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International Journal of Molecular... Feb 2023In spite of consistent progress at the level of basic research and of clinical treatment, acute myeloid leukemia (AML) still represents an unmet clinical need for adult... (Review)
Review
In spite of consistent progress at the level of basic research and of clinical treatment, acute myeloid leukemia (AML) still represents an unmet clinical need for adult and pediatric patients. To improve the outcomes of these patients, it is necessary to identify new therapeutic targets. IL3RA (CD123, alpha subunit of the interleukin 3 receptor) is a cell membrane protein overexpressed in several hematologic malignancies, including AML blastic plasmocytoid dendritic cell neoplasms (BPDCN). Given the higher expression of CD123 on leukemic cells compared to normal hematopoietic cells and its low/absent expression on normal hematopoietic stem cells, it appears as a suitable and attractive target for therapy. Various drugs targeting CD123 have been developed and evaluated at clinical level: interleukin-3 conjugated with diphtheria toxin; naked neutralizing anti-CD123 antibodies; drug-antibody conjugates; bispecific antibodies targeting both CD123 and CD3; and chimeric antigen receptor (CAR) T cells engineered to target CD123. Some of these agents have shown promising results at the clinical level, including tagraxofusp (CD123 conjugated with diphtheria toxin) for the treatment of BPDCN and IMGN632 (anti-CD123 drug-conjugate), and flotetuzumab (bispecific anti-CD123 and anti-CD3 monoclonal antibody) for the treatment of AML. However, the therapeutic efficacy of CD123-targeting treatments is still unsatisfactory and must be improved through new therapeutic strategies and combined treatments with other antileukemic drugs.
Topics: Adult; Child; Humans; Antineoplastic Agents; Combined Modality Therapy; Dendritic Cells; Diphtheria Toxin; Immunoconjugates; Leukemia, Myeloid, Acute
PubMed: 36769040
DOI: 10.3390/ijms24032718 -
Archives of Razi Institute Jan 2021The changes in temperature levels can potentially affect the toxins in terms of stability and immunological properties via alteration of their structures. Diphtheria...
The changes in temperature levels can potentially affect the toxins in terms of stability and immunological properties via alteration of their structures. Diphtheria Toxin (DT) is highly considered by scientists since its mechanism of action is similar to those of most bacterial toxins, such as botulinum, tetanus, and anthrax. The protection of conformational B-cell epitopes is critically important in the process of diphtheria vaccine production. This study aimed to evaluate the conformational changes of the DT structure at three different temperature levels (27˚C, 37˚C, and 47˚C) using molecular dynamic simulations. Secondary structures were analyzed in YASARA software. According to the results, significant decreases were observed in percentages of the β-sheets, turns, and the helices of the DT structure at 47˚C in comparison with those at 27˚C and 37˚C. Furthermore, the tertiary structure of the DT was compared at different temperatures using the contact map. Accordingly, the results showed that the root-mean-square deviation of the DT structure increased upon temperature rising. In addition, amino acids D68, G128, G171, C186, and K534-S535 at 27˚C and 37˚C, as well as amino acids G26, P38, S291, T267, H384, A356, and V518 at 47˚C showed higher root mean square fluctuation values. The finding demonstrated that the stability of the DT structure decreased at high temperature (47˚C). The solvent-accessible surface area diagram showed that the hydrophobicity of the DT structure increased via temperature rising, and the amino acid residues belonging to B-cell epitopes extended through increasing temperature. However, B-cell epitopes belonging to the junction region of chains A and B were only present at 37˚C. The results of this study are expected to be applicable for determining a suitable temperature level for the production process of the diphtheria vaccine.
Topics: Diphtheria Toxin; Epitopes, B-Lymphocyte; Molecular Dynamics Simulation; Protein Structure, Secondary; Temperature
PubMed: 33403838
DOI: 10.22092/ari.2019.127251.1377 -
Molecular and Cellular Biology Jul 1983We investigated two phenotypically distinct types of diphtheria toxin-resistant mutants of Chinese hamster cells and compared their resistance with that of naturally... (Comparative Study)
Comparative Study
We investigated two phenotypically distinct types of diphtheria toxin-resistant mutants of Chinese hamster cells and compared their resistance with that of naturally resistant mouse cells. All are resistant due to a defect in the process of internalization and delivery of toxin to its target in the cytosol, elongation factor 2. By cell hybridization studies, analysis of cross-resistance, and determination of specific binding sites for 125I-labeled diphtheria toxin, we showed that these cell strains fall into two distinct complementation groups. The Dipr group encompasses Chinese hamster strains that are resistant only to diphtheria toxin, as well as mouse LM cells. These strains possess a normal complement of high-affinity binding sites for diphtheria toxin, but these receptors are unable to deliver active toxin fragment A to the cytosol. Cells of the DPVr group have a broader spectrum of resistance, including Pseudomonas exotoxin A and several enveloped viruses as well as diphtheria toxin. In these studies, which investigate the resistance of these cells to diphtheria toxin, we demonstrate that they possess a reduced number of specific binding sites for this toxin and behave, phenotypically, like cells treated with the proton ionophore monensin. Their resistance is related to a defect in a mechanism required for release of active toxin from the endocytic vesicle.
Topics: Ammonium Chloride; Animals; Binding Sites; Biological Transport, Active; Cells, Cultured; Cricetinae; Cricetulus; Diphtheria Toxin; Drug Resistance; Female; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Mice; Monensin; Mutation; Ovary; Receptors, Cell Surface; Receptors, Cholinergic
PubMed: 6888380
DOI: 10.1128/mcb.3.7.1283-1294.1983 -
The Biochemical Journal Feb 1992Natural diphtheria toxin is synthesized as a single polypeptide chain that is activated by cleavage into an A- and a B-fragment, which are linked by a disulphide bond....
Natural diphtheria toxin is synthesized as a single polypeptide chain that is activated by cleavage into an A- and a B-fragment, which are linked by a disulphide bond. In the present work the ability of independently translated A- and B-fragments to associate was investigated. Low amounts of A- and B-fragments synthesized in vitro were mixed under conditions that allowed formation of a disulphide bridge between the fragments. Under these conditions toxin was reconstituted in close to 100% yield and found to be as toxic to Vero cells as natural diphtheria toxin. Efficient association between the A- and B-fragment was dependent on the formation of a disulphide bridge. Reconstituted toxin obtained from one [35S]methionine-labelled fragment and one unlabelled fragment proved useful in translocation studies. Addition of a number of different polypeptides to the N- and C-termini of either fragment did not, in most cases, prevent reconstitution. The ready reconstitution allows easy manipulations with the toxin to form targeted molecules and to develop diphtheria toxin as a vector for translocation of peptides to the cytosol. The fact that the reconstituted toxin does not need to be nicked with proteinases to be active allows experimentation with proteinase-sensitive constructs.
Topics: Amino Acid Sequence; Apolipoprotein A-I; Biological Transport; Cell Membrane; Diphtheria Toxin; Disulfides; Hydrogen-Ion Concentration; Molecular Sequence Data; Peptide Fragments; Peptides; Recombinant Fusion Proteins; Staphylococcal Protein A; Tetrahydrofolate Dehydrogenase
PubMed: 1536643
DOI: 10.1042/bj2810619 -
Scientific Reports Apr 2017Diphtheria toxin kills human cells because it delivers its enzyme domain DTA into their cytosol where it inhibits protein synthesis. After receptor-mediated uptake of...
Diphtheria toxin kills human cells because it delivers its enzyme domain DTA into their cytosol where it inhibits protein synthesis. After receptor-mediated uptake of the toxin, DTA translocates from acidic endosomes into the cytosol, which might be assisted by host cell factors. Here we investigated the role of Hsp90 and its co-chaperones during the uptake of native diphtheria toxin into human cells and identified the components of the Hsp90 machinery including Hsp90, Hsp70, Cyp40 and the FK506 binding proteins FKBP51 and FKBP52 as DTA binding partners. Moreover, pharmacological inhibition of the chaperone activity of Hsp90 and Hsp70 and of the peptidyl-prolyl cis/trans isomerase (PPIase) activity of Cyps and FKBPs protected cells from intoxication with diphtheria toxin and inhibited the pH-dependent trans-membrane transport of DTA into the cytosol. In conclusion, these host cell factors facilitate toxin uptake into human cells, which might lead to development of novel therapeutic strategies against diphtheria.
Topics: Animals; CHO Cells; Carrier Proteins; Cell Membrane; Cells, Cultured; Cricetulus; Cytosol; Diphtheria Toxin; Enzyme Activation; HSP90 Heat-Shock Proteins; HeLa Cells; Host-Pathogen Interactions; Humans; Hydrogen-Ion Concentration; Models, Biological; Molecular Chaperones; Peptidylprolyl Isomerase; Protein Binding; Protein Transport; Proteolysis
PubMed: 28377614
DOI: 10.1038/s41598-017-00780-x -
Proceedings of the National Academy of... Jan 1980We have used flow dialysis to demonstrate binding of ATP and related compounds to diphtheria toxin. The results define a new site on the toxin molecule (the P site),...
We have used flow dialysis to demonstrate binding of ATP and related compounds to diphtheria toxin. The results define a new site on the toxin molecule (the P site), which has distinctly different properties from the NAD+-binding site of the fragment A moiety. The relative affinities of various compounds for the P site are similar to their capacities to inhibit toxin attachment to cell surfaces and its action on cells. This suggests that the P site may correspond to the binding site for cell surface receptors. Affinity of nucleotides for the toxin depends strongly on the number of phosphates, although both nucleoside and phosphate moieties contribute to the interaction. A substantial fraction of the toxin in any given preparation did not bind ATP in a rapidly reversible manner and was not retained on ATP-Sepharose. This fraction, which varied in magnitude from preparation to preparation, was isolated and shown to contain an endogenous, firmly bound nucleotide or nucleotide-like compound. The presence of this compound may explain some of the physical heterogeneity within individual preparations of purified toxin as well as variations in physical and biological properties among various preparations.
Topics: Adenine Nucleotides; Adenosine Triphosphate; Binding, Competitive; Chromatography, Affinity; Chromatography, Gel; Dialysis; Diphtheria Toxin; Receptors, Drug
PubMed: 6928618
DOI: 10.1073/pnas.77.1.267 -
Toxins Nov 2020Diphtheria toxin, an exotoxin secreted by that causes disease in humans by inhibiting protein synthesis, enters the cell via receptor-mediated endocytosis. The...
Diphtheria toxin, an exotoxin secreted by that causes disease in humans by inhibiting protein synthesis, enters the cell via receptor-mediated endocytosis. The subsequent endosomal acidification triggers a series of conformational changes, resulting in the refolding and membrane insertion of the translocation (T-)domain and ultimately leading to the translocation of the catalytic domain into the cytoplasm. Here, we use X-ray crystallography along with circular dichroism and fluorescence spectroscopy to gain insight into the mechanism of the early stages of pH-dependent conformational transition. For the first time, we present the high-resolution structure of the diphtheria toxin at a mildly acidic pH (5-6) and compare it to the structure at neutral pH (7). We demonstrate that neither catalytic nor receptor-binding domains change their structure upon this acidification, while the T-domain undergoes a conformational change that results in the unfolding of the TH2-3 helices. Surprisingly, the TH1 helix maintains its conformation in the crystal of the full-length toxin even at pH 5. This contrasts with the evidence from the new and previously published data, obtained by spectroscopic measurements and molecular dynamics computer simulations, which indicate the refolding of TH1 upon the acidification of the isolated T-domain. The overall results imply that the membrane interactions of the T-domain are critical in ensuring the proper conformational changes required for the preparation of the diphtheria toxin for the cellular entry.
Topics: Binding Sites; Catalytic Domain; Circular Dichroism; Crystallography, X-Ray; Diphtheria Toxin; Hydrogen-Ion Concentration; Molecular Dynamics Simulation; Mutation; Protein Binding; Protein Unfolding; Spectrometry, Fluorescence; Structure-Activity Relationship
PubMed: 33171806
DOI: 10.3390/toxins12110704 -
Molecular Oncology May 2017CD19 is expressed on normal and neoplastic B cells and is a promising target for immunotherapy. However, there is still an unmet need to further develop novel...
CD19 is expressed on normal and neoplastic B cells and is a promising target for immunotherapy. However, there is still an unmet need to further develop novel therapeutic drugs for the treatment of the refractory/relapsing human CD19 tumors. We have developed a diphtheria toxin-based anti-human CD19 immunotoxin for targeting human CD19 tumors. We have constructed three isoforms of the CD19 immunotoxin: monovalent, bivalent, and foldback diabody. In vitro binding affinity and efficacy analysis demonstrated that the bivalent isoform had the highest binding affinity and in vitro efficacy. The in vivo efficacy of the CD19 immunotoxins was assessed using human CD19 JeKo-1 tumor-bearing NOD/SCID IL-2 receptor γ (NSG) mouse model. In these animals, CD19 immunotoxins significantly prolonged the median survival from 31 days in controls to 34, 36, and 40 days in animals receiving the monovalent isoform, foldback diabody isoform, and bivalent isoform, respectively. The bivalent CD19 immunotoxin is a promising therapeutic drug candidate for targeting relapsing/refractory human CD19 tumors.
Topics: Animals; Antigens, CD19; Cell Survival; DNA; Diphtheria Toxin; Dose-Response Relationship, Drug; Humans; Immunotoxins; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasms; Time Factors; Tumor Cells, Cultured
PubMed: 28306193
DOI: 10.1002/1878-0261.12056 -
European Journal of Biochemistry Dec 1987The membrane insertion of diphtheria toxin and of its B chain mutants crm 45, crm 228 and crm 1001 has been followed by hydrophobic photolabelling with photoactivatable...
The membrane insertion of diphtheria toxin and of its B chain mutants crm 45, crm 228 and crm 1001 has been followed by hydrophobic photolabelling with photoactivatable phosphatidylcholine analogues. It was found that diphtheria toxin binds to the lipid bilayer surface at neutral pH while at low pH both its A and B chains also interact with the hydrocarbon chains of phospholipids. The pH dependence of photolabelling of the two protomers is different: the pKa of fragment B is around 5.9 while that of fragment A is around 5.2. The latter value correlates with the pH of half-maximal intoxication of cells incubated with the toxin in acidic mediums. These results suggest that fragment B penetrates into the bilayer first and assists the insertion of fragment A and that the lipid insertion of fragment B is not the rate-controlling step in the process of membrane translocation of diphtheria toxin. crm 45 behaves as diphtheria toxin in the photolabelling assay but, nonetheless, it is found to be three orders of magnitude less toxic than diphtheria toxin on acid-treated cells, suggesting that the 12-kDa COOH-terminal segment of diphtheria toxin is important not only for its binding to the cell receptor but also for the membrane translocation of the toxin. It is suggested that crm 1001 is non-toxic because of a defect in its membrane translocation which occurs at a lower extent and at a lower pH than that of the native toxin; as a consequence crm 1001 may be unable to escape from the endosome lumen into the cytoplasm before the fusion of the endosome with lysosomes.
Topics: Animals; Cell Line; Cell Survival; Diphtheria Toxin; Hydrogen-Ion Concentration; Lipid Bilayers; Lipid Metabolism; Peptide Fragments; Permeability; Phosphatidylcholines; Photochemistry; Temperature
PubMed: 3691512
DOI: 10.1111/j.1432-1033.1987.tb13655.x -
Trends in Molecular Medicine Feb 2024Diphthamide, a complex modification on eukaryotic translation elongation factor 2 (eEF2), assures reading-frame fidelity during translation. Diphthamide and enzymes for... (Review)
Review
Diphthamide, a complex modification on eukaryotic translation elongation factor 2 (eEF2), assures reading-frame fidelity during translation. Diphthamide and enzymes for its synthesis are conserved in eukaryotes and archaea. Originally identified as target for diphtheria toxin (DT) in humans, its clinical relevance now proves to be broader than the link to pathogenic bacteria. Diphthamide synthesis enzymes (DPH1 and DPH3) are associated with cancer, and DPH gene mutations can cause diphthamide deficiency syndrome (DDS). Finally, new analyses provide evidence that diphthamide may restrict propagation of viruses including SARS-CoV-2 and HIV-1, and that DPH enzymes are targeted by viruses for degradation to overcome this restriction. This review describes how diphthamide is synthesized and functions in translation, and covers its clinical relevance in human development, cancer, and infectious diseases.
Topics: Humans; Peptide Elongation Factor 2; Clinical Relevance; Diphtheria Toxin; Neoplasms; Histidine
PubMed: 38097404
DOI: 10.1016/j.molmed.2023.11.008