Rational Design and Evaluation of Mammalian Ribonuclease Cytotoxins

Abstract

Mammalian pancreatic-type ribonucleases (ptRNases) comprise an enzyme family that is remarkably well suited for therapeutic exploitation. ptRNases are robust and prodigious catalysts of RNA cleavage that can naturally access the cytosol. Instilling cytotoxic activity requires endowing them with the ability to evade a cytosolic inhibitor protein while retaining other key attributes. These efforts have informed our understanding of ptRNase-based cytotoxins, as well as the action of protein-based drugs with cytosolic targets. Here, we address the most pressing problems encountered in the design of cytotoxic ptRNases, along with potential solutions. In addition, we describe assays that can be used to evaluate a successful design in vitro, in cellulo, and in vivo. The emerging information validates the continuing development of ptRNases as chemotherapeutic agents.

Bovine Brain Ribonuclease Is the Functional Homolog of Human Ribonuclease 1

“…d Value is for the complex with bovine ribonuclease inhibitor, determined as described (31,35,36). e Value was determined with circular dichroism spectroscopy (75).…”

“…Inhibitor Dissociation Rate-The equilibrium dissociation rates of the RI⅐ribonuclease complexes were determined as described (31,35,36). Briefly, RI and 2Ј,7Ј-diethylfluoresceinlabeled ribonucleases were mixed in equimolar ratios, and the resulting solution was incubated at 25°C for 5 min.…”

“…Cellular Internalization of Ribonucleases-The uptake of BODIPY-labeled ribonucleases into nonadherent mammalian cells was monitored by flow cytometry, as described (35). Human K-562 cells were grown in RPMI media (Invitrogen) containing FBS (10% v/v) and penicillin/streptomycin (Invitrogen).…”

Assignments of RNase A by ADAPT-NMR and enhancer

“…RNase A catalyzes the cleavage of the P–O 5′ bond of RNA with a k cat / K M value that can exceed 10 9 M −1 s −1 and exhibits high thermostability ( T m 62 °C). (For a review, see: Lomax et al, 2012.) The presumed biological function of RNase A is catalysis of the depolymerization of ingested RNA.…”

“…Recently, homologs and variants of RNase A have shown potential as cancer chemotherapeutic agents (Lomax et al 2012). Enabling RNase A to achieve its clinical potential is likely to require a thorough understanding of its interactions with cellular molecules, including cell-surface glycans and the cytosolic ribonuclease inhibitor protein.…”