Lysosomotropism of basic cathepsin K inhibitors contributes to increased cellular potencies against off-target...

Area of Science:

• Biochemistry
• Pharmacology
• Cell Biology

Background:

• Lysosomal cysteine protease cathepsin K is a therapeutic target for osteoporosis.
• Aryl-piperazine cathepsin K inhibitors demonstrate high selectivity against purified enzymes but exhibit altered potency in cell-based assays.

Purpose of the Study:

• To investigate the discrepancy between purified enzyme potency and cell-based activity of aryl-piperazine cathepsin K inhibitors.
• To understand the impact of lysosomal trapping on the functional selectivity of these inhibitors.

Main Methods:

• Enzyme occupancy assays using purified enzymes and cell-based systems.
• Assessment of lysosomal accumulation and lysosomotropic properties.
• Evaluation of off-target activities in cell-based cathepsin S antigen presentation and cathepsin K bone resorption assays.

Main Results:

• Aryl-piperazine inhibitors, such as CRA-013783, accumulate in lysosomes due to their basic, lipophilic nature (lysosomal trapping).
• This lysosomal trapping leads to significantly higher potency in cell-based occupancy assays compared to purified enzyme assays.
• Off-target activities were increased for basic inhibitors in cell-based assays, while functional cathepsin K activity remained unaffected.

Conclusions:

• The basic and lipophilic nature of aryl-piperazine cathepsin K inhibitors causes lysosomal trapping, reducing their functional selectivity in cellular environments.
• Lysosomal trapping complicates the prediction of therapeutic efficacy based solely on purified enzyme assays.
• Nonbasic analogues may offer improved functional selectivity, warranting further investigation for osteoporosis therapy.