Kinetic Characterization of Inhibition of Cathepsins L and S by Peptides With Anticancer Potential

Cysteine cathepsins have been suggested as attractive therapeutic targets due to their critical role in several pathologies. In particular, inhibitors of cysteine cathepsins reduce the viability of tumor cells. The present study uses enzyme kinetics to characterize the interaction of human cathepsins L and S with their peptide substrate acetyl-QLLR-7-amino-4-methylcoumarin (Ac-QLLR-AMC) and peptide inhibitors with anti-tumor activity: FFSFGGAL (CS-PEP1) and acetyl-PLVE-fluoromethyl-ketone (Ac-PLVE-fmk). Due to multiple cellular locations of cathepsins, our study is conducted under different pH conditions, simulating lysosomal and cytosolic environments (pH 4.6 and 6.5-7.0). Catalytic activities of both cathepsins are higher at pH 6.5-7.0 compared to pH 4.6. Affinities for the substrate or inhibitor CS-PEP1 are higher for cathepsin L than S independent of pH, but show different pH sensitivities, reciprocating different pI's of the cathepsins. Mixed inhibition by CS-PEP1 is demonstrated for both cathepsins. While preincubation of cathepsins with CS-PEP1 does not enhance the inhibition, Ac-PLVE-fmk inactivates both cathepsins in the preincubation medium. A strong increase in the inactivation rate is observed with increasing pH in the interval including pK _a of the active site cysteine residues of cathepsins, in agreement with the irreversible modification by mono-fluoromethyl ketones of the catalytic thiolate anion. At pH 4.6, cathepsin L has a higher affinity for Ac-PLVE-fmk, but a slower rate of the irreversible modification compared to cathepsin S. Our findings highlight opportunities for differential targeting of L and S cathepsins by peptide inhibitors in different cellular compartments, providing directions for cathepsin- and location-specific drug design.