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

Mutants of the Angiotensin Converting-Enzyme 2 (ACE2) carboxypeptidase possessing enhanced hydrolytic activity and specificity hold potential to beneficially modulate the Angiotensin receptor (ATR) therapeutic axis with increased efficacy and reduced potential side effects relative to wild type ACE2. In pursuing this goal, we established a yeast display-based liquid chromatography screen that enabled use of directed evolution to identify ACE2 mutants with improved target peptide substrate, Angiotensin-II (Ang-II), activity and specificity relative to Apelin-13, an off-target peptide substrate. Screening yeast-displayed ACE2 active site residue saturation mutant libraries revealed three substitution-tolerant positions that can be mutated to enhance ACE2's activity profile. Double mutant libraries combining substitutions at these positions, M360, T371 and Y510, yielded candidate improved ACE2 mutants that were recombinantly expressed and purified at 1 mg/L yield and > 90% homogeneity. Relative to wild type, the leading mutant, T371L/Y510Ile, has seven-fold increased kcat toward Ang-II and six-fold decreased kcat/Km for Apelin-13 hydrolysis. In single substrate hydrolysis assays featuring physiologically relevant substrate concentrations T371L/Y510Ile hydrolyzes more Ang-II than wild type with concomitant Ang-II:Apelin-13 specificity improvements reaching 30-fold. Additionally, T371L/Y510Ile hydrolyzed Ang-II at rates greater than wild type, with Apelin-13 hydrolysis reductions of up to 80 percent, in multiplex assays containing a mixture of peptides relevant to the ATR therapeutic axis. Our efforts have delivered ATR axis-acting therapeutic candidates with relevance to established and unexplored ACE2 therapeutic applications and demonstrate the feasibility of developing ACE2 variants for use in biomedical contexts unrelated to the ATR axis such as localized activation of peptide-based prodrugs.

Proteomics workflow for APP/Aβ TOMAHAQ analysis in endosomal and lysosomal fractions v2

The ability to detect processing of APP to the Ab amyloid peptide is challenging. This protocols describes methods for analysis of Ab "half-tryptic" peptides from purified organelles (endosomes and lysosomes). The targeted proteomics approach using TOMAHAQ coupled with Tomahto, which is an API for use on a Thermo orbitrap instrument that facilitates detection of trigger peptides and fragmentation of target peptide reporter ions.

Proteomics workflow for APP/Aβ TOMAHAQ analysis in endosomal and lysosomal fractions v1

The ability to detect processing of APP to the Ab amyloid peptide is challenging. This protocols describes methods for analysis of Ab "half-tryptic" peptides from purified organelles (endosomes and lysosomes). The targeted proteomics approach using TOMAHAQ coupled with Tomahto, which is an API for use on a Thermo orbitrap instrument that facilitates detection of trigger peptides and fragmentation of target peptide reporter ions.

Site-Specific Fluorogenic Protein Labelling Agent for Bioconjugation

Many clinically relevant therapeutic agents are formed from the conjugation of small molecules to biomolecules through conjugating linkers. In this study, two novel conjugating linkers were prepared, comprising a central coumarin core, functionalized with a dimaleimide moiety at one end and a terminal alkyne at the other. In our first design, we developed a protein labelling method that site-specifically introduces an alkyne functional group to a dicysteine target peptide tag that was genetically fused to a protein of interest. This method allows for the subsequent attachment of azide-functionalized cargo in the facile synthesis of novel protein-cargo conjugates. However, the fluorogenic aspect of the reaction between the linker and the target peptide was less than we desired. To address this shortcoming, a second linker reagent was prepared. This new design also allowed for the site-specific introduction of an alkyne functional group onto the target peptide, but in a highly fluorogenic and rapid manner. The site-specific addition of an alkyne group to a protein of interest was thus monitored in situ by fluorescence increase, prior to the attachment of azide-functionalized cargo. Finally, we also demonstrated that the cargo can also be attached first, in an azide/alkyne cycloaddition reaction, prior to fluorogenic conjugation with the target peptide-fused protein.

Controlling the block sequence of multi-block oligomer ligands for neutralization of a target peptide

A series of block oligomers consisting of the same monomer composition but a different block sequence was prepared via reversible addition–fragmentation chain transfer (RAFT) polymerization to screen high affinity ligands for a toxic peptide.