Protease inhibition faces challenges due to specificity and safety concerns. Innovative approaches, including non-natural peptide inhibitors (NNPIs) and antibody-conjugated delivery (APICs), show promise in targeting cancers and diseases, enabling precise, effective, and safer treatments through tailored, cell-specific therapies.
Protease inhibition presents significant challenges due to the essential physiological roles of proteases and their structural similarities, which complicate the design of highly specific inhibitors. Despite being critical players in diseases such as cancer, autoimmune disorders, and osteoporosis, many protease inhibitors have failed clinical trials due to safety concerns. For instance, inhibitors targeting cathepsin S for autoimmune diseases and cathepsin K for osteoporosis were halted due to adverse effects, highlighting the need for innovative approaches to deliver these inhibitors safely and effectively. Recent research has advanced the understanding of protease inhibition, particularly with cathepsin S, which plays a pivotal role in lymphoma progression.
It was found to regulate interactions between lymphoma cells and T cells, promoting tumor growth by altering antigen presentation. Inhibiting cathepsin S enhanced the immune system’s ability to recognize and destroy lymphoma cells by shifting antigen presentation to favor CD8+ T cell activation. Using this knowledge, researchers developed a prototype inhibitor based on a modified peptide structure with a Michael acceptor, designed to covalently bind to the enzyme’s active site. While this design showed initial promise, it required further refinement to improve potency and specificity. To address these limitations, a non-natural peptide inhibitor (NNPI) library was created, employing a screening approach inspired by site saturation mutagenesis.
This strategy involved systematically testing amino acid modifications to identify potent inhibitors tailored to specific proteases, including cathepsins S, B, K, and L. These efforts successfully produced highly potent and selective inhibitors, demonstrating that the screening approach could exploit the natural substrate preferences of different proteases. However, NNPIs’ inability to cross cellular membranes posed a delivery challenge. This was resolved by developing antibody-peptide inhibitor conjugates (APICs), which leverage antibodies to deliver inhibitors specifically to target cells. APICs effectively transported inhibitors into lysosomes, enabling precise protease inhibition and altered antigen presentation. For example, cathepsin S inhibition enhanced immune recognition and elimination of lymphoma cells, while cathepsin B inhibition reduced the invasiveness of breast cancer cells. These results suggest that APICs hold great promise for treating cancers like lymphoma, breast, and pancreatic cancer, as well as other diseases such as autoimmune disorders and osteoporosis. By enabling targeted delivery of protease inhibitors, this approach addresses safety concerns and has the potential to extend to undruggable proteases and non-protease targets, representing a transformative advance in precision medicine.
By Gabriele Imperato, Department of Pharmacological and Biomolecular Sciences “Rodolfo Paoletti”, University of Milan.
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