# Redirecting the Peptide Cleavage Causes Protease Inactivation

## TL;DR

Scientists designed new inhibitors that irreversibly disable a key enzyme, human cathepsin B, by redirecting its normal cleavage process.

## Contribution

A novel irreversible inhibition mechanism for cysteine proteases using redirected cleavage and carbamate warheads is introduced.

## Key findings

- Inhibitors with N-terminal carbamate warheads cause irreversible inactivation of human cathepsin B.
- Redirected cleavage forms non-canonical covalent complexes that resist hydrolysis.
- Inhibitors show second-order rate constants exceeding 10,000 M⁻¹s⁻¹.

## Abstract

Cysteine and serine proteases cleave peptides through covalent catalysis by generating a transient adduct with the N‐terminal part of the substrate after releasing its C‐terminal part. We demonstrate the unique redirection of this event leading to strong enzyme inactivation. For targeting human cathepsin B, a cysteine protease of significant therapeutic importance, we designed tailored peptidomimetics with a variety of dipeptide fragments directed toward the occluding loop and equipped with numerous N‐terminal carbamate warheads. The carbamate deprotonation catalyzed by the active site thiolate initiates the redirected cleavage. The C‐terminal part of the inhibitors remains covalently attached to the protease. Hydrolysis of such carbamoyl‐enzyme complexes is catalytically unsupported rendering inhibition irreversible. This novel mechanism of action comprises a significant extension of the covalent drug space.

Cysteine proteases catalyze substrate cleavage by a two‐step acyl transfer mechanism. Newly designed peptidic inhibitors of human cathepsin B with N‐terminal carbamate warheads undergo a redirected cleavage and generate non‐canonical covalent complexes. Representatives of this chemotype exhibit second order rate constants of inactivation greater than 10 000 M−1s−1.