Conditionally Active Polypeptides via Net Charge Tuning
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods struggle to develop polypeptides that are conditionally active, meaning they are less active under normal conditions but more active under aberrant conditions, which is crucial for reducing side effects and enhancing therapeutic efficacy, especially in environments like tumor microenvironments.
Innovation Solution
The method involves increasing the net charge of parent polypeptides by introducing charged amino acid residues, such as Aspartic acid, Glutamic acid, Arginine, and Lysine, to create conditionally active polypeptides that exhibit decreased activity at normal physiological conditions and increased activity at aberrant conditions, using techniques like directed mutagenesis and site-directed mutagenesis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If polypeptides are made highly active under normal conditions, then therapeutic efficacy is improved, but side effects increase due to activity in non-target tissues
Solution Approach 1:
The patent applies local quality by engineering polypeptides with pH-dependent activity characteristics. The polypeptides are designed to have high activity specifically in the acidic tumor microenvironment (pH 6.5-7.0) while maintaining low activity at normal physiological pH (7.2-7.4). This is achieved through directed mutagenesis to introduce amino acid substitutions that create pH-sensitive ionization states, enabling the polypeptide to selectively affect target tissues (tumors) while sparing normal tissues, thus resolving the contradiction between therapeutic efficacy and side effects
Solution Approach 2:
The patent employs parameter changes by modifying the pH sensitivity of polypeptides through amino acid substitution. By changing the ionization parameters of specific residues (e.g., introducing histidine, aspartic acid, or glutamic acid at strategic positions), the polypeptide's activity becomes contingent on pH conditions. This allows the same polypeptide to exhibit high activity in acidic tumor environments while remaining inactive at neutral physiological pH, thereby achieving selective therapeutic action with minimized side effects
2Adaptability or versatility
If polypeptides are evolved for conditional activity through multiple amino acid substitutions, then activity selectivity is improved, but protein stability may deteriorate
Solution Approach 1:
The patent applies local quality by introducing amino acid substitutions specifically at the active site or regulatory regions of the polypeptide while leaving the core structural domains unchanged. This localized mutagenesis approach allows the polypeptide to gain pH-dependent activity selectivity without compromising the overall structural stability. The substitutions are strategically placed to affect only the functional properties needed for conditional activity, thereby achieving activity selectivity while maintaining protein stability
Solution Approach 2:
The patent employs partial action by introducing a limited number of targeted amino acid substitutions rather than extensive mutagenesis. By making only the necessary minimal changes (e.g., 1-3 strategic substitutions) to achieve pH-dependent activity, the patent avoids the stability issues that would arise from multiple random mutations. This partial mutagenesis approach achieves the desired activity selectivity while preserving the native protein's structural integrity and stability
Data Source
AI summary
A method of preparing a conditionally active polypeptide from a parent polypeptide, comprising steps of evolving a DNA encoding the parent polypeptide by increasing a net charge of the parent polypeptide using one or more techniques selected from increasing a total number of codons of charged amino acid residues in the DNA and decreasing a total number of codons of uncharged amino acid residues in the DNA to create mutant DNAs; expressing the mutant DNAs to obtain mutant polypeptides; and selecting the conditionally active polypeptide from the mutant polypeptides which exhibits a decrease in activity in a first assay at a first value of a condition compared to the same activity in a second assay at a second value of the same condition. The conditionally active polypeptide, pharmaceutical compositions containing same, nanoparticle and drug conjugates thereof and uses thereof are also provided.