Principal Investigator Ronald Raines
By understanding the underlying cellular processes, we are able to endow an otherwise innocuous human enzyme with clinically useful cytotoxicity. Ribonucleases catalyze the cleavage of RNA. Early-on, we made contributions to chemical enzymology (e.g., general acid–base catalysis and enzymatic processivity) and protein folding (e.g., prolyl peptide-bond isomerization and disulfide-bond formation) by using ribonuclease A as a model system. Then, we reasoned that this cationic enzyme, which can enter human cells (which are anionic), could become cytotoxic if it were able to evade a cytosolic inhibitor protein. By determining the structure of the human enzyme·inhibitor complex and making rational changes to a handful of amino-acid residues, we have created ribonucleases that evade the inhibitor and are indeed toxic to human cells, verifying our hypothesis. We have also demonstrated that ribonucleases have a marked preference for killing cancerous cells due to a nanomolar affinity for Globo H, which is a cell-surface hexasaccharide that is a human cancer antigen.
An inhibitor-evading variant of the human ribonuclease is in a Phase I clinical trial as a cancer chemotherapeutic agent. Over 50 patients have been treated with our ribonuclease at the University of Texas MD Anderson Cancer Center and the University of Wisconsin Carbone Cancer Center, and many of these patients have achieved stable disease. To enable on‑going mechanistic analyses, we have used CRISPR/Cas9 to knockout the inhibitor protein in HeLa cells, making them defenseless against invading ribonucleases. We are also determining whether natural N‑glycosylation enables the ribonuclease in human serum to evade the inhibitor protein and provide an innate immunity against cancer, and we are developing new small-molecule fluorogenic probes to illuminate the pathway taken by extracellular ribonucleases towards intracellular RNA.