Editorial

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Open AccessEditorial

Toxin and Immunotoxin Based Therapeutic Approaches

by Massimo Bortolotti, Letizia Polito and Andrea Bolognesi

Toxins 2022, 14(1), 63; https://doi.org/10.3390/toxins14010063 - 17 Jan 2022

Cited by 7 | Viewed by 2105

Abstract

The concept of “magic bullets”, i [...] Full article

(This article belongs to the Special Issue Toxin and Immunotoxin Based Therapeutic Approaches)

Research

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16 pages, 5477 KiB

Open AccessArticle

Sequence, Structure, and Binding Site Analysis of Kirkiin in Comparison with Ricin and Other Type 2 RIPs

by Stefania Maiello, Rosario Iglesias, Letizia Polito, Lucía Citores, Massimo Bortolotti, José M. Ferreras and Andrea Bolognesi

Toxins 2021, 13(12), 862; https://doi.org/10.3390/toxins13120862 - 03 Dec 2021

Cited by 3 | Viewed by 2419

Abstract

Kirkiin is a new type 2 ribosome-inactivating protein (RIP) purified from the caudex of Adenia kirkii with a cytotoxicity compared to that of stenodactylin. The high toxicity of RIPs from Adenia genus plants makes them interesting tools for biotechnology and therapeutic applications, particularly [...] Read more.

Kirkiin is a new type 2 ribosome-inactivating protein (RIP) purified from the caudex of Adenia kirkii with a cytotoxicity compared to that of stenodactylin. The high toxicity of RIPs from Adenia genus plants makes them interesting tools for biotechnology and therapeutic applications, particularly in cancer therapy. The complete amino acid sequence and 3D structure prediction of kirkiin are here reported. Gene sequence analysis revealed that kirkiin is encoded by a 1572 bp open reading frame, corresponding to 524 amino acid residues, without introns. The amino acid sequence analysis showed a high degree of identity with other Adenia RIPs. The 3D structure of kirkiin preserves the overall folding of type 2 RIPs. The key amino acids of the active site, described for ricin and other RIPs, are also conserved in the kirkiin A chain. Sugar affinity studies and docking experiments revealed that both the 1α and 2γ sites of the kirkiin B chain exhibit binding activity toward lactose and D-galactose, being lower than ricin. The replacement of His246 in the kirkiin 2γ site instead of Tyr248 in ricin causes a different structure arrangement that could explain the lower sugar affinity of kirkiin with respect to ricin. Full article

(This article belongs to the Special Issue Toxin and Immunotoxin Based Therapeutic Approaches)

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17 pages, 4361 KiB

Open AccessArticle

DT389-YP7, a Recombinant Immunotoxin against Glypican-3 That Inhibits Hepatocellular Cancer Cells: An In Vitro Study

by Hamid Hashemi Yeganeh, Mohammad Heiat, Marek Kieliszek, Seyed Moayed Alavian and Ehsan Rezaie

Toxins 2021, 13(11), 749; https://doi.org/10.3390/toxins13110749 - 22 Oct 2021

Cited by 16 | Viewed by 2425

Abstract

Hepatocellular carcinoma (HCC) is one of the high-metastatic types of cancer, and metastasis occurs in one-third of patients with HCC. To maintain the effectiveness of drug compounds on cancer cells and minimize their side effects on normal cells, it is important to use [...] Read more.

Hepatocellular carcinoma (HCC) is one of the high-metastatic types of cancer, and metastasis occurs in one-third of patients with HCC. To maintain the effectiveness of drug compounds on cancer cells and minimize their side effects on normal cells, it is important to use new approaches for overcoming malignancies. Immunotoxins (ITs), an example of such a new approach, are protein-structured compounds consisting of toxic and binding moieties which can specifically bind to cancer cells and efficiently induce cell death. Here, we design and scrutinize a novel immunotoxin against an oncofetal marker on HCC cells. We applied a truncated diphtheria toxin (DT389) without binding domain as a toxin moiety to be fused with a humanized YP7 scFv against a high-expressed Glypican-3 (GPC3) antigen on the surface of HCC cells. Cytotoxic effects of this IT were investigated on HepG2 (GPC3⁺) and SkBr3 (GPC3⁻) cell lines as positive- and negative-expressed GPC3 antigens. The dissociation constant (Kd) was calculated 11.39 nM and 18.02 nM for IT and YP7 scfv, respectively, whereas only IT showed toxic effects on the HepG2 cell line, and decreased cell viability (IC50 = 848.2 ng/mL). Changing morphology (up to 85%), cell cycle arrest at G2 phase (up to 13%), increasing intracellular reactive oxygen species (ROSs) (up to 50%), inducing apoptosis (up to 38% for apoptosis and 23% for necrosis), and an almost complete inhibition of cell movement were other effects of immunotoxin treatment on HepG2 cells, not on SkBr3 cell line. These promising results reveal that this new recombinant immunotoxin can be considered as an option as an HCC inhibitor. However, more extensive studies are needed to accomplish this concept. Full article

(This article belongs to the Special Issue Toxin and Immunotoxin Based Therapeutic Approaches)

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15 pages, 2529 KiB

Open AccessArticle

Cytotoxicity Effect of Quinoin, Type 1 Ribosome-Inactivating Protein from Quinoa Seeds, on Glioblastoma Cells

by Rossella Rotondo, Sara Ragucci, Salvatore Castaldo, Maria Antonietta Oliva, Nicola Landi, Paolo V. Pedone, Antonietta Arcella and Antimo Di Maro

Toxins 2021, 13(10), 684; https://doi.org/10.3390/toxins13100684 - 25 Sep 2021

Cited by 12 | Viewed by 2516

Abstract

Ribosome-inactivating proteins (RIPs) are found in several edible plants and are well characterized. Many studies highlight their use in cancer therapy, alone or as immunoconjugates, linked to monoclonal antibodies directed against target cancer cells. In this context, we investigate the cytotoxicity of quinoin, [...] Read more.

Ribosome-inactivating proteins (RIPs) are found in several edible plants and are well characterized. Many studies highlight their use in cancer therapy, alone or as immunoconjugates, linked to monoclonal antibodies directed against target cancer cells. In this context, we investigate the cytotoxicity of quinoin, a novel type 1 RIP from quinoa seeds, on human continuous and primary glioblastoma cell lines. The cytotoxic effect of quinoin was assayed on human continuous glioblastoma U87Mg cells. Moreover, considering that common conventional glioblastoma multiforme (GBM) cell lines are genetically different from the tumors from which they derive, the cytotoxicity of quinoin was subsequently tested towards primary cells NULU and ZAR (two cell lines established from patients’ gliomas), also in combination with the chemotherapeutic agent temozolomide (TMZ), currently used in glioblastoma treatment. The present study demonstrated that quinoin (2.5 and 5.0 nM) strongly reduced glioblastoma cells’ growth. The mechanisms responsible for the inhibitory action of quinoin are different in the tested primary cell lines, reproducing the heterogeneous response of glioblastoma cells. Interestingly, primary cells treated with quinoin in combination with TMZ were more sensitive to the treatment. Overall, our data highlight that quinoin could represent a novel tool for glioblastoma therapy and a possible adjuvant for the treatment of the disease in combination with TMZ, alone or as possible immunoconjugates/nanoconstructs. Full article

(This article belongs to the Special Issue Toxin and Immunotoxin Based Therapeutic Approaches)

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13 pages, 2913 KiB

Open AccessArticle

Pseudomonas Exotoxin A Based Toxins Targeting Epidermal Growth Factor Receptor for the Treatment of Prostate Cancer

by Alexandra Fischer, Isis Wolf, Hendrik Fuchs, Anie Priscilla Masilamani and Philipp Wolf

Toxins 2020, 12(12), 753; https://doi.org/10.3390/toxins12120753 - 28 Nov 2020

Cited by 12 | Viewed by 3094

Abstract

The epidermal growth factor receptor (EGFR) was found to be a valuable target on prostate cancer (PCa) cells. However, EGFR inhibitors mostly failed in clinical studies with patients suffering from PCa. We therefore tested the targeted toxins EGF-PE40 and EGF-PE24mut consisting of the [...] Read more.

The epidermal growth factor receptor (EGFR) was found to be a valuable target on prostate cancer (PCa) cells. However, EGFR inhibitors mostly failed in clinical studies with patients suffering from PCa. We therefore tested the targeted toxins EGF-PE40 and EGF-PE24mut consisting of the natural ligand EGF as binding domain and PE40, the natural toxin domain of Pseudomonas Exotoxin A, or PE24mut, the de-immunized variant thereof, as toxin domains. Both targeted toxins were expressed in the periplasm of E.coli and evoked an inhibition of protein biosynthesis in EGFR-expressing PCa cells. Concentration- and time-dependent killing of PCa cells was found with IC₅₀ values after 48 and 72 h in the low nanomolar or picomolar range based on the induction of apoptosis. EGF-PE24mut was found to be about 11- to 120-fold less toxic than EGF-PE40. Both targeted toxins were more than 600 to 140,000-fold more cytotoxic than the EGFR inhibitor erlotinib. Due to their high and specific cytotoxicity, the EGF-based targeted toxins EGF-PE40 and EGF-PE24mut represent promising candidates for the future treatment of PCa. Full article

(This article belongs to the Special Issue Toxin and Immunotoxin Based Therapeutic Approaches)

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18 pages, 8423 KiB

Open AccessArticle

Primary Sequence and 3D Structure Prediction of the Plant Toxin Stenodactylin

by Rosario Iglesias, Letizia Polito, Massimo Bortolotti, Manuela Pedrazzi, Lucía Citores, José M. Ferreras and Andrea Bolognesi

Toxins 2020, 12(9), 538; https://doi.org/10.3390/toxins12090538 - 21 Aug 2020

Cited by 5 | Viewed by 2969

Abstract

Stenodactylin is one of the most potent type 2 ribosome-inactivating proteins (RIPs); its high toxicity has been demonstrated in several models both in vitro and in vivo. Due to its peculiarities, stenodactylin could have several medical and biotechnological applications in neuroscience and cancer [...] Read more.

Stenodactylin is one of the most potent type 2 ribosome-inactivating proteins (RIPs); its high toxicity has been demonstrated in several models both in vitro and in vivo. Due to its peculiarities, stenodactylin could have several medical and biotechnological applications in neuroscience and cancer treatment. In this work, we report the complete amino acid sequence of stenodactylin and 3D structure prediction. The comparison between the primary sequence of stenodactylin and other RIPs allowed us to identify homologies/differences and the amino acids involved in RIP toxic activity. Stenodactylin RNA was isolated from plant caudex, reverse transcribed through PCR and the cDNA was amplificated and cloned into a plasmid vector and further analyzed by sequencing. Nucleotide sequence analysis showed that stenodactylin A and B chains contain 251 and 258 amino acids, respectively. The key amino acids of the active site described for ricin and most other RIPs are also conserved in the stenodactylin A chain. Stenodactylin amino acid sequence shows a high identity degree with volkensin (81.7% for A chain, 90.3% for B chain), whilst when compared with other type 2 RIPs the identity degree ranges from 27.7 to 33.0% for the A chain and from 42.1 to 47.7% for the B chain. Full article

(This article belongs to the Special Issue Toxin and Immunotoxin Based Therapeutic Approaches)

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Review

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15 pages, 1584 KiB

Open AccessReview

Immunotoxin Screening System: A Rapid and Direct Approach to Obtain Functional Antibodies with Internalization Capacities

by Shusei Hamamichi, Takeshi Fukuhara and Nobutaka Hattori

Toxins 2020, 12(10), 658; https://doi.org/10.3390/toxins12100658 - 15 Oct 2020

Cited by 14 | Viewed by 5245

Abstract

Toxins, while harmful and potentially lethal, have been engineered to develop potent therapeutics including cytotoxins and immunotoxins (ITs), which are modalities with highly selective targeting capabilities. Currently, three cytotoxins and IT are FDA-approved for treatment of multiple forms of hematological cancer, and additional [...] Read more.

Toxins, while harmful and potentially lethal, have been engineered to develop potent therapeutics including cytotoxins and immunotoxins (ITs), which are modalities with highly selective targeting capabilities. Currently, three cytotoxins and IT are FDA-approved for treatment of multiple forms of hematological cancer, and additional ITs are tested in the clinical trials or at the preclinical level. For next generation of ITs, as well as antibody-mediated drug delivery systems, specific targeting by monoclonal antibodies is critical to enhance efficacies and reduce side effects, and this methodological field remains open to discover potent therapeutic monoclonal antibodies. Here, we describe our application of engineered toxin termed a cell-based IT screening system. This unique screening strategy offers the following advantages: (1) identification of monoclonal antibodies that recognize cell-surface molecules, (2) selection of the antibodies that are internalized into the cells, (3) selection of the antibodies that induce cytotoxicity since they are linked with toxins, and (4) determination of state-specific activities of the antibodies by differential screening under multiple experimental conditions. Since the functional monoclonal antibodies with internalization capacities have been identified successfully, we have pursued their subsequent modifications beyond antibody drug conjugates, resulting in development of immunoliposomes. Collectively, this screening system by using engineered toxin is a versatile platform, which enables straight-forward and rapid selection for discovery of novel functional antibodies. Full article

(This article belongs to the Special Issue Toxin and Immunotoxin Based Therapeutic Approaches)

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Other

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10 pages, 1615 KiB

Open AccessBrief Report

Novel Binding Mechanisms of Fusion Broad Range Anti-Infective Protein Ricin A Chain Mutant-Pokeweed Antiviral Protein 1 (RTAM-PAP1) against SARS-CoV-2 Key Proteins in Silico

by Yasser Hassan, Sherry Ogg and Hui Ge

Toxins 2020, 12(9), 602; https://doi.org/10.3390/toxins12090602 - 17 Sep 2020

Cited by 4 | Viewed by 6973

Abstract

The deadly pandemic named COVID-19, caused by a new coronavirus (SARS-CoV-2), emerged in 2019 and is still spreading globally at a dangerous pace. As of today, there are no proven vaccines, therapies, or even strategies to fight off this virus. Here, we describe [...] Read more.

The deadly pandemic named COVID-19, caused by a new coronavirus (SARS-CoV-2), emerged in 2019 and is still spreading globally at a dangerous pace. As of today, there are no proven vaccines, therapies, or even strategies to fight off this virus. Here, we describe the in silico docking results of a novel broad range anti-infective fusion protein RTAM-PAP1 against the various key proteins of SARS-CoV-2 using the latest protein-ligand docking software. RTAM-PAP1 was compared against the SARS-CoV-2 B38 antibody, ricin A chain, a pokeweed antiviral protein from leaves, and the lectin griffithsin using the special CoDockPP COVID-19 version. These experiments revealed novel binding mechanisms of RTAM-PAP1 with a high affinity to numerous SARS-CoV-2 key proteins. RTAM-PAP1 was further characterized in a preliminary toxicity study in mice and was found to be a potential therapeutic candidate. These findings might lead to the discovery of novel SARS-CoV-2 targets and therapeutic protein structures with outstanding functions. Full article

(This article belongs to the Special Issue Toxin and Immunotoxin Based Therapeutic Approaches)

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