Discovering Enzyme Activators

There are four research areas in the Department of Pharmaceutical Chemistry. Discovering enzyme activators is a research challenge within chemical biology and medicinal chemistry.

The challenge

Most chemical probes and drug discovery efforts focus on inhibiting enzymes. But while blocking an enzyme’s function indicates if it is necessary for a cellular activity, selective activation would indicate whether it is sufficient. In addition, many biological pathways are highly regulated, with a sequential cascade of signals leading to the activation of dormant proenzymes. Discovering direct activators of such latent enzymes would help to reveal how their ignition occurs naturally and suggest new drug leads that, by circumventing upstream mutations, are less vulnerable to resistance.

Examples of our research and methods include

Activating executioner caspases to fight cancer

human procaspase-3

Human procaspase-3, an inactive proenzyme. Molecular image made with UCSF Chimera developed by UCSF Resource for Biocomputing, Visualization, and Informatics.

Like most of the hundreds of human protease enzymes, the 12 members of the caspase family are expressed and stored as proenzymes that are activated only in the wake of specific signaling cascades.

Indeed, a subset of caspases, the executioner caspases -3, -6, and -7, are especially important to keep under control. Upon proteolytic activation by other caspases and proteases, they directly generate the protein-cleaving demolition of apoptosis—the programmed self-destruction of aberrant or unneeded cells that fails to occur in cancers and over-occurs in neuro-degenerative disease.

Department research seeks to directly and selectively activate executioner caspases via chemical probes and protein engineering to determine their specific mechanisms, contributions, and targets in the cellular implosion process. An ultimate goal would be to discover and utilize caspase activators as more direct and effective chemotherapies against cancer cells.

Discovering nanofibril scaffold mimics

Toward that end, researchers used high-throughput screening to discover and refine a synthetic compound dubbed 1541B that activates procaspase-3 with high specificity, independent of upstream signaling. 1541B induced apoptosis in multiple cell lines, including cancerous ones. Indeed, the results indicated that activation of procaspase-3 alone is sufficient to induce rapid apoptosis.

model of proscapase-3 activation

Proposed model of proscapase-3 activation: a trace amount of active caspase-3 enzymes cleave and activate precursors upon colocalization with proenzymes clustering on nanofibril surfaces.

Department researchers were surprised to find that 1541B molecules self-assemble into nanofibrils. Electron microscopy revealed that procaspase-3 binds in clusters to the nanofibrils, a process that appears to mimic protein scaffolding complexes that recruit and activate upstream procaspases.

Further analysis of the mechanism found that trace amounts of active caspase-3 enzyme can cleave and activate its precursors clustered on the nanofibrils in a self-amplifying chain reaction.

Eventually, the nanofibril scaffold might be used to design other novel proenzyme activators and offers another way for researchers to manipulate and study enzyme function. In addition, the finding has implications for Alzheimer’s disease, as amyloid beta fibrils were found to similarly stimulate procaspase-3 activation and thus hasten neuronal cell death.