Thomas J. Rutkoski

Evasion of Ribonuclease Inhibitor as a Determinant of Ribonuclease Cytotoxicity

Onconase (ONC) is an amphibian member of the bovine pancreatic ribonuclease (RNase A) superfamily that exhibits innate antitumoral activity. ONC has been granted both orphan-drug and fast-track status by the U.S. Food and Drug Administration for the treatment of malignant mesothelioma, and is poised to become the first chemotherapeutic agent based on a ribonuclease. Investigations into the mechanism of ribonuclease-based cytotoxicity have elucidated several important determinants for cytotoxicity, including efficient deliverance of ribonucleolytic activity to the cytosol and preservation of conformation stability. Nevertheless, the most striking similarity between ONC and bovine seminal ribonuclease, another naturally cytotoxic ribonuclease, is their insensitivity to inhibition by the potent cytosolic ribonuclease inhibitor protein (RI). RI typically binds to its ribonuclease ligands with femtomolar affinity--an extraordinary feat considering the modest sequence identity among the bound ribonucleases. Mammalian ribonucleases such as RNase A or its human homologue, RNase 1, have the potential to be more attractive chemotherapeutic agents than ONC owing to their higher catalytic activity, low potential for immunogenicity, favorable tissue distribution, and high therapeutic index, but are limited by their sensitivity to RI. These non-toxic mammalian ribonucleases can be transformed into potent cytotoxins by engendering them with RI-evasion using protein engineering strategies such as site-directed mutagenesis, multimerization, fusion to a targeting moiety, and chemical modification. In several instances, these engineered ribonucleases exhibit greater cytotoxicity in vitro than does ONC. Herein, we review the biochemical characteristics of RIribonuclease complexes and progress towards the development of mammalian ribonuclease-based chemotherapeutics through the elicitation of RI-evasion.

Structure, dynamics, and specificity of endoglucanase D from Clostridium cellulovorans.

The enzymatic degradation of cellulose is a critical step in the biological conversion of plant biomass into an abundant renewable energy source. An understanding of the structural and dynamic features that cellulases utilize to bind a single strand of crystalline cellulose and hydrolyze the β-1,4-glycosidic bonds of cellulose to produce fermentable sugars would greatly facilitate the engineering of improved cellulases for the large-scale conversion of plant biomass. Endoglucanase D (EngD) from Clostridium cellulovorans is a modular enzyme comprising an N-terminal catalytic domain and a C-terminal carbohydrate-binding module, which is attached via a flexible linker. Here, we present the 2.1-Å-resolution crystal structures of full-length EngD with and without cellotriose bound, solution small-angle X-ray scattering (SAXS) studies of the full-length enzyme, the characterization of the active cleft glucose binding subsites, and substrate specificity of EngD on soluble and insoluble polymeric carbohydrates. SAXS data support a model in which the linker is flexible, allowing EngD to adopt an extended conformation in solution. The cellotriose-bound EngD structure revealed an extended active-site cleft that contains seven glucose-binding subsites, but unlike the majority of structurally determined endocellulases, the active-site cleft of EngD is partially enclosed by Trp162 and Tyr232. EngD variants, which lack Trp162, showed a significant reduction in activity and an alteration in the distribution of cellohexaose degradation products, suggesting that Trp162 plays a direct role in substrate binding.

Antitumor Activity of Ribonuclease Multimers Created by Site-Specific Covalent Tethering

Site-specific cross-linking can generate homogeneous multimeric proteins of defined valency. Pancreatic-type ribonucleases are an especially attractive target, as their natural dimers can enter mammalian cells, evade the cytosolic ribonuclease inhibitor (RI), and exert their toxic ribonucleolytic activity. Here, we report on the use of eight distinct thiol-reactive cross-linking reagents to produce dimeric and trimeric conjugates of four pancreatic-type ribonucleases. Both the site of conjugation and, to a lesser extent, the propinquity of the monomers within the conjugate modulate affinity for RI, and hence cytotoxicity. Still, the cytotoxicity of the multimers is confounded in vitro by their increased hydrodynamic radius, which attenuates cytosolic entry. A monomeric RI-evasive variant of bovine pancreatic ribonuclease (RNase A) inhibits the growth of human prostate and lung tumors in mice. An RI-evasive trimeric conjugate inhibits tumor growth at a lower dose and with less frequent administration than does the monomer. This effect is attributable to an enhanced persistence of the trimers in circulation. On a molecular basis, the trimer is ∼300-fold more efficacious and as well tolerated as erlotinib, which is in clinical use for the treatment of lung cancer. These data encourage the development of mammalian ribonucleases for the treatment of human cancers.

Site-specific PEGylation endows a mammalian ribonuclease with antitumor activity.

Mammalian ribonucleases are emerging as cancer chemotherapeutic agents. Their cationicity engenders cell permeability, and their enzymatic activity destroys the biochemical information encoded by RNA. The pharmacologic potential of ribonucleases is, however, obviated by their high sensitivity to a cytosolic inhibitor protein (RI) and their small size, which limits their residence in serum. We reasoned that site specific conjugation of a poly(ethylene glycol) (PEG) chain could both reduce sensitivity to RI and increase serum half-life. We found that appending a PEG moiety can enable bovine pancreatic ribonuclease (RNase A) to evade RI, depending on the site of conjugation and the length and branching of the chain. Although a pendant PEG moiety decreases antiproliferative activity in vitro, PEGylation discourages renal clearance in vivo and leads to nearly complete tumor growth inhibition in a mouse xenograft model. These data demonstrate that a pendant PEG moiety can be beneficial to the action of proteins that act within the cytosol, and that strategic site-specific PEGylation can endow a mammalian ribonuclease with potent antitumor activity.

Effectiveness and limitations of local structural entropy optimization in the thermal stabilization of mesophilic and thermophilic adenylate kinases

Local structural entropy (LSE) is a descriptor for the extent of conformational heterogeneity in short protein sequences that is computed from structural information derived from the Protein Data Bank. Reducing the LSE of a protein sequence by introducing amino acid mutations can result in fewer conformational states and thus a more stable structure, indicating that LSE optimization can be used as a protein stabilization method. Here, we describe a series of LSE optimization experiments designed to stabilize mesophilic and thermophilic adenylate kinases (AKs) and report crystal structures of LSE‐optimized AK variants. In the mesophilic AK, thermal stabilization by LSE reduction was effective but limited. Structural analyses of the LSE‐optimized mesophilic AK variants revealed a strong correlation between LSE and the apolar buried surface area. Additional mutations designed to introduce noncovalent interactions between distant regions of the polypeptide resulted in further stabilization. Unexpectedly, optimizing the LSE of the thermophilic AK resulted in a decrease in thermal stability. This destabilization was reduced when charged residues were excluded from the possible substitutions during LSE optimization. These observations suggest that stabilization by LSE reduction may result from the optimization of local hydrophobic contacts. The limitations of this process are likely due to ignorance of other interactions that bridge distant regions in a given amino acid sequence. Our results illustrate the effectiveness and limitations of LSE optimization as a protein stabilization strategy and highlight the importance and complementarity of local conformational stability and global interactions in protein thermal stability. Proteins 2014; 82:2631–2642.