Protease-Cleavable Polypeptides With Dual Recognition for Tissue Selectivity
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Solution Overview
Problem
There is a need to identify new substrates for proteases that can be utilized in therapeutic, diagnostic, and prophylactic applications, as existing substrates are resistant to cleavage by certain proteases and lack specificity in physiological reactions.
Innovation Solution
Development of isolated polypeptides comprising substrates with multiple cleavable moieties (CM1 and CM2) that are specifically cleavable by proteases such as MMP2, MMP9, MMP14, and MT-SP1, with improved cleavability and in vivo stability, allowing for targeted activation of therapeutic and diagnostic agents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing substrates are used for protease applications, then the substrate structure is simple and well-characterized, but the substrates are resistant to cleavage by certain proteases and lack specificity in physiological reactions
Solution Approach 1:
The patent applies composite materials by creating hybrid substrates that combine peptide sequences from different sources (e.g., natural peptide sequences engineered with non-natural amino acids, or combinations of different amino acid residues). These composite substrates exhibit improved cleavability by specific proteases while maintaining structural stability and specificity, resolving the contradiction between enhanced protease recognition and structural simplicity.
2Measurement precision
If substrates are designed to be highly specific to certain proteases, then cleavage specificity is improved, but the substrates may lack broad applicability across different protease types
Solution Approach 1:
The patent implements universality by designing substrate structures that can be recognized and cleaved by multiple protease types. By incorporating amino acid sequences with conserved recognition motifs that appeal to broader protease families, or by creating modular substrates where different domains can be swapped to target specific proteases, the substrates achieve both specificity and versatility across different protease applications.
Solution Approach 2:
The patent applies local quality by modifying specific regions of the substrate molecule to enhance recognition by target proteases while leaving other regions unchanged. By introducing specific amino acid substitutions or additions at key recognition sites (such as P1, P1', or other protease interaction domains), the substrates achieve high specificity for certain proteases without compromising their overall structure or applicability to different protease systems.
3Reliability
If multiple amino acid residues are introduced to improve protease cleavage, then cleavability is enhanced, but the complexity of substrate identification and characterization increases
Solution Approach 1:
The patent applies segmentation by dividing the substrate into distinct functional domains or modules, each with a specific role (e.g., protease recognition domain, cleavage site, and reporter/domain of interest). By systematically varying amino acid residues in specific positions within these segmented domains and using combinatorial library approaches, the patent enhances cleavage efficiency while maintaining organized structures that facilitate identification and characterization through standardized assays and sequencing methods.
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 polypeptides demonstrate enhanced cleavability and in vivo stability, enabling targeted activation in diseased tissues with minimal activation in healthy tissues, thereby improving therapeutic efficacy and diagnostic accuracy.
Implementation Method 1
Proteases are enzymes that catalyze the hydrolysis of peptide bonds between amino acid residues
Implementation Method 2
Proteases are enzymes that catalyze the hydrolysis of peptide bonds
Data Source
AI summary
Isolated polypeptides that include a cleavable moiety that is a substrate for at least two proteases (e.g., MT-SP1 and an MMP) are disclosed. Activatable molecules including the isolated polypeptides are disclosed. Methods of making and using the isolated polypeptides and activatable molecules including the isolated polypeptides in a variety of therapeutic, diagnostic, and prophylactic applications are disclosed.