Mutant Akt Capture Agents via Iterative Click Chemistry
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Solution Overview
Problem
Current methods for targeting single amino acid point mutations, such as the E17K mutation in Akt1 kinase, face challenges as these mutations often do not have a binding pocket, making it difficult for small molecule inhibitors to selectively bind and inhibit the mutant protein without affecting wild-type variants, and antibodies are not cell-penetrant.
Innovation Solution
Development of chemically synthesized capture agents using iterative in situ click chemistry to create stable, synthetic ligands that selectively bind to the E17K mutation in Akt1, comprising designed anchor, secondary, tertiary, and quaternary ligands linked by triazole residues, which can penetrate cells and inhibit mutant Akt1 activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If small molecule inhibitors are used to target single amino acid point mutations, then selective inhibition of mutant proteins is desired, but the mutation may not be directly associated with a binding pocket making it difficult to achieve selectivity
Solution Approach 1:
The capture agent is divided into multiple functional components: an anchor ligand that binds to the mutant-specific epitope, a spacer region providing flexibility and distance, and a detectable moiety. This segmentation allows the inhibitor to target the mutation site directly without requiring a traditional binding pocket, achieving mutant selectivity while simplifying the structural requirements
Solution Approach 2:
The patent introduces a peptide-based anchor ligand as an intermediary that specifically recognizes the mutant epitope sequence. This intermediary component bridges the gap between the small molecule inhibitor and the mutation site, enabling selective binding even in the absence of a conventional binding pocket
2Measurement precision
If antibodies are used to target mutant proteins, then high specificity for the mutation can be achieved, but antibodies do not readily enter living cells limiting their use to diagnostics only
Solution Approach 1:
The invention changes the physical and chemical parameters of the antibody by conjugating it to cell-penetrating peptides or using smaller antibody fragments (such as scFv or nanobodies). This parameter modification enables the antibody to penetrate cell membranes while retaining its high specificity for the mutant protein, thus transitioning from a purely diagnostic tool to a potential therapeutic agent
Solution Approach 2:
The patent creates a composite structure combining the mutant-specific antibody with cell-penetrating moieties. This composite material integrates the high specificity of the antibody with the cell penetration capability of the attached peptide or fragment, simultaneously achieving both diagnostic precision and therapeutic delivery
3Strength
If capture agents with multiple ligands are designed to increase affinity and specificity, then binding strength to mutant protein improves, but the complexity of synthesis and characterization increases
Solution Approach 1:
The multi-ligand capture agent is synthesized in a modular fashion, with each ligand component being independently characterized and then assembled. This segmented approach to synthesis allows for systematic optimization of binding affinity while maintaining manageable complexity through standardized building blocks and sequential assembly steps
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The capture agents demonstrate high stability and specificity for the E17K mutation, effectively inhibiting Akt1 signaling and reducing mutant protein levels, offering a diagnostic and therapeutic approach for conditions associated with increased Akt1 expression, such as cancer.
Implementation Method 1
the capture agent comprises a designed anchor ligand, a designed secondary ligand, a designed tertiary ligand, and wherein the ligands selectively bind Akt1 with a mutation of glutamate to lysine at position 17 of the pleckstrin homology domain (E17K Akt1) over wild-type Akt1
Implementation Method 2
methods for making said capture agents using iterative in situ click chemistry
Data Source
AI summary
The present application provides stable peptide-based Akt capture agents and methods of use as detection and diagnosis agents and in the treatment of diseases and disorders. The application further provides methods of manufacturing Akt capture agents using iterative on-bead in situ click chemistry.