Onconase

Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200-250 mg/L)...

Onconase is an RNase of the ribonuclease A superfamily that is purified from the Northern leopard frog (Rana pipiens). It targets several types of malignant tumors, digests cytoplasmic transfer RNA (tRNA), and causes tumor cell apoptosis. Onconase has been employed in clinical trials as an antitumor drug, and has revealed its valuable clinical activity in several types of tumors, particularly pleural mesothelioma. However, its inefficiency in targeting tumor cells and its non‑specific toxicity in normal tissues have diminished its clinical benefits...

Antibody-drug conjugates (ADCs) have evolved as a new class of potent cancer therapeutics. We here report on the development of ADCs with specificity for the B-cell lineage specific (surface) antigen CD22 being expressed in the majority of hematological malignancies. As targeting moiety a previously generated humanized anti-CD22 single-chain variable fragment (scFv) derivative from the monoclonal antibody RFB4 was reengineered into a humanized IgG1 antibody format (huRFB4). Onconase (ranpirnase), a clinically active pancreatic-type ribonuclease, was employed as cytotoxic payload moiety...

Biotherapeutics have many promising applications, such as anti-cancer treatments, immune suppression, and vaccines. However, due to their biological nature, some biotherapeutics can be challenging to rapidly express and screen for activity through traditional recombinant methods. For example, difficult-to-express proteins may be cytotoxic or form inclusion bodies during expression, increasing the time, labor, and difficulty of purification and downstream characterization. One potential pathway to simplify the expression and screening of such therapeutics is to utilize cell-free protein synthesis...

Human glioblastoma multiforme is one of the most aggressive malignant brain tumor types, and the mean survival time of patients with a brain tumor is <2 years when traditional therapies are administered. Thus, numerous studies have focused on the development of novel treatments for brain tumors. Frog ribonucleases, such as Onconase and Rana catesbeiana ribonuclease (RC-RNase), exert antitumor effects on various tumor cells, including cervical cancer, breast cancer, hepatoma, leukemia, pancreatic cancer and prostate cancer cells...

Many ribonucleases (RNases) are able to inhibit the reproduction of viruses in infected cell cultures and laboratory animals, but molecular mechanisms of their antiviral activity remain unclear. The review observes the most known RNases which possess established antiviral effects, actually intracellular RNases (RNase L, MCPIPI protein, eosinophylic RNases) as well as exogenously applied ones (RNase A, BS-RNase, onconase, binase, synthetic RNases). Attention is given on two important but not always obligatory aspects in molecule of RNases, which have antiviral properties: catalytic activity and ability to the dimerization...

Gliomas are rarely curable malignant brain tumors arising from normal glial cells. The scorpion-derived small peptide, chlorotoxin (CTX), can selectively bind malignant gliomas. In the present study, a CTX-conjugated onconase (Onc), a small cytotoxic ribonuclease, was prepared as a potential anti-glioma drug. In this conjugate, recombinant CTX was covalently linked with recombinant Onc by reversible disulfide linkage. The chemically conjugated CTX-Onc showed much higher cytotoxicity to the cultured glioma U251 and SHG-44 cells than the physical mixture of CTX and Onc (CTX + Onc)...

Onconase® (ONC) is a member of the RNase super-family that is secreted in oocytes and early embryos of Rana pipiens. Over the last years, research interest about this small and basic frog RNase, also called ranpirnase, constantly increased because of its high cytotoxicity and anticancer properties. Onconase is currently used in clinical trials for cancer therapy; however, the precise mechanisms determining cytotoxicity in cancer cells have not yet been fully investigated. In the present manuscript, we evaluate the antitumoral property of onconase in pancreatic adenocarcinoma cells and in non-tumorigenic cells as a control...

Cytotoxic ribonucleases such as the leopard frog derivative Ranpirnase (Onconase(®)) have emerged as a valuable new class of cancer therapeutics. Clinical trials employing single agent Ranpirnase in cancer patients have demonstrated significant clinical activity and surprisingly low immunogenicity. However, dose-limiting toxicity due to unspecific uptake of the RNase into non-cancerous cells is reached at relatively low concentrations of > 1 mg/m(2). We have in the present study generated a dimeric anti-EGFR Ranpirnase-diabody fusion protein capable to deliver two Ranpirnase moieties per molecule to EGFR-positive tumour cells...

Antibody therapy of solid cancers is well established, but suffers from unsatisfactory tumor penetration of large immunoglobulins or from low serum retention of antibody fragments. Oncolytic viruses are in advanced clinical development showing excellent safety, but suboptimal potency due to limited virus spread within tumors. Here, by developing an immunoRNase-encoding oncolytic adenovirus, we combine viral oncolysis with intratumoral genetic delivery of a small antibody-fusion protein for targeted bystander killing of tumor cells (viro-antibody therapy)...

Onconase (ONC) is a member of a ribonuclease superfamily that has cytostatic activity against malignant mesothelioma (MM). The objective of this investigation was to develop bovine serum albumin (BSA)-chitosan based hybrid nanoformulations for the efficient delivery of ONC to MM while minimizing the exposure to normal tissues. Taguchi orthogonal array L9 type design was used to formulate ONC loaded BSA nanocarriers (ONC-ANC) with a mean particle size of 15.78 ± 0.24 nm (ζ = -21.89 ± 0.11 mV). The ONC-ANC surface was hybridized using varying chitosan concentrations ranging between 0...

Comparative studies on homologous proteins can provide knowledge on how limited changes in the primary structure find their expression in large effects on catalytic activity, stability or the folding behavior. For more than half a century, members of the ribonuclease A superfamily have been the subject of a myriad of studies on protein folding and stability. Both the unfolding and refolding kinetics as well as the structure of several folding intermediates of ribonuclease A have been characterized in detail...

Although major advancements in antitumor treatment have been observed, several B cell-derived malignancies still remain incurable. A promising approach that involves targeting RNA either by the use of specific antisense oligonucleotides or cytostatic/cytotoxic ribonucleases (RNases) is being promoted. Two amphibian RNases, onconase (ONC; ranpirnase) and, more recently, r-amphinase (r-Amph), have already been tested, but thus far, mostly on solid tumors. In this study, for the first time we provide comprehensive data on ex vivo and in vivo cytotoxic activity of ONC or r-Amph against cancer cells from different B cell lymphoid malignancies, together with their detailed mode of antitumor action...

Ranpirnase (Onconase) is a frogspawn-derived disulfide-rich peptide with ribonuclease activity that may be used for tumor treatment. In the present study, we established an efficient approach for preparing mature ranpirnase which may be used for research and therapeutic purposes. The designed ranpirnase precursors carried a 6xHis-tag and were recombinantly expressed in Escherichia coli . After S-sulfonation, the precursors were purified by immobilized metal-ion affinity chromatography. Following removal of the tag by aminopeptidase cleavage, cyclization and in vitro oxidative refolding, the mature ranpirnase was obtained with considerable yield, and the yield of mature ranpirnase was ~50-60 mg per liter cultures...

Ranpirnase (onconase; ONC) is an endoribonuclease obtained from the frog Rana pipiens. This enzyme exhibits anticancer properties mediated by degradation of cellular RNA and induction of apoptosis. In this study we assessed cytotoxicity of ONC in combination with currently used anticancer drugs on a human diffuse large B-cell lymphoma (DLBCL)-derived cell line (Toledo). Cytotoxic activity was measured by the exclusion of propidium iodide assay while apoptosis was assessed using the annexin-V binding method...

The structure of onconase C30A/C75A double mutant has been determined at 1.12Å resolution. The structure has high structural homology to other onconase structures. The changes being results of mutation are relatively small, distributed asymmetrically around the two mutated positions, and they are observed not only in the mutation region but expanded to entire molecule. Different conformation of Lys31 side chain that influences the hydrogen bonding network around catalytic triad is probably responsible for lower catalytic efficiency of double mutant...

Pancreatic-type ribonucleases show clinical promise as chemotherapeutic agents but are limited in efficacy by the inefficiency of their uptake by human cells. Cellular uptake can be increased by the addition of positive charges to the surface of ribonucleases, either by site-directed mutagenesis or by chemical modification. This observation has led to the hypothesis that ribonuclease uptake by cells depends on electrostatics. Here, we use a combination of experimental and computational methods to ascertain the contribution of electrostatics to the cellular uptake of ribonucleases...

Peptidyl-prolyl isomerization reactions can make for rate-limiting steps in protein folding due to their high activation energy. Onconase, an unusually stable ribonuclease A homologue from the Northern leopard frog, contains four trans proline residues in its native state. During the refolding from its guanidine hydrochloride unfolded state, which includes the formation of a folding intermediate, the slowest of the three phases has earlier been attributed to a cis-to-trans peptidyl-prolyl isomerization reaction...

Unfolding by chemical denaturants and the linear extrapolation method are widely used to determine the free energy of proteins. Ribonuclease 3 from bullfrog shows an extraordinary behavior in guanidinium hydrochloride in comparison to its homologues ribonuclease A and onconase with a high transition midpoint of denaturation but an apparently low cooperativity. The analysis of the interdependence of thermal, urea-, and guanidine hydrochloride-induced unfolding revealed that whereas addition of urea resulted in the expected destabilization of all three proteins, guanidine hydrochloride acted diversely: in contrast to ribonuclease A and onconase, both of which were destabilized as expected, low concentrations of guanidine hydrochloride significantly stabilize ribonuclease 3 from bullfrog...

Onconase® is a highly cytotoxic amphibian homolog of Ribonuclease A. Here, we describe the construction of circularly permuted Onconase® variants by connecting the N- and C-termini of this enzyme with amino acid residues that are recognized and cleaved by the human immunodeficiency virus protease. Uncleaved circularly permuted Onconase® variants are unusually stable, non-cytotoxic and can internalize in human T-lymphocyte Jurkat cells. The structure, stability and dynamics of an intact and a cleaved circularly permuted Onconase® variant were determined by Nuclear Magnetic Resonance spectroscopy and provide valuable insight into the changes in catalytic efficiency caused by the cleavage...