UCSF

Most proteases are expressed as inactive precursors, known as zymogens. Studies to explore zymogen activation can further elucidate biological regulatory mechanisms and lead to novel chemical biology tools to trigger them. Caspases, or cysteine aspartyl proteases, are expressed as such inactive precursors, or procaspases, which become activated in fate-determining transformations, such as apoptosis. Procaspases are naturally activated upon binding to scaffolding complexes or as a result of cleavage by upstream proteases in response to extrinsic or intrinsic cellular signals. Removal of an N-terminal prodomain and an additional cleavage to yield a large and small subunit generates the mature enzyme.

A small molecule, termed 1541, was identified from a high-throughput screen to facilitate procaspase-3 maturation. Unexpectedly, 1541 assembles into nanofibrils that bind procaspase-3, however, the proenzyme alone cannot auto-process. A trace amount of mature caspase-3 contaminant dominates procaspase-3 activity measurements. Thus, 1541 acts as a scaffolding moiety to promote induced proximity of procaspase-3 with caspase-3 to enhance autocatalytic maturation of the proenzyme. Similar to known signaling complexes, proteogenic fibrils may serve as a platform for procaspase maturation. These studies suggest that increasing the activity of an upstream protease or promoting colocalization of the proenzyme with an active protease are reasonable approaches to facilitate procaspase-3 activation.

1541 represents a first-in-class, self-assembling, small-molecule nanofibril, which catalyzes procaspase-3 activation. A focused library of 1541-analogs was generated to evaluate the structural requirements for procaspase binding and activation. An initial screen of the library against procaspase-3, -6, and -7 identified compounds that can selectively activate each procaspase. These active molecules also form fibrils, yet demonstrate selective interactions with the zymogen and mature caspases. Thus, procaspase binding and activation can be tuned based on the substituents of the small molecule.

Surprisingly, 1541 also induces apoptosis in a manner dependent upon the fibrils. While 1541 promotes cell death and caspase activity in most cell lines tested, additional fibril-forming analogs appear to have more selective toxicity profiles. Cell death induced by the fibrils is independent of both the intrinsic and extrinsic cellular pathways. Nevertheless, the cellular activity of these fibrils is unlikely due to direct binding of procaspases.