A novel EGFR-specific recombinant ricin-panitumumab (scFv) immunotoxin against breast and colorectal cancer cell lines; in silico and in vitro analyses.

The Epidermal Growth Factor Receptor (EGFR) has been of high importance as it is over expressed in a wide diversity of epithelial cancers, promoting cell proliferation and survival pathways. Recombinant immunotoxins (ITs) have emerged as a promising targeted therapy for cancer treatment. In this study, we aimed to investigate the antitumor activity of a novel recombinant immunotoxin designed against EGFR. Using an in silico approach, we confirmed the stability of the RTA-scFv fusion protein. The immunotoxin was successfully cloned and expressed in the pET32a vector, and the purified protein was analyzed by electrophoresis and western blotting. In vitro evaluations were conducted to assess the biological activities of the recombinant proteins (RTA-scFv, RTA, scFv). The novel immunotoxin demonstrated significant anti-proliferative and pro-apoptotic effects against cancer cell lines. The MTT cytotoxicity assay revealed a decrease in cell viability in the treated cancer cell lines. Additionally, Annexin V/Propidium iodide staining followed by flow cytometry analysis showed a significant induction of apoptosis in the cancer cell lines, with half maximal inhibitory concentration (IC50) values of 81.71 nM for MDA-MB-468 and 145.2 nM for HCT116 cells (P < 0.05). Furthermore, the EGFR-specific immunotoxin exhibited non-allergenic properties. The recombinant protein demonstrated high affinity binding to EGFR. Overall, this study presents a promising strategy for the development of recombinant immunotoxins as potential candidates for the treatment of EGFR-expressing cancers.

Directing ricin-based immunotoxins with targeting affibodies and KDEL signal peptide to cancer cells effectively induces apoptosis and tumor suppression.

The plant toxin ricin, especially its cytotoxic A chain (RTA), can be genetically engineered with targeting ligands to develop specific anti-cancer recombinant immunotoxins (RITs). Here, we used affibody molecules targeting two cancer biomarkers, the receptors HER2 and EGFR, along with the KDEL signal peptide to construct two cancer-specific ricin-based RITs, HER2Afb-RTA-KDEL and EGFRAfb-RTA-KDEL. The affibodies successfully provided target-specificity and subsequent receptor-mediated endocytosis and the KDEL signal peptide routed the RITs through the retrograde transport pathway, effectively delivering RTA to the cytosol as well as avoiding the alternate recycling pathway that typical cancer cells frequently have. The in vivo efficacy of RITs was enhanced by introducing the albumin binding domain (AlBD) to construct AlBD/HER2Afb/RTA-KDEL. Systemic administration of AlBD-containing RITs to tumor-bearing mice significantly suppressed tumor growth without any noticeable side-effects. Collectively, combining target-selective affibody molecules, a cytotoxic RTA, and an intracellularly designating peptide, we successfully developed cancer-specific and efficacious ricin-based RITs. This approach can be applied to develop novel protein-based "magic bullets" to effectively suppress tumors that are resistant to conventional anti-cancer drugs.

Analysis of the Sequence Preference of Saporin by Deep Sequencing.

Ribosome-inactivating proteins (RIPs) are RNA:adenosine glycosidases that inactivate eukaryotic ribosomes by depurinating the sarcin-ricin loop (SRL) in 28S rRNA. The GAGA sequence at the top of the SRL or at the top of a hairpin loop is assumed to be their target motif. Saporin is a RIP widely used to develop immunotoxins for research and medical applications, but its sequence specificity has not been investigated. Here, we combine the conventional aniline cleavage assay for depurinated nucleic acids with high-throughput sequencing to study sequence-specific depurination of oligonucleotides caused by saporin. Our data reveal the sequence preference of saporin for different substrates and show that the GAGA motif is not efficiently targeted by this protein, neither in RNA nor in DNA. Instead, a preference of saporin for certain hairpin DNAs was observed. The observed sequence-specific activity of saporin may be relevant to antiviral or apoptosis-inducing effects of RIPs. The developed method could also be useful for studying the sequence specificity of depurination by other RIPs or enzymes.

A ricin-based peptide BRIP from Hordeum vulgare inhibits Mpro of SARS-CoV-2.

COVID-19 pandemic caused by SARS-CoV-2 led to the research aiming to find the inhibitors of this virus. Towards this world problem, an attempt was made to identify SARS-CoV-2 main protease (Mpro) inhibitory peptides from ricin domains. The ricin-based peptide from barley (BRIP) was able to inhibit Mpro in vitro with an IC50 of 0.52 nM. Its low and no cytotoxicity upto 50 µM suggested its therapeutic potential against SARS-CoV-2. The most favorable binding site on Mpro was identified by molecular docking and steered molecular dynamics (MD) simulations. The Mpro-BRIP interactions were further investigated by evaluating the trajectories for microsecond timescale MD simulations. The structural parameters of Mpro-BRIP complex were stable, and the presence of oppositely charged surfaces on the binding interface of BRIP and Mpro complex further contributed to the overall stability of the protein-peptide complex. Among the components of thermodynamic binding free energy, Van der Waals and electrostatic contributions were most favorable for complex formation. Our findings provide novel insight into the area of inhibitor development against COVID-19.

Cytotoxicity, Apoptosis, Migration Inhibition, and Autophagy-Induced by Crude Ricin from Ricinus communis Seeds in A549 Lung Cancer Cell Lines.

BACKGROUND Ricin protein derived from Ricinus communis seeds is known to have a high toxicity to humans and animals. Several studies revealed that ricin, belonging to ribosome inactivating protein-I, has cytotoxic properties against various types of cancer cell lines. MATERIAL AND METHODS Crude ricin (CR) from the seeds of R. communis was investigated for its cytotoxicity on the A549 cancer cell lines using the MTS assay, and the cell death mechanism was explored using flow cytometry and Western blot methods. The cell migration was measured using a scratch/wound-healing method and the autophagy activity was explored using Western blotting. RESULTS CR showed cytotoxicity against A549 cancer cell lines, with an IC50 of 40.94 ppm. CR induced apoptosis and necrosis, but apoptosis occurred more frequently than necrosis. Apoptosis induced by CR was mediated by the activation of caspase-9 and caspase-3. CR inhibited cell migration in a concentration- and time-dependent manner, with the highest effect occurred at the concentration of 1.0 ppm. The autophagic experiment showed that CR inhibited autophagy in A549 lung cancer cells by decreasing Beclin-1 levels while increasing Atg5 levels in a concentration-dependent manner and CR decreased LC3-II level while increasing p62 level. Cisplatin treatment also inhibited autophagy as it exhibited the same effect on those autophagic proteins as CR. CONCLUSIONS Our findings suggest that CR might be a potential candidate for anticancer drugs, but further study is needed to verify its anticancer properties.

Discovery of RTA ricin subunit inhibitors: a computational study using PM7 quantum chemical method and steered molecular dynamics.

Ricin is a potent toxin derived from the castor bean plant and comprises two subunits, RTA and RTB. Because of its cytotoxicity, ricin has alarmed world authorities for its potential use as a chemical weapon. Ricin also affects castor bean agribusiness, given the risk of animal and human poisoning. Over the years, many groups attempted to propose small-molecules that bind to the RTA active site, the catalytic chain. Despite such efforts, there is still no effective countermeasure against ricin poisoning. The computational study carried out in the present work renews the discussion about small-molecules that may inhibit this toxin. Here, a structure-based virtual screening protocol capable of discerning active RTA inhibitors from inactive ones was performed to screen over 2 million compounds from the ZINC database to find novel scaffolds that strongly bind into the active site of the RTA. Besides, a novel score method based on ligand undocking force profiles and semi-empirical quantum chemical calculations provided insights into the rescore of docking poses. Summing up, the filtering steps pointed out seven main compounds, with the SCF00-451 as a promising candidate to inhibit the killing activity of such potent phytotoxin.

Identification of novel potential ricin inhibitors by virtual screening, molecular docking, molecular dynamics and MM-PBSA calculations: a drug repurposing approach.

Ricin is a potent cytotoxin with no available antidote. Its catalytic subunit, RTA, damages the ribosomal RNA (rRNA) of eukaryotic cells, preventing protein synthesis and eventually leading to cell death. The combination between easiness of obtention and high toxicity turns ricin into a potential weapon for terrorist attacks, urging the need of discovering effective antidotes. On this context, we used computational techniques, in order to identify potential ricin inhibitors among approved drugs. Two libraries were screened by two different docking algorithms, followed by molecular dynamics simulations and MM-PBSA calculations in order to corroborate the docking results. Three drugs were identified as potential ricin inhibitors: deferoxamine, leucovorin and plazomicin. Our calculations showed that these compounds were able to, simultaneously, form hydrogen bonds with residues of the catalytic site and the secondary binding site of RTA, qualifying as potential antidotes against intoxication by ricin.Communicated by Ramaswamy H. Sarma.

Context-sensitivity of isosteric substitutions of non-Watson-Crick basepairs in recurrent RNA 3D motifs.

Sequence variation in a widespread, recurrent, structured RNA 3D motif, the Sarcin/Ricin (S/R), was studied to address three related questions: First, how do the stabilities of structured RNA 3D motifs, composed of non-Watson-Crick (non-WC) basepairs, compare to WC-paired helices of similar length and sequence? Second, what are the effects on the stabilities of such motifs of isosteric and non-isosteric base substitutions in the non-WC pairs? And third, is there selection for particular base combinations in non-WC basepairs, depending on the temperature regime to which an organism adapts? A survey of large and small subunit rRNAs from organisms adapted to different temperatures revealed the presence of systematic sequence variations at many non-WC paired sites of S/R motifs. UV melting analysis and enzymatic digestion assays of oligonucleotides containing the motif suggest that more stable motifs tend to be more rigid. We further found that the base substitutions at non-Watson-Crick pairing sites can significantly affect the thermodynamic stabilities of S/R motifs and these effects are highly context specific indicating the importance of base-stacking and base-phosphate interactions on motif stability. This study highlights the significance of non-canonical base pairs and their contributions to modulating the stability and flexibility of RNA molecules.

Intracellular Transport of Ribosome-Inactivating Proteins Depends on Annexin 13.

In the present study, we assessed the role of annexin 13 membrane-binding protein (ANXA13) in the intracellular transport of vesicles containing type II ribosome-inactivating proteins (RIP-IIs). A modified human intestinal epithelial cell line HT29 was used, in which the expression of ANXA13 was significantly reduced. The cytotoxic effect of ricin and viscumin was evaluated by modification of 28S ribosome RNA. The observed differences in the activity of toxins on the parental and modified HT29 lines indicate that ANXA13 plays a different role in the intracellular transport of vesicles containing the RIP-IIs.

Relationship between the Expression Level of PSMD11 and Other Proteasome Proteins with the Activity of Ricin and Viscumin.

The role of proteasome proteins and proteins of the ERAD system in the cytotoxicity of type II ribosome-inactivating proteins ricin and viscumin was investigated. For this, the cell line of colorectal adenocarcinoma HT29, as well as the HT29-sh002 line obtained on its basis, were used. On the basis on the proteome analysis of these lines and the estimation of the proportion of inactivated ribosomes, it was shown that the contribution of the proteasome to the degradation of the catalytic subunits of toxins is different. The role of the Cdc37 co-chaperone in maintaining the stability of A subunit of viscumin in the cytoplasm is shown.

Induction of Male Sterility in Tobacco by Anther-Specific Expression of the Gene for Ricin Enzymatic Subunit A Chain RTA.

Targeted gene expression in plants allows us to further study biological traits of interest, such as reproductive and developmental processes. Here, the tobacco TA29 anther-specific promoter was used to direct the expression of the ricin enzymatic subunit A (RTA) in transgenic tobacco plants, phenotypic analysis of the resulting positive transgenic tobacco (Nicotiana tabacum L.) plants demonstrated that RTA expression led to a reduction in pistil length and shriveling of anthers, as well as the grayish-brown color of anthers, the reduced pollen viability and male sterility. For the first time, a plant-derived ricin gene enzymatic subunit A (RTA) expression system under the tissue-specific promoter was demonstrated to be sensitive and efficient in controlling plant sterility and creating male-sterile materials. Consequently, it could be used to control other agronomic traits and produce hybrid seeds in plants in the future.

Ricin and Ricinus communis in pharmacology and toxicology-from ancient use and "Papyrus Ebers" to modern perspectives and "poisonous plant of the year 2018".

While probably originating from Africa, the plant Ricinus communis is found nowadays around the world, grown for industrial use as a source of castor oil production, wildly sprouting in many regions, or used as ornamental plant. As regards its pharmacological utility, a variety of medical purposes of selected parts of the plant, e.g., as a laxative, an anti-infective, or an anti-inflammatory drug, have been described already in the sixteenth century BC in the famous Papyrus Ebers (treasured in the Library of the University of Leipzig). Quite in contrast, on the toxicological side, the native plant has become the "poisonous plant 2018" in Germany. As of today, a number of isolated components of the plant/seeds have been characterized, including, e.g., castor oil, ricin, Ricinus communis agglutinin, ricinin, nudiflorin, and several allergenic compounds. This review mainly focuses on the most toxic protein, ricin D, classified as a type 2 ribosome-inactivating protein (RIP2). Ricin is one of the most potent and lethal substances known. It has been considered as an important bioweapon (categorized as a Category B agent (second-highest priority)) and an attractive agent for bioterroristic activities. On the other hand, ricin presents great potential, e.g., as an anti-cancer agent or in cell-based research, and is even explored in the context of nanoparticle formulations in tumor therapy. This review provides a comprehensive overview of the pharmacology and toxicology-related body of knowledge on ricin. Toxicokinetic/toxicodynamic aspects of ricin poisoning and possibilities for analytical detection and therapeutic use are summarized as well.

Anticancer Activities of Ricin-Liposome Complexes on SKMEL-28 Cells.

Background: Ricin has been reported as a potential chemical for cancer treatment. However, so far, the application of ricin in cancer treatment is very limited because of its non-specificity. Methods: In this study, ricin were conjugated/ encapsulated with DOTAP/DOPE liposome to form ricin-liposome complexes (ricin-lipososme1, ricin-liposome2, ricin-liposome3 and ricin-liposome4). Characteristics of ricin-liposome complexes were analyzed and their effects on survival, apoptosis, migration, invasion and tumor formation of SKMEL-28 melanoma cells were examined by carrying out the MTT assay, apoptosis assay, scratch wound healing assay, invasion assay and soft-agar colony formation assay, respectively. Results: Ricin-liposome complexes had even size-distribution with average size of around 340 nm. These ricin-liposome complexes were able to penetrate into the cells via endocytosis with the highest ability of the ricinliposome3. It also showed that ricin-liposome3 expressed very high toxicity with the IC50 of 62.4 ng/mL and followed by ricin-liposome4 (286.4 mg/mL), ricin-liposome2 (417.5 ng/mL), and ricin-liposome1 (604.3 ng/mL) to SKMEL-28 cells at 36 hours post treatment. At the concentrations of IC10 (10.1 ng/mL), ricin-liposome3 strongly induced necrosis and apoptosis of SKMEL-28 cells up to 25.6% and 11.4%, respectively. Moreover, ricin-liposome3 expressed great anticancer properties by decreasing the migration, invasion and tumor formation abilities of SKMEL-28 cells of 7.5 folds, 4.3 folds and 5.9 folds, respectively, compared with those of control SKMEL-28 cells. Conclusion: The obtained results from our study suggest that although ricin is listed as one of the most poisonous substances in nature, it can be used in the complex forms with liposome to increase its specificity to apply in treatment of melanoma and other cancers.

Antiproliferative potential from aqueous Viscum album L. preparations and their main constituents in comparison with ricin and purothionin on human cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Mistletoe has been used since ancient times in Europe mostly for medicinal purposes. Since 1917, mistletoe preparations have been applied in cancer therapy and today are the most frequently used complementary medicine in tumor treatment. The main cytotoxic constituents of Viscum album are lectins and viscotoxins. AIM OF THE STUDY: The aim of this in vitro study was to investigate the antiproliferative potential of Viscum album preparations from different host trees and to assess the impact of mistletoe lectin 1 (ML-1) and viscotoxin A (VT-A) in comparison to a structurally similar lectin and thionin. MATERIALS AND METHODS: By means of widely accepted 2D Alamar Blue Assay, based on population counting of living cells using a fluorescent cell viability dye, the potential impact to inhibit tumor cell of the mistletoe preparations (Iscucin®) and their single compounds (ML-1 and VT-A) on the cell growth of six human cancer cell lines were evaluated. Also the mixture of ML-1 and VT-A corresponding to the contents in the specific mistletoe preparations were monitored. Ricin and purothionin were used as reference lectin and reference thionin, respectively. RESULTS: The lung carcinoma cell line HCC827 was very sensitive to the Iscucin® preparations. Very strong antiproliferative effects were found with Iscucin®Salicis and Tiliae and a strong with Iscucin®Crataegi, Mali and Populi. The IC50 concentrations of the Iscucin® preparations correlated with their respective ML-1 contents, but the ML-1 levels were much lower than the IC50 concentration of isolated ML-1 (1 ng/ml - 56 ng/ml). ML-1 was much more effective than ricin. Iscucin® preparations, ML-1 and ricin showed antiproliferative activity on human tumor cells. VT-A and purothionin had no effect on cell viability in the concentration ranges tested. CONCLUSION: The complete mistletoe extract is more potent to inhibit tumor cell proliferation than isolated ML-1 at an equivalent concentration level. Phenolic compounds found in all Iscucin® preparations might contribute to uphold the cytotoxic activity of ML-1 by antioxidative action. However, further studies are necessary to evaluate the role of VT-A and possible synergistic actions to the antiproliferative effect of aqueous mistletoe extracts.

Genome-wide CRISPR Analysis Identifies Substrate-Specific Conjugation Modules in ER-Associated Degradation.

The ubiquitin proteasome system (UPS) maintains the integrity of the proteome by selectively degrading misfolded or mis-assembled proteins, but the rules that govern how conformationally defective proteins in the secretory pathway are selected from the structurally and topologically diverse constellation of correctly folded membrane and secretory proteins for efficient degradation by cytosolic proteasomes is not well understood. Here, we combine parallel pooled genome-wide CRISPR-Cas9 forward genetic screening with a highly quantitative and sensitive protein turnover assay to discover a previously undescribed collaboration between membrane-embedded cytoplasmic ubiquitin E3 ligases to conjugate heterotypic branched or mixed ubiquitin (Ub) chains on substrates of endoplasmic-reticulum-associated degradation (ERAD). These findings demonstrate that parallel CRISPR analysis can be used to deconvolve highly complex cell biological processes and identify new biochemical pathways in protein quality control.

Construction and characterization of the recombinant immunotoxin RTA-4D5-KDEL targeting HER2/neu-positive cancer cells and locating the endoplasmic reticulum.

The specific targeting of immunotoxins enables their wide application in cancer therapy. The A-chain of the ricin protein (RTA) is an N-glycosidase that catalyzes the removal of adenine from the 28S rRNA, preventing protein translation and leading to cell death. Ricin is highly toxic but can only exert its toxic effects from within the cytoplasm. In this study, we linked the anti-HER2 single-chain variable fragment 4D5 scFv and the endoplasmic reticulum-targeting peptide KDEL to the C-terminal of the RTA to construct immunotoxin RTA-4D5-KDEL. In vitro experiments showed that the anticancer effect of RTA-4D5-KDEL towards ovarian cancer cells SKOV-3 increased 440-fold and 28-fold relative to RTA and RTA-4D5, respectively. RTA-4D5-KDEL had a strong inhibitory effect on HER2-overexpressing SKOV-3 cells and caused little damage to normal HEK-293 cells and H460 lung cancer cells. Immunofluorescence experiments showed that the immunotoxin RTA-4D5 could specifically bind to SKOV-3 cells, but not to normal cells HEK-293. The immunotoxin RTA-4D5-KDEL could rapidly localize the recombinant protein to the endoplasmic reticulum. These results suggest that the recombinant immunotoxin RTA-4D5-KDEL has a strong inhibitory effect on ovarian cancer cells that overexpress HER2 but little harm to the normal cells.

Expression of novel fusion antiviral proteins ricin a chain-pokeweed antiviral proteins (RTA-PAPs) in Escherichia coli and their inhibition of protein synthesis and of hepatitis B virus in vitro.

BACKGROUND: Ricin A chain (RTA) and Pokeweed antiviral proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential production amenability and sub-toxic antiviral value of novel fusion proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral protein isoform 1 from seeds (RTA-PAPS1) was produced in an E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and protein synthesis inhibitory activity assayed by comparison to the production of a control protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. The fusion protein was further optimized using in silico tools, produced in an E. coli in vivo expression system, purified by a three-step process from soluble lysate and confirmed in a protein synthesis inhibition activity assay. RESULTS: Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded protein was bioactive with a 50% protein synthesis inhibitory concentration (IC50) of 0.06 nM (3.63 ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03 nM (1.82 ng/ml) and a therapeutic index of > 21,818 with noticeable steric hindrance. Results also showed that the optimized protein ricin A chain mutant-Pokeweed antiviral protein isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function on protein synthesis inhibition activity, with an IC50 of 0.03 nM (1.82 ng/ml), and with minimal, if any, steric hindrance. CONCLUSIONS: Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant gain of function on protein synthesis inhibition and anti-HBV activities in vitro with a high therapeutic index and, thus, merit further development as potential potent antiviral agents against chronic HBV infection to be used as a standalone or in combination with existent therapies.

Selective CXCR4+ Cancer Cell Targeting and Potent Antineoplastic Effect by a Nanostructured Version of Recombinant Ricin.

Under the unmet need of efficient tumor-targeting drugs for oncology, a recombinant version of the plant toxin ricin (the modular protein T22-mRTA-H6) is engineered to self-assemble as protein-only, CXCR4-targeted nanoparticles. The soluble version of the construct self-organizes as regular 11 nm planar entities that are highly cytotoxic in cultured CXCR4+ cancer cells upon short time exposure, with a determined IC50 in the nanomolar order of magnitude. The chemical inhibition of CXCR4 binding sites in exposed cells results in a dramatic reduction of the cytotoxic potency, proving the receptor-dependent mechanism of cytotoxicity. The insoluble version of T22-mRTA-H6 is, contrarily, moderately active, indicating that free, nanostructured protein is the optimal drug form. In animal models of acute myeloid leukemia, T22-mRTA-H6 nanoparticles show an impressive and highly selective therapeutic effect, dramatically reducing the leukemia cells affectation of clinically relevant organs. Functionalized T22-mRTA-H6 nanoparticles are then promising prototypes of chemically homogeneous, highly potent antitumor nanostructured toxins for precise oncotherapies based on self-mediated intracellular drug delivery.

Modelling toxin effects on protein biosynthesis in eukaryotic cells.

We present a rather generic model for toxin (ricin) inhibition of protein biosynthesis in eukaryotic cells. We also study reduction of the ricin toxic effects with application of antibodies against the RTB subunit of ricin molecules. Both species initially are delivered extracellularly. The model accounts for the pinocytotic and receptor-mediated toxin endocytosis and the intact toxin exocytotic removal out of the cell. The model also includes the lysosomal toxin destruction, the intact toxin motion to the endoplasmic reticulum (ER) for separation of its molecules into the RTA and RTB subunits, and the RTA chain translocation into the cytosol. In the cytosol, one portion of the RTA undergoes degradation via the ERAD. The other its portion can inactivate ribosomes at a large rate. The model is based on a system of deterministic ODEs. The influence of the kinetic parameters on the protein concentration and antibody protection factor is studied in detail.

Hyperuricaemia, Xanthine Oxidoreductase and Ribosome-Inactivating Proteins from Plants: The Contributions of Fiorenzo Stirpe to Frontline Research.

The enzymes called ribosome-inactivating proteins (RIPs) that are able to depurinate  nucleic acids and arrest vital cellular functions, including protein synthesis, are still a frontline  research field, mostly because of their promising medical applications. The contributions of Stirpe  to the development of these studies has been one of the most relevant. After a short biographical  introduction, an overview is offered of the main results obtained by his investigations during last  55 years on his main research lines: hyperuricaemia, xanthine oxidoreductase and RIPs.