Natural and Engineered Cytotoxic Ribonucleases: Therapeutic Potential

Rybak, Susanna M.; Newton, Dianne L.

doi:10.1006/excr.1999.4718

Cited by 86 publications

(76 citation statements)

References 93 publications

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“…At its N-terminus it contains a short peptide (Xpress peptide, Invitrogen) linked downstream to an a⁄nity tag of six histidine residues. Between V H and V L chains of the scFv a 15-residue linker, made up of glycine and serine (G 4 S) 3 , is interposed, and a six-residue spacer is inserted between the antibody fragment and the ribonuclease to minimize hindrance between the two moieties of the chimeric protein. The scFv was linked to the N-terminus of HPRNase as this is more solvent-accessible than the C-terminus [19].…”

Section: Construction and Puri¢cation Of Erb-hprmentioning

confidence: 99%

“…The key to the IR strategy is that RNases, although per se not cytotoxic, become cytotoxic when they are internalized by the target cell, i.e. the cell that displays on its surface the epitope recognized by the antibody moiety [3]. This is why the most suitable epitopes for the selection of an antibody for IR are cell receptors, since the anti-receptor antibody can mimic the receptor ligand and be internalized.…”

Section: Introductionmentioning

confidence: 99%

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A new RNase‐based immunoconjugate selectively cytotoxic for ErbB2‐overexpressing cells

et al. 2002

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We report a new tumor-directed immunoRNase, a chimeric protein made up of an antibody fragment (single-chain Fv fragment) directed to ErbB2, a cell surface receptor, and a non-toxic, human ribonuclease, which upon cell internalization becomes cytotoxic. The immunoRNase is active as a ribonuclease, specifically binds and selectively kills ErbB2-positive cells. ErbB2 is one of the most specific tumor-associated antigens identified so far, overexpressed on tumor cells of different origin. Its choice as target antigen and that of a non-toxic, human RNase as the killer moiety makes this immunoRNase a new, potentially attractive anticancer agent. ß

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Section: Construction and Puri¢cation Of Erb-hprmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A new RNase‐based immunoconjugate selectively cytotoxic for ErbB2‐overexpressing cells

et al. 2002

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“…In contrast, variants of a single protein, the N-terminal domain of the repressor protein from bacteriophage , have been used to demonstrate a definite link between conformational stability and metabolic turnover (18). More recently, the conformational stability of bovine pancreatic trypsin inhibitor variants was shown to correlate with the yield of intact protein produced in a heterologous system (19).RNase A homologs elicit diverse biological activities, including specific toxicity to cancer cells (20,21). Onconase™, which is a homolog of RNase A in the Northern leopard frog, is now in Phase III clinical trials for the treatment of malignant mesothelioma.…”

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confidence: 99%

“…RNase A homologs elicit diverse biological activities, including specific toxicity to cancer cells (20,21). Onconase™, which is a homolog of RNase A in the Northern leopard frog, is now in Phase III clinical trials for the treatment of malignant mesothelioma.…”

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confidence: 99%

Conformational Stability Is a Determinant of Ribonuclease A Cytotoxicity

Klink¹,

Raines²

2000

Journal of Biological Chemistry

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Onconase™, a homolog of bovine pancreatic ribonuclease A (RNase A) with high conformational stability, is cytotoxic and has efficacy as a cancer chemotherapeutic agent. Unlike wild-type RNase A, the G88R variant is toxic to cancer cells. Here, variants in which disulfide bonds were removed from or added to G88R RNase A were used to probe the relationship between conformational stability and cytotoxicity in a methodical manner. The conformational stability of the C40A/G88R/C95A and C65A/C72A/G88R variants is less than that of G88R RNase A. In contrast, a new disulfide bond that links the N and C termini (residues 4 and 118) increases the conformational stability of G88R RNase A and C65A/C72A/ G88R RNase A. These changes have little effect on the ribonucleolytic activity of the enzyme or on its ability to evade the cytosolic ribonuclease inhibitor protein. The changes do, however, have a substantial effect on toxicity toward human erythroleukemia cells. Specifically, conformational stability correlates directly with cytotoxicity as well as with resistance to proteolysis. These data indicate that conformational stability is a key determinant of RNase A cytotoxicity and suggest that cytotoxicity relies on avoiding proteolysis. This finding suggests a means to produce new cancer chemotherapeutic agents based on mammalian ribonucleases. The free energy difference between the native and unfolded states of a protein is small-typically 5-15 kcal/mol (1, 2). In their native state, many proteins are less susceptible to proteolytic degradation than when unfolded (3). In the unfolded state, the steric protection of peptide bonds provided by the compact native state is lost (4). For example, bovine pancreatic ribonuclease A (RNase A) 1 (EC 3.1.27.5 (5, 6)) is degraded more readily by proteases in the presence of denaturants or elevated temperatures (7-10) and less readily when glycosylated (11).The rates of intracellular protein turnover vary by 10 3 -fold (12). Apparently, some proteins are better able to thwart the proteolytic machinery (13,14). These proteins remain intact and retain activity longer within the cell. A correlation between the conformational stability of a protein and its catabolism was reported over 20 years ago (15). Since then, studies using unrelated proteins have either supported (16) or contradicted (14, 17) this correlation. Drawing conclusions from these studies is problematic because the proteins were divergent in characteristics that have been implicated in metabolic turnover. In contrast, variants of a single protein, the N-terminal domain of the repressor protein from bacteriophage , have been used to demonstrate a definite link between conformational stability and metabolic turnover (18). More recently, the conformational stability of bovine pancreatic trypsin inhibitor variants was shown to correlate with the yield of intact protein produced in a heterologous system (19).RNase A homologs elicit diverse biological activities, including specific toxicity to cancer cells (20,21). Onconase™, which ...

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“…Now homologs of RNase A are becoming important new chemotherapeutics (2)(3)(4). For example, low levels of bovine seminal ribonuclease (BS-RNase) 1 are cytotoxic (5,6).…”

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confidence: 99%

A Ribonuclease A Variant with Low Catalytic Activity but High Cytotoxicity

Bretscher¹,

Abel²,

Raines³

2000

Journal of Biological Chemistry

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OnconaseTM , a homolog of ribonuclease A (RNase A) with low ribonucleolytic activity, is cytotoxic and has efficacy as a cancer chemotherapeutic. Here variants of RNase A were used to probe the interplay between ribonucleolytic activity and evasion of the cytosolic ribonuclease inhibitor protein (RI) in the cytotoxicity of ribonucleases. K41R/G88R RNase A is a less active catalyst than G88R RNase A but, surprisingly, is more cytotoxic. Like Onconase TM , the K41R/G88R variant has a low affinity for RI, which apparently compensates for its low ribonucleolytic activity. In contrast, K41A/G88R RNase A, which has the same affinity for RI as does the K41R/ G88R variant, is not cytotoxic. The nontoxic K41A/G88R variant is a much less active catalyst than is the toxic K41R/G88R variant. These data indicate that maintaining sufficient ribonucleolytic activity in the presence of RI is a requirement for a homolog or variant of RNase A to be cytotoxic. This principle can guide the design of new chemotherapeutics based on homologs and variants of RNase A.Ribonuclease A (RNase A; EC 3.1.27.5 (1)) was perhaps the most studied enzyme of the twentieth century. Now homologs of RNase A are becoming important new chemotherapeutics (2-4). For example, low levels of bovine seminal ribonuclease (BS-RNase) 1 are cytotoxic (5, 6). More significantly, Onconase TM (ONC), which is isolated from the frog Rana pipiens (7), is currently in Phase III human clinical trials for the treatment of malignant mesothelioma. In addition, ONC inhibits HIV-1 replication in chronically infected human cells (8). Understanding the mechanism of ribonuclease cytotoxicity is vital for the further development of ribonucleases as chemotherapeutics.Ribonuclease-mediated cytotoxicity is known to depend on several factors. Ribonucleases must enter the cell and reach the cytosol, where RNA degradation leads ultimately to cell death (9, 10). Indeed, injecting ribonucleases directly into Xenopus oocytes increases their cytotoxicity (11,12). In the cytosol, ribonucleases encounter the ribonuclease inhibitor protein (RI). RI constitutes Ն0.01% of protein in the cytosol (13,14) and inactivates ribonucleases by forming a tight complex that prevents RNA substrates from entering the active site (Fig. 1A) (15).Ribonucleolytic activity is requisite for the cytotoxicity of BS-RNase and ONC (16,17). Yet despite its relatively high ribonucleolytic activity, RNase A is not cytotoxic (9, 18). The cytotoxicity of BS-RNase and ONC has been attributed to the ability of BS-RNase A and ONC to evade RI (19,20). RI is a potent inhibitor of RNase A with K i near 10 Ϫ14 M (21, 22). In contrast, ONC (estimated K i Ն 10 Ϫ6 M (23)) and BS-RNase (24) escape inhibition by RI.BS-RNase and ONC use different strategies to evade RI. BS-RNase forms a homodimer, which is stabilized by two intersubunit disulfide bridges. This dimeric form has a much lower affinity for RI than does the free monomer (24). ONC evades RI as a monomer. Only 3 of the 24 RNase A residues that contact RI are conserved in ONC...

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Natural and Engineered Cytotoxic Ribonucleases: Therapeutic Potential

Cited by 86 publications

References 93 publications

A new RNase‐based immunoconjugate selectively cytotoxic for ErbB2‐overexpressing cells

A new RNase‐based immunoconjugate selectively cytotoxic for ErbB2‐overexpressing cells

Conformational Stability Is a Determinant of Ribonuclease A Cytotoxicity

A Ribonuclease A Variant with Low Catalytic Activity but High Cytotoxicity

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