Christopher Bachran

Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development

To study the role of the diphthamide modification on eukaryotic elongation factor 2 (eEF2), we generated an eEF2 Gly717Arg mutant mouse, in which the first step of diphthamide biosynthesis is prevented. Interestingly, the Gly717-to-Arg mutation partially compensates the eEF2 functional loss resulting from diphthamide deficiency, possibly because the added +1 charge compensates for the loss of the +1 charge on diphthamide. Therefore, in contrast to mouse embryonic fibroblasts (MEFs) from OVCA1−/− mice, eEF2G717R/G717R MEFs retain full activity in polypeptide elongation and have normal growth rates. Furthermore, eEF2G717R/G717R mice showed milder phenotypes than OVCA1−/− mice (which are 100% embryonic lethal) and a small fraction survived to adulthood without obvious abnormalities. Moreover, eEF2G717R/G717R/OVCA1−/− double mutant mice displayed the milder phenotypes of the eEF2G717R/G717R mice, suggesting that the embryonic lethality of OVCA1−/− mice is due to diphthamide deficiency. We confirmed that the diphthamide modification is essential for eEF2 to prevent −1 frameshifting during translation and show that the Gly717-to-Arg mutation cannot rescue this defect.

The toxin component of targeted anti-tumor toxins determines their efficacy increase by saponins.

Tumor‐targeting protein toxins are composed of a toxic enzyme coupled to a specific cell binding domain that targets cancer‐associated antigens. The anti‐tumor treatment by targeted toxins is accompanied by dose‐limiting side effects. The future prospects of targeted toxins for therapeutic use in humans will be determined by reduce side effects. Certain plant secondary metabolites (saponins) were shown to increase the efficacy of a particular epidermal growth factor receptor (EGFR)‐targeted toxin, paralleled by a tremendous decrease of side effects.

Anthrax Toxin-Mediated Delivery of the Pseudomonas Exotoxin A Enzymatic Domain to the Cytosol of Tumor Cells via Cleavable Ubiquitin Fusions

ABSTRACT Anthrax toxin proteins from Bacillus anthracis constitute a highly efficient system for delivering cytotoxic enzymes to the cytosol of tumor cells. However, exogenous proteins delivered to the cytosol of cells are subject to ubiquitination on lysines and proteasomal degradation, which limit their potency. We created fusion proteins containing modified ubiquitins with their C-terminal regions fused to the Pseudomonas exotoxin A catalytic domain (PEIII) in order to achieve delivery and release of PEIII to the cytosol. Fusion proteins in which all seven lysines of wild-type ubiquitin were retained while the site cleaved by cytosolic deubiquitinating enzymes (DUBs) was removed were nontoxic, apparently due to rapid ubiquitination and proteasomal degradation. Fusion proteins in which all lysines of wild-type ubiquitin were substituted by arginine had high potency, exceeding that of a simple fusion lacking ubiquitin. This variant was less toxic to nontumor tissues in mice than the fusion protein lacking ubiquitin and was very efficient for tumor treatment in mice. The potency of these proteins was highly dependent on the number of lysines retained in the ubiquitin domain and on retention of the C-terminal ubiquitin sequence cleaved by DUBs. It appears that rapid cytosolic release of a cytotoxic enzyme (e.g., PEIII) that is itself resistant to ubiquitination is an effective strategy for enhancing the potency of tumor-targeting toxins. IMPORTANCE Bacterial toxins typically have highly efficient mechanisms for cellular delivery of their enzymatic components. Cytosolic delivery of therapeutic enzymes and drugs is an important topic in molecular medicine. We describe anthrax toxin fusion proteins containing ubiquitin as a cytosolic cleavable linker that improves the delivery of an enzyme to mammalian cells. The ubiquitin linker allowed modulation of potency in cells and in mice. This effective strategy for enhancing the intracellular potency of an enzyme may be useful for the cytosolic delivery and release of internalized drugs. Bacterial toxins typically have highly efficient mechanisms for cellular delivery of their enzymatic components. Cytosolic delivery of therapeutic enzymes and drugs is an important topic in molecular medicine. We describe anthrax toxin fusion proteins containing ubiquitin as a cytosolic cleavable linker that improves the delivery of an enzyme to mammalian cells. The ubiquitin linker allowed modulation of potency in cells and in mice. This effective strategy for enhancing the intracellular potency of an enzyme may be useful for the cytosolic delivery and release of internalized drugs.

Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis.

Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGFα). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGFα). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.

Tumor Targeting and Drug Delivery by Anthrax Toxin

Anthrax toxin is a potent tripartite protein toxin from Bacillus anthracis. It is one of the two virulence factors and causes the disease anthrax. The receptor-binding component of the toxin, protective antigen, needs to be cleaved by furin-like proteases to be activated and to deliver the enzymatic moieties lethal factor and edema factor to the cytosol of cells. Alteration of the protease cleavage site allows the activation of the toxin selectively in response to the presence of tumor-associated proteases. This initial idea of re-targeting anthrax toxin to tumor cells was further elaborated in recent years and resulted in the design of many modifications of anthrax toxin, which resulted in successful tumor therapy in animal models. These modifications include the combination of different toxin variants that require activation by two different tumor-associated proteases for increased specificity of toxin activation. The anthrax toxin system has proved to be a versatile system for drug delivery of several enzymatic moieties into cells. This highly efficient delivery system has recently been further modified by introducing ubiquitin as a cytosolic cleavage site into lethal factor fusion proteins. This review article describes the latest developments in this field of tumor targeting and drug delivery.

Abstract 5601: Efficient tumor therapy by anthrax toxin fusion proteins that contain cytolethal distending toxin B.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnCytolethal distending toxin (Cdt) is produced by several Gram-negative bacterial species. It is composed of three subunits, CdtA, CdtB, and CdtC, with CdtB being the catalytic subunit. We fused CdtB from Haemophilus ducreyi with the N-terminal 254 amino acids of Bacillus anthracis toxin lethal factor (LFn) to produce a novel, potent anti-tumor drug. CdtB is transported into the cytosol of targeted cells via the efficient delivery mechanism of anthrax toxin. The fusion protein efficiently killed various human tumor cell lines by inducing a complete cell cycle arrest in the G2/M phase and subsequently inducing apoptosis. Despite the described role of CdtB as a DNase, we detected a prominent phosphatase activity of CdtB, similar to phosphatase and tensin homolog (PTEN) activity. Animal studies showed very low toxicity of a systemically-applied tumor-specific treatment and impressive anti-tumor effects, resulting in a 90% cure rate. This study demonstrates the great potential of a combination of efficient drug delivery by a modified anthrax toxin system and the enzymatic activity of CdtB, and argues for the continued development of this novel anti-cancer drug.nnCitation Format: Christopher Bachran, Suzanne Abdelazim, Radka Hasikova, Shihui Liu, Stephen H. Leppla. Efficient tumor therapy by anthrax toxin fusion proteins that contain cytolethal distending toxin B. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5601. doi:10.1158/1538-7445.AM2013-5601