Ribonuclease: From kcat/KM to the Clinic

Abstract

The vast majority of extant drugs are small molecules that block the flow of biochemical information. Enzymes also have the potential to disrupt this flow. For example, ribonuclease A, which spawned the first enzymatic reaction mechanism and seminal advances in protein chemistry, is an efficient catalyst of RNA cleavage. We have discovered that this highly cationic enzyme naturally enters the cytosol of mammalian cells. By enabling ribonuclease A to evade a femtomolar cytosolic inhibitor protein, we have endowed it with the ability to cleave cellular RNA and thereby kill human cells. A variety of chemical and biological tools, especially “fluorogenic labels”, have enabled us to reveal the kinetic mechanism by which ribonuclease A exerts its cytotoxic activity. Importantly, ribonucleases have a marked preference for killing cancerous cells due to a nanomolar affinity for Globo H, a cell-surface glycan that is a human cancer antigen. An inhibitor-evading variant of the human homolog of ribonuclease A is in a Phase I clinical trial as a cancer chemotherapeutic agent. Patients have been treated with this enzyme at the University of Texas MD Anderson Cancer Center and the University of Wisconsin Carbone Cancer Center, and have achieved stable disease. Meanwhile, we have discovered that the cytosolic inhibitor protein is phosphorylated by enzymes of the ERK pathway, increasing its already prodigious affinity for ribonucleases. The clinical ribonuclease exhibits strongly synergistic toxicity with ERK-pathway inhibitors (e.g., trametinib and dabrafenib) towards lung cancer cells (including a KRASG12C variant) and melanoma cells (including BRAFV600E variants). Thus, decades of basic research on ribonuclease A are now poised to yield a drug. Support or Funding Information This work is supported by Grant R01 CA073808 (NIH). Structure of human ribonuclease (blue) bound to its natural inhibitor protein (red). The complex (Kd = 10−17 M) is the tightest known between biomolecules. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.