Efficient Delivery of Structurally Diverse Protein Cargo into Mammalian Cells by a Bacterial Toxin

Auger, Anick; Park, Minyoung; Nitschké, Felix; Minassian, Lori M.; Beilhartz, Greg L.; Minassian, Berge A.; Melnyk, Roman A.

doi:10.1021/acs.molpharmaceut.5b00233

Cited by 46 publications

(58 citation statements)

References 56 publications

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“…Targeting intracellular targets with protein-based inhibitors is currently not a therapeutic option due to the practical limitations of immunogenicity and the lack of efficient means for delivering proteins in to cells in vivo. However, progress continues to be made on methods for intracellular delivery of mRNA or proteins [30][31][32], and strategies to reduce immunogenicity, including the use of mirror-image proteins [33], have been developed. Thus, in the future, we are hopeful that UbVs may become bona fide drug candidates, but at present, they are best viewed as tool compounds that can enable drug discovery.…”

Section: Discussionmentioning

confidence: 99%

Potent and selective inhibition of pathogenic viruses by engineered ubiquitin variants

et al. 2017

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The recent Middle East respiratory syndrome coronavirus (MERS-CoV), Ebola and Zika virus outbreaks exemplify the continued threat of (re-)emerging viruses to human health, and our inability to rapidly develop effective therapeutic countermeasures. Many viruses, including MERS-CoV and the Crimean-Congo hemorrhagic fever virus (CCHFV) encode deubiquitinating (DUB) enzymes that are critical for viral replication and pathogenicity. They bind and remove ubiquitin (Ub) and interferon stimulated gene 15 (ISG15) from cellular proteins to suppress host antiviral innate immune responses. A variety of viral DUBs (vDUBs), including the MERS-CoV papain-like protease, are responsible for cleaving the viral replicase polyproteins during replication, and are thereby critical components of the viral replication cycle. Together, this makes vDUBs highly attractive antiviral drug targets. However, structural similarity between the catalytic cores of vDUBs and human DUBs complicates the development of selective small molecule vDUB inhibitors. We have thus developed an alternative strategy to target the vDUB activity through a rational protein design approach. Here, we report the use of phage-displayed ubiquitin variant (UbV) libraries to rapidly identify potent and highly selective protein-based inhibitors targeting the DUB domains of MERS-CoV and CCHFV. UbVs bound the vDUBs with high affinity and specificity to inhibit deubiquitination, deISGylation and in the case of MERS-CoV also viral replicative polyprotein processing. Co-crystallization studies further revealed critical molecular interactions between UbVs and MERS-CoV or CCHFV vDUBs, accounting for the observed binding specificity and high affinity. Finally, expression of UbVs during MERS-CoV infection reduced infectious progeny titers by more than four orders of magnitude, demonstrating the remarkable potency of UbVs as antiviral agents. Our results thereby establish a strategy to produce protein-based inhibitors that could protect against a diverse range of viruses by providing UbVs via mRNA or protein delivery technologies or through transgenic techniques.

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Section: Discussionmentioning

confidence: 99%

Potent and selective inhibition of pathogenic viruses by engineered ubiquitin variants

et al. 2017

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“…Amylase is the only enzyme known to digest Lafora bodies. We have already shown that we are able to deliver amylase to cells in culture in this fashion and that amylase remains functional after crossing the cell membrane (Auger et al ., ). This disease, which we are particularly familiar with, exemplifies the immense possibilities ahead for all our patients with any form of PME.…”

Section: Protein Replacement Therapymentioning

confidence: 96%

“…It was shown that the toxicity of this toxin can be completely eliminated by a point mutation, whilst retaining its translocation properties. Finally, it was shown that a cargo protein, almost of any size, can be attached to this toxin and co-delivered to cells (so far in cell culture experiments only) with maintained function of the delivered protein (Auger et al, 2015). There are other post-genetic era therapeutic approaches in the works, and indeed a lot of excitement that the expression of certain genes will follow Mendelian genetics.…”

Section: Protein Replacement Therapymentioning

confidence: 99%

Post‐modern therapeutic approaches for progressive myoclonus epilepsy

Minassian

2016

Epileptic Disorders

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While the PME are arguably the severest epilepsies and neurological disorders, the vast majority are monogenic. Additionally, many affect straightforward biochemical pathways. Finally, by definition, they occur in previously healthy and well-developed brains. As such, their therapies should be easier than in complex, albeit often less severe, neurological developmental disorders where the complex, poorly understood, and extremely difficult-to-correct, neural network of the brain is affected. This last article reviews the latest cutting edge technologies in monogenic disease therapy, with some examples provided applicable to a number of disease. It aims to give a sense of where we are and how much closer we are, to the goal of making an actual organic difference.

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“…DT consists of a catalytically-active A fragment (DTA) and a B fragment (DTB) that includes both a receptor binding domain (DTR) and a translocation domain (DTT). DTB alone can bind its surface receptor, the heparin-binding epidermal growth factor-like growth factor (HB-EGF), and transfer a wide range of protein cargos to the cell cytosol via a receptor-mediated endocytic mechanism (Auger et al, 2015). The introduction of two amino acid substitutions (K51E and E148K) into DTA resulted in a non-toxic, catalytically-inactive A-fragment, herein referred to as DTa.…”

Section: Rrsp Into Cellsmentioning

confidence: 99%

“…Assessing the therapeutic potential of RRSP is however precluded by the fact that RRSP is a 56-kDa domain of a larger protein toxin that alone does not readily diffuse across biological Vidimar et al / Page 5 5 membranes, thus preventing its access to intracellular RAS. Recently, we demonstrated that the translocation machinery of diphtheria toxin (DT) can be engineered to deliver a broad diversity of passenger proteins into target cells (Auger et al, 2015;Park et al, 2018). In the present study, we exploit the receptor-targeting and membrane translocation properties of DT to deliver RRSP into target cells.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of tumor growth by a novel engineered chimeric toxin that cleaves activated mutant and wild-type RAS

Vidimar¹,

Beilhartz

Park

et al. 2019

Preprint

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One Sentence Summary: Engineered chimeric toxin halts tumor growth in vivo via RAS cleavage Vidimar et al / Page 2 2 SummaryDespite nearly four decades of effort, broad inhibition of oncogenic RAS using small molecule approaches has proven to be a major challenge. Here we describe the development of a novel pan-RAS biologic inhibitor comprised of the RAS-RAP1-specific endopeptidase fused to the protein delivery machinery of diphtheria toxin. We show that this engineered chimeric toxin irreversibly cleaves and inactivates intracellular RAS at low picomolar concentrations terminating downstream signaling in receptor-bearing cells. Further, we demonstrate in vivo target engagement and reduction of tumor burden in three mouse xenograft models driven by either wild-type or mutant RAS. Intracellular delivery of a potent anti-RAS biologic through a receptor-mediated mechanism represents a promising new approach to developing RAS therapeutics against a broad array of cancers. SignificanceRAS oncoproteins have long been considered among the most elusive drug targets in cancer research. At issue is the lack of accessible drug binding sites and the extreme affinity for its GTP substrate. Covalent inhibitors against the KRAS G12C mutant have shown early clinical promise, however, targeting the other oncogenic RAS mutants across three RAS isoforms has proven challenging. Inhibition of activated wild-type RAS in the absence of canonical RAS mutations is also highly desirable in certain tumors. Here, we demonstrate delivery of an extremely potent pan-RAS and RAP1 cleaving enzyme in therapeutic quantities to specific receptor-bearing cells in vitro and in vivo. We aim to advance this approach to engineer the first targeted pan-RAS inhibitor for cancer therapy.Vidimar et al / Page 3

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Efficient Delivery of Structurally Diverse Protein Cargo into Mammalian Cells by a Bacterial Toxin

Cited by 46 publications

References 56 publications

Potent and selective inhibition of pathogenic viruses by engineered ubiquitin variants

Potent and selective inhibition of pathogenic viruses by engineered ubiquitin variants

Post‐modern therapeutic approaches for progressive myoclonus epilepsy

Inhibition of tumor growth by a novel engineered chimeric toxin that cleaves activated mutant and wild-type RAS

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