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bioRxiv · 10.1101/2022.01.03.474817

Directed evolution of angiotensin-converting enzyme 2 (ACE2) variants with enhanced peptidase activity profiles for novel therapeutic applications

Heinzelman, P.·Romero, P. A.
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Abstract

Angiotensin Converting-Enzyme 2 (ACE2) is currently being investigated for its ability to beneficially modulate the Angiotensin receptor (ATR) therapeutic axis to treat multiple human diseases, but its broad substrate scope and diverse physiological roles limit its potential as a therapeutic agent. In this work we engineer ACE2 variants with enhanced hydrolytic activity and specificity toward Angiotensin-II (Ang-II) that may lead to new therapeutic candidates with improved efficacy and reduced off-target side effects. We established a yeast display-based liquid chromatography screen that enabled use of directed evolution to discover ACE2 variants with improved Ang-II activity and specificity relative to the off-target peptide substrates Apelin-13 and Angiotensin-I (Ang-I). We screened ACE2 active site libraries to reveal three substitution-tolerant positions (M360, T371 and Y510) that can be mutated to enhance ACE2s activity profile and followed up on these hits with focused double mutant libraries to further improve the enzyme. Relative to wildtype ACE2, our top variant (T371L/Y510Ile) displayed a sevenfold increase in Ang-II turnover number (kcat), a sixfold diminished catalytic efficiency (kcat/Km) on Apelin-13, and an overall decreased activity on other ACE2 substrates that were not directly assayed in directed evolution screen. At physiologically relevant substrate concentrations, T371L/Y510Ile hydrolyzes more Ang-II than wildtype ACE2 with concomitant Ang-II:Apelin-13 specificity improvements reaching 30-fold. Our efforts have delivered ATR axis-acting therapeutic candidates with relevance to both established and unexplored ACE2 therapeutic applications and provide a foundation for further ACE2 engineering efforts. Significance StatementThe Angiotensin Converting Enzyme 2 (ACE2) carboxypeptidase is being clinically trialed for treatment of acute respiratory distress syndrome (ARDS) related to traumatic injuries and viral infections. We developed an enzyme engineering platform that enabled discovery of ACE2 variants possessing enhanced peptide hydrolysis activity and specificity profiles relevant to ARDS treatment. Beyond their potential utility as ARDS therapeutics, our improved ACE2 variants could be further developed for new unexplored ACE2 therapeutic contexts such as treating Alzheimers Disease.

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Heinzelman, P., Romero, P. A.. 2022-01-03. Directed evolution of angiotensin-converting enzyme 2 (ACE2) variants with enhanced peptidase activity profiles for novel therapeutic applications. https://doi.org/10.1101/2022.01.03.474817

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