Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

By reason of their cytotoxicity, ribonucleases (RNases) are potential anti-tumor drugs. Particularly members from the RNase A and RNase T1 superfamilies have shown promising results. Among these enzymes, Onconase, an RNase from the Northern Leopard frog, is furthest along in clinical trials. A general model for the mechanism of the cytotoxic action of RNases includes the interaction of the enzyme with the cellular membrane, internalization, translocation to the cytosol, and degradation of ribonucleic acid. The interplay of these processes as well as the role of the thermodynamic and proteolytic stability, the catalytic activity, and the capability of the RNase to evade the intracellular RNase inhibitor has not yet been fully elucidated. This paper discusses the various approaches to exploit RNases as cytotoxic agents.
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PMID:Natural and engineered ribonucleases as potential cancer therapeutics. 1690 46

Ranpirnase [Onconase] is an amphibian oocyte/early embryo ribonuclease (RNase) of 105 amino acids in length that is capable of controlling tumour growth by degrading RNA within cancer cells, resulting in inhibition of protein synthesis and arresting mitosis in G(1 )phase. It represents the first successful isolation, purification and characterisation of the oocytic/early embryonic factor that is capable of controlling cell growth activities of the early embryonic tissues. Alfacell Corporation is currently conducting clinical trials of ranpirnase in patients with unresectable malignant mesothelioma and non-small-cell lung cancer. The company may initiate phase II clinical trials in breast cancer and oesophageal cancer in 2006. Alfacell expanded a research agreement with the National Cancer Institute in September 2002, allowing the NCI to examine the effects of ranpirnase as a radiation enhancer. However, investigation in this use of ranpirnase now appears to be discontinued. Alfacell is conducting a confirmatory phase IIIb registration trial of ranpirnase plus doxorubicin versus doxorubicin alone in more than 360 patients with unresectable malignant mesothelioma, and will assess survival as the primary endpoint. The targeted treatment group in this trial represents 90% of malignant mesothelioma patients at the time of diagnosis. The trial is being conducted in the US, Canada, Poland, Italy, Germany, Australia, New Zealand, Russia, Romania, Mexico and Brazil. In April 2006, a total of 210 events (patient deaths) was reached, representing two-thirds of the required events for the study. Results from the protocol-specified first interim analysis based on one-third of the required events have been reported and the company has the option to conduct a second interim analysis of the data at any point after 210 events. A final analysis will be undertaken at 316 events. Alfacell completed a phase III trial of single-agent ranpirnase in patients with unresectable malignant mesothelioma in April 1999. The efficacy of ranpirnase was compared with that of doxorubicin (head-to-head). The primary objectives were overall survival, progression-free survival and quality of life. In preclinical studies, ranpirnase demonstrated significant activity against neuroblastoma, rhabdomyosarcoma and chemotherapy-resistant variants of these cancer cells. Development for these indications has been discontinued. Preclinical investigations conducted by Alfacell showed synergistic antitumour effects between ranpirnase and proteasome inhibitors. However, development is this area has been discontinued. Alfacell announced in May 2003 that it would be providing ranpirnase to the federal severe acute respiratory syndrome (SARS) testing programme for evaluation against the human coronavirus implicated in the disease. No further development has been reported. Alfacell has received nine US and four European patents for ranpirnase. Patents issued in the US range from the 1996-issued patent (No. 5 559 212) covering the amino acid sequence of ranpirnase, to the patent (No. 6 175 003 B1) issued in January 2001 protecting the gene sequences of the compound plus another genetically engineered variant, effectively protecting the company's proprietary technology. In August 2002, Alfacell received a US patent (No. 6 423 515 B1) entitled 'Methods of Making Nucleic Acids Encoding Ribonucleases'. This patent is effective until 2020.
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PMID:Ranpirnase: amphibian ribonuclease A, P-30 protein-alfacell. 1732 10

Onconase is an RNase with a very specific property because it is selectively toxic to transformed cells. This toxin is thought to recognize cell surface receptors, and the protection conferred by metabolic poisons against Onconase toxicity indicated that this RNase relies on endocytic uptake to kill cells. Nevertheless, its internalization pathway has yet to be unraveled. We show here that Onconase enters cells using AP-2/clathrin-mediated endocytosis. It is then routed, together with transferrin, to the receptor recycling compartment. Increasing the Onconase concentration in this structure using tetanus toxin light chain expression enhanced Onconase toxicity, indicating that recycling endosomes are a key compartment for Onconase cytosolic delivery. This intracellular destination is specific to Onconase because other (and much less toxic) RNases follow the default pathway to late endosomes/lysosomes. Drugs neutralizing endosomal pH increased Onconase translocation efficiency from purified endosomes during cell-free translocation assays by preventing Onconase dissociation from its receptor at endosomal pH. Consistently, endosome neutralization enhanced Onconase toxicity up to 100-fold. Onconase translocation also required cytosolic ATP hydrolysis. This toxin therefore shows an unusual entry process that relies on clathrin-dependent endocytic uptake and then neutralization of low endosomal pH for efficient translocation from the endosomal lumen to the cytosol.
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PMID:Intracellular pathway of Onconase that enables its delivery to the cytosol. 1737 40

Besides Onconase (ONC) and its V11/N20/R103-variant, oocytes of the Northern Leopard frog (Rana pipiens) contain another homologue of ribonuclease A, which we named Amphinase (Amph). Four variants (Amph-1-4) were isolated and sequenced, each 114 amino acid residues in length and N-glycosylated at two positions. Sequence identities (a) among the variants and (b) versus ONC are 86.8-99.1% and 38.2-40.0%, respectively. When compared with other amphibian ribonucleases, a typical pattern of cysteine residues is evident but the N-terminal pyroglutamate residue is replaced by a six-residue extension. Amph variants have relatively weak ribonucleolytic activity that is insensitive to human ribonuclease inhibitor protein (RI). Values of k(cat)/K(M) with hypersensitive fluorogenic substrates are 10(4) and 10(2)-fold lower than the maximum values exhibited by ribonuclease A and ONC, respectively, and there is little cytosine/uracil or adenine/guanine discrimination at the B(1) or B(2) subsites, respectively. Amph variants have cytotoxic activity toward A-253 carcinoma cells that requires intact ribonucleolytic activity. The glycan component has little or no influence over single-stranded RNA cleavage, RI evasion or cytotoxicity. The crystal structures of natural and recombinant Amph-2 (determined at 1.8 and 1.9 A resolution, respectively) reveal that the N terminus is unlikely to play a catalytic role (but an unusual alpha2-beta1 loop may do so) and the B(2) subsite is rudimentary. At the active site, structural features that may contribute to the enzyme's low ribonucleolytic activity are the fixture of Lys14 in an obstructive position, the accompanying ejection of Lys42, and a lack of constraints on the conformations of Lys42 and His107.
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PMID:Enzymatic and structural characterisation of amphinase, a novel cytotoxic ribonuclease from Rana pipiens oocytes. 1756 Jun 6

Onconase (ONC) is a cytotoxic ribonuclease of the pancreatic ribonuclease A superfamily isolated from oocytes or early embryos of the Northern leopard frog (Rana pipiens). It shows anticancer activity and currently is in Phase IIIb clinical trial for unresectable malignant mesothelioma. We generated several variants of ONC possessing mutations in selected structural regions of the molecule that have altered ribonucleolytic activity and/or conformational stability. The relationship between the stability and ribonucleolytic activity of these variants and their cytostatic and cytotoxic properties was investigated on several tumor cell lines. Similar to ONC, all variants were inducing reproductive cell death detected by reduced clonogenicity. The surviving cells proliferated at reduced rates as reflected by diminished size of colonies and prolongation of G(0/1) phase of the cell cycle. Some cells were undergoing apoptosis. The cytotoxic and cytostatic effects of ONC and its variants were predominantly determined by their catalytic activity rather than by conformational stability.
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PMID:The interdependence between catalytic activity, conformational stability, and cytotoxicity of onconase. 1763 63

Onconase (ONC), an antitumor ribonuclease from oocytes of a frog Rana pipiens, capable of inducing apoptosis in many cell lines is synergistic with several other anticancer drugs. Since cytotoxic effects of numerous drugs are modulated by reactive oxygen intermediates (ROI), we have studied effects of ONC on the intracellular level of oxidants in several normal cell types as well as tumor cell lines. It is demonstrated for the first time that ONC substantially decreases the content of ROI in all cell lines studied. This effect depends on the ribonucleolytic activity of the enzyme and is due to both, decreased rate of ROI generation and accelerated rate of their degradation. Onconase decreases the mitochondrial transmembrane potential and consequently, generation of ATP. Simultaneously the enzyme decreases the expression of an antiapoptotic protein Bcl-2, and upregulates the proapoptotic Bax protein. These finding are consistent with the enzyme propensity to induce apoptosis. The observed antioxidant activity of ONC may be an important element of its cytotoxicity towards cancer cells. The enzyme seems to exert its biological activities by interfering with the redox system of cellular regulation.
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PMID:Onconase, an anti-tumor ribonuclease suppresses intracellular oxidative stress. 1767 95

The cytotoxic RNase, Onconase (ONC), isolated from amphibian oocytes, was used to study its effect on the radiation response in A549 human NSCLC in vitro and in vivo. In cell culture studies, we found that ONC increased the radiation response by ONC-induced inhibition of O2 consumption (QO2). The occurrence of apoptosis was increased by ONC and was dependent on dosages and time exposure (measured by a Tunnel in situ cell death detection assay). Moreover, ONC inhibited sublethal damage repair (SLDR), confirmed by a split dose experiment. In animal studies, ONC significantly increased the radiation-induced tumor growth delay of A549 tumors in vivo. Using a non-invasive DCE-MRI technology, ONC-induced changes of perfusion were observed in A549 tumors. We concluded that the ONC-induced enhancement in tumor oxygenation was mainly due to the reduction in QO2 rather than an increase in tumor blood flow. This investigation suggests important potential clinical uses of ONC for the treatment of NSCLC cancer patients.
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PMID:Possible mechanisms of improved radiation response by cytotoxic RNase, Onconase, on A549 human lung cancer xenografts of nude mice. 1772 47

Onconase, a cytotoxic and antitumor RNase inhibits viral replication in chronically HIV-1-infected human cells under sub lethal concentrations. Cellular tRNA has been implicated as the target for onconase action. We have recently shown that onconase cleaves selectively at GG residues in the UGG context in the variable loop and D-arm of the tRNA substrates. We therefore examined onconase cleavage specificity in in vitro transcribed tRNA(Lys3), which is the primer for HIV-1 reverse transcription but does not have UGG anywhere in its sequence. Onconase was found to cleave tRNA(Lys3) predominantly at the GG residues in the GGG triplet present in the variable loop. Mutations at this site did not effect onconase cleavages. Interestingly thus, onconase seems to cleave predominantly in the variable loop of tRNA(Lys3) regardless of the sequence context implying possible contribution of even structural determinants for its selective cleavages.
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PMID:Onconase action on tRNA(Lys3), the primer for HIV-1 reverse transcription. 1788 4

Onconase is an extremely stable member of the RNase A superfamily. The increase in the thermodynamic stability by 20 kJ x mol(-1) in comparison to RNase A was expected to result in altered folding behavior. Despite the lack of cis-Pro residues in native Onconase, refolding at low concentrations of guanidine hydrochloride was complex and showed three kinetic phases (fast, medium, and slow), with rate constants differing by a factor of about 10 each. None of the phases could be accelerated by peptidyl-prolyl cis-trans isomerases, pointing to the absence of kinetic phases that are limited by Pro isomerization. The detailed analysis by various probes indicates that the burial of the N-terminal Trp3, which is associated with the restoration of the active site, occurs in the slow phase, i.e. in the last step of refolding. Evidently, in contrast to the folding of RNase A, there is no catalytically active native-like intermediate in the folding of Onconase.
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PMID:Identification of three phases in Onconase refolding. 1794 37

Onconase (ONC) is a homolog of bovine pancreatic ribonuclease (RNase A) from the frog Rana pipiens. ONC displays antitumoral activity and is in advanced clinical trials for the treatment of cancer. Here, we report the first atomic structures of ONC-nucleic acid complexes: a T89N/E91A ONC-5'-AMP complex at 1.65 A resolution and a wild-type ONC-d(AUGA) complex at 1.90 A resolution. The latter structure and site-directed mutagenesis were used to reveal the atomic basis for substrate recognition and turnover by ONC. The residues in ONC that are proximal to the scissile phosphodiester bond (His10, Lys31, and His97) and uracil nucleobase (Thr35, Asp67, and Phe98) are conserved from RNase A and serve to generate a similar bell-shaped pH versus k(cat)/K(M) profile for RNA cleavage. Glu91 of ONC forms two hydrogen bonds with the guanine nucleobase in d(AUGA), and Thr89 is in close proximity to that nucleobase. Installing a neutral or cationic residue at position 91 or an asparagine residue at position 89 virtually eliminated the 10(2)-fold guanine:adenine preference of ONC. A variant that combined such substitutions, T89N/E91A ONC, actually preferred adenine over guanine. In contrast, installing an arginine residue at position 91 increased the guanine preference and afforded an ONC variant with the highest known k(cat)/K(M) value. These data indicate that ONC discriminates between guanine and adenine by using Coulombic interactions and a network of hydrogen bonds. The structure of the ONC-d(AUGA) complex was also used to probe other aspects of catalysis. For example, the T5R substitution, designed to create a favorable Coulombic interaction between ONC and a phosphoryl group in RNA, increased ribonucleolytic activity by twofold. No variant, however, was more toxic to human cancer cells than wild-type ONC. Together, these findings provide a cynosure for understanding catalysis of RNA cleavage in a system of high medicinal relevance.
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PMID:Structural basis for catalysis by onconase. 1800 69


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