Binding domain mapping and compounds, compositions, complexes, methods, and kits related thereto
Inventors
Liotta, Lance • Kunkel, Alessandra Luchini • Still, Amanda • Paige, Mikell
Assignees
George Mason University • George Mason Research Foundation Inc
Publication Number
US-12331096-B2
Publication Date
2025-06-17
Expiration Date
2040-01-03
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Abstract
The present disclosure relates to compounds, complexes, compositions, kits and methods for determining interacting and/or binding sites between e.g., proteins, proteins and nucleic acids, proteins and small molecules, or intrachain protein domains. The disclosure provides rapid and direct positive identification of the contact interface region between such molecules, and can be applied to individual interacting pairs, as well as large-scale or global interactions.
Core Innovation
The invention provides compounds, complexes, compositions, kits, and methods for determining interacting or binding sites between proteins, proteins and nucleic acids, proteins and small molecules, or intrachain protein domains. These compounds act as organic molecule masking pigments that bind to exposed regions of a protein in solution, enabling rapid and direct identification of contact interface regions between such molecules. The approach works for individual interacting pairs as well as large-scale or global interactions.
The invention addresses the problem that protein-protein interactions (PPIs), despite their importance in disease states, have been difficult targets for drug discovery due to large, flat, and featureless interaction interfaces and a lack of precise interface structural information. Existing structural and non-crystallographic techniques are time-consuming, require large amounts of protein or tagging, and are prone to false positives or artifacts. Therefore, there is a need for new and effective compounds, compositions, and methods to determine protein interaction interfaces.
The invention proposes the use of organic molecule masking pigments, exemplified by compounds having Formula (I) and related azo coupling products, that bind with high affinity and slow off-rates to small exposed protein regions (~3 amino acids). These compounds mask protease cleavage sites on exposed surfaces of native, folded proteins or complexes, preventing digestion at painted sites and revealing interaction interfaces upon dissociation and protease treatment. Methods include preparing such compounds by azo coupling reactions and applying them to proteins to form complexes, enabling determination of inaccessible interaction sites and facilitating inhibitor design.
Claims Coverage
The patent contains three independent claims covering peptides and methods related to disrupting PD-1/PD-L1 interaction.
Peptide comprising specified amino acid sequences
A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2 (CYRAMISYGGADYKRITC), SEQ ID NO:4 (CLKYDAPAFTVTC), or SEQ ID NO:7 (CLNWYRMSPSNQTDKLAAC).
Method for preventing or disrupting PD-1/PD-L1 interaction using peptide
A method for preventing or disrupting the interaction between PD-1 and PD-L1 by contacting PD-1 or PD-L1 with the peptide of the first inventive feature before, concomitant with, or after their interaction.
Individual peptides with the specified amino acid sequence
Peptides individually comprising the amino acid sequence CYRAMISYGGADYKRITC (SEQ ID NO:2), CLKYDAPAFTVTC (SEQ ID NO:4), or CLNWYRMSPSNQTDKLAAC (SEQ ID NO:7).
The claims cover peptides with specific amino acid sequences that inhibit PD-1/PD-L1 interactions and include methods of using these peptides to prevent or disrupt such interactions.
Stated Advantages
Provides rapid and direct positive identification of molecular contact interface regions between proteins or between proteins and other molecules.
Enables high-resolution (“protein painting”) coverage of protein surfaces at a scale of approximately 3 amino acids.
The compounds bind with high affinity and slow off-rates, remain bound after denaturation, and stabilize proteins against denaturation.
Allows identification of interaction sites without requiring crystallography or large amounts of protein or genetic tagging.
Facilitates discovery and design of inhibitors targeting protein interaction sites.
Documented Applications
Elucidation of protein-protein, protein-drug, and protein-ligand interactions.
Mapping of protein folding and protein binding domains during folding.
Detection and characterization of protein-nucleic acid interactions, including identification of miRNA binding sites.
Identification of protein misfolding sequences and exposed binding sites.
Production of antibodies specific for protein-ligand interaction regions or interfaces.
Drug discovery for inhibitors that block protein-protein interactions, exemplified by inhibitors of PD-1/PD-L1 and YAP/ZO interactions.
Diagnostic kits for determining protein-ligand binding events in mixtures for applications such as personalized medicine.
Therapeutic methods for preventing or disrupting PD-1/PD-L1 interaction in diseases such as cancer or viral infections using the disclosed peptides.
Therapeutic methods for preventing or disrupting YAP/ZO interaction in diseases such as cancer and wound healing using the disclosed peptides.
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