Massively parallel, computationally guided design of a proenzyme

Abstract

Significance Proteins have shown promise as therapeutics and diagnostics, but their effectiveness is limited by our inability to spatially target their activity. To overcome this limitation, we developed a computationally guided method to design inactive proenzymes or zymogens, which are activated through cleavage by a protease. Since proteases are differentially expressed in various tissues and disease states, including cancer, these proenzymes could be targeted to the desired microenvironment. We tested our method on the therapeutically relevant protein carboxypeptidase G2 (CPG2). We designed Pro-CPG2s that are inhibited by 80 to 98% and are partially to fully reactivatable following protease treatment. The developed methodology, with further refinements, could pave the way for routinely designing protease-activated protein-based therapeutics and diagnostics that act in a spatially controlled manner.

Strategies for Conditional Regulation of Proteins

“…One of the applications of this irreversible covalent chemistry is the development of enzyme-activable therapeutics. Bulky moieties are introduced on the POI to preserve it in an inert state until it encounters an enzyme that cleaves off these moieties to activate the POI. − Antibodies are also regulated in a similar fashion by introducing cleavable bulky moieties on the paratope (Figure a). − On the other hand, certain proteins or peptides are required to be protected from proteolytic activity. Peptide macrocyclization and site-specific conjugation (PEGylation, etc.)…”

Unleashing the Power of Proenzyme Delivery for Targeted Therapeutic Applications Using Biodegradable Lipid Nanoparticles

“…The reversible chemical modification of protein provides a facile yet versatile approach to designing endogenously activated proenzymes. 10 These methodologies involve the temporary inactivation of enzymes through the attachment of removable ligands either at the active site or at a site critical for the conformation of enzyme. 43 The enzyme remains inactive until the occurrence of specific endogenous stimuli such as pH change, the presence of distinct metabolites, or other cellular biochemical markers.…”

“…In addition, computational biology and bioinformatics are expected to play a pivotal role in the refinement of proenzyme conformation, thereby optimizing their design with unprecedented efficiency. 10 We anticipate that proenzymes will be designed with enhanced specificity and adaptability, which will be particularly transformative for developing targeted biotherapeutics and genome editing.…”

“…While effective, this method is laborious and exhibits limited versatility. 10 An alternative, using genetic code expansion, allows for the site-specific incorporation of unnatural amino acids for protein activity regulation. 11,12 However, this strategy often suffers from low protein expression efficiency and limited structural diversity.…”

“…This involves introducing an inhibitory domain to “cage” the enzymatic activity, which is released under specific conditions via a protease-cleavable linker. While effective, this method is laborious and exhibits limited versatility . An alternative, using genetic code expansion, allows for the site-specific incorporation of unnatural amino acids for protein activity regulation. , However, this strategy often suffers from low protein expression efficiency and limited structural diversity.…”