Identification of a peptide motif that potently inhibits two functionally distinct subunits of Shiga toxin

Watanabe-Takahashi, Miho; Tamada, Masakazu; Senda, Masuo; Hibino, M.; Shimizu, Eihan; Okuta, Akiko; Miyazawa, Atsuo; Senda, Toshiya; Nishikawa, Kiyotaka

doi:10.1038/s42003-021-02068-3

Cited by 14 publications

(15 citation statements)

References 29 publications

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“…The main chain of Arg8 of R3A-mono also electrostatically interacts with Asp70 of the B-subunit. Overall, we find that the binding patterns of these shorter peptides are almost identical to that of MMβA-mono 18 . Combined with results from the competition assay, these data demonstrate that these shorter peptides efficiently bind to the A-subunit of Stx2a, and R2A-mono is the minimal essential motif needed for binding.…”

Section: Resultsmentioning

confidence: 57%

“…In this study, we identified R2A-mono as the minimal essential motif of MMβA-mono that binds to the A-subunit of Stx2a. Using X-ray crystal structural analysis, we clearly show direct binding between R2A-mono and the A-subunit, in a manner that is identical to that of the Arg8-Arg9-Ala10 region of full-length MMβA-mono 18 . The critical role of Arg8-Arg9 in Stx2a binding was further confirmed by MD simulations with MMβA-mono, which indicated in all trials that Arg8 and Arg9 electrostatically interact with A-subunit residues Glu167 and Asp94, respectively.…”

Section: Discussionmentioning

confidence: 83%

“…MMβA-mono is a 10-residue peptide with the sequence, Met-Ala-Met-Met-βAla-Arg-Arg-Arg-Arg-Ala, in which βAla is synthetic amino acid. This peptide has been shown to bind exclusively to the Stx2a A-subunit (apparent Kd = 0.05 µM), but not to the B-subunit pentamer 18 . To determine a minimal essential motif of MMβA-mono that binds to the Stx2a A-subunit, we prepared a series of shorter peptides based on the sequence of MMβA-mono and measured their ability to inhibit binding of MMβA-mono to the A-subunit using the AlphaScreen assay.…”

Section: Resultsmentioning

confidence: 98%

“…Affinity-based screening of this library identified a series of tetravalent peptides that bind to the B-subunit pentamer with high affinity and inhibit Stx toxicity in vitro and in vivo 16 , 17 . One of these tetravalent peptides, MMβA-tet, which has the synthetic amino acid βAla in its motif, inhibits both Stx1a and Stx2a with the greatest potency 18 . Unexpectedly, we further found that a monomeric peptide with the same motif as MMβA-tet (referred to as MMβA-mono), also inhibits cytotoxicity of Stx1a and Stx2a, although it cannot exert the clustering effect nor bind to the Stx B-subunit pentamer.…”

Section: Introductionmentioning

confidence: 99%

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A unique peptide-based pharmacophore identifies an inhibitory compound against the A-subunit of Shiga toxin

Watanabe-Takahashi

Senda

Yoshino

et al. 2022

Sci Rep

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Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli (EHEC), can cause fatal systemic complications. Recently, we identified a potent inhibitory peptide that binds to the catalytic A-subunit of Stx. Here, using biochemical structural analysis and X-ray crystallography, we determined a minimal essential peptide motif that occupies the catalytic cavity and is required for binding to the A-subunit of Stx2a, a highly virulent Stx subtype. Molecular dynamics simulations also identified the same motif and allowed determination of a unique pharmacophore for A-subunit binding. Notably, a series of synthetic peptides containing the motif efficiently inhibit Stx2a. In addition, pharmacophore screening and subsequent docking simulations ultimately identified nine Stx2a-interacting molecules out of a chemical compound database consisting of over 7,400,000 molecules. Critically, one of these molecules markedly inhibits Stx2a both in vitro and in vivo, clearly demonstrating the significance of the pharmacophore for identifying therapeutic agents against EHEC infection.

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Section: Resultsmentioning

confidence: 57%

Section: Discussionmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

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A unique peptide-based pharmacophore identifies an inhibitory compound against the A-subunit of Shiga toxin

Watanabe-Takahashi

Senda

Yoshino

et al. 2022

Sci Rep

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“…Finally, HUS is rarely fatal whereas cancer, e.g., pancreatic, always is. Any potential circulating VT-induced renal pathology may prove treatable [172,173] Intratumoural VT1 administration into a Gb 3 expressing tumour provides a further means to reduce risk. Although circulating VT will concentrate in a Gb 3 positive tumour [92,103], high affinity receptor binding within the tumour following judicious i.t.…”

Section: Riskmentioning

confidence: 99%

Therapeutic Uses of Bacterial Subunit Toxins

Lingwood

2021

Toxins

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The B subunit pentamer verotoxin (VT aka Shiga toxin-Stx) binding to its cellular glycosphingolipid (GSL) receptor, globotriaosyl ceramide (Gb3) mediates internalization and the subsequent receptor mediated retrograde intracellular traffic of the AB5 subunit holotoxin to the endoplasmic reticulum. Subunit separation and cytosolic A subunit transit via the ER retrotranslocon as a misfolded protein mimic, then inhibits protein synthesis to kill cells, which can cause hemolytic uremic syndrome clinically. This represents one of the most studied systems of prokaryotic hijacking of eukaryotic biology. Similarly, the interaction of cholera AB5 toxin with its GSL receptor, GM1 ganglioside, is the key component of the gastrointestinal pathogenesis of cholera and follows the same retrograde transport pathway for A subunit cytosol access. Although both VT and CT are the cause of major pathology worldwide, the toxin–receptor interaction is itself being manipulated to generate new approaches to control, rather than cause, disease. This arena comprises two areas: anti neoplasia, and protein misfolding diseases. CT/CTB subunit immunomodulatory function and anti-cancer toxin immunoconjugates will not be considered here. In the verotoxin case, it is clear that Gb3 (and VT targeting) is upregulated in many human cancers and that there is a relationship between GSL expression and cancer drug resistance. While both verotoxin and cholera toxin similarly hijack the intracellular ERAD quality control system of nascent protein folding, the more widespread cell expression of GM1 makes cholera the toxin of choice as the means to more widely utilise ERAD targeting to ameliorate genetic diseases of protein misfolding. Gb3 is primarily expressed in human renal tissue. Glomerular endothelial cells are the primary VT target but Gb3 is expressed in other endothelial beds, notably brain endothelial cells which can mediate the encephalopathy primarily associated with VT2-producing E. coli infection. The Gb3 levels can be regulated by cytokines released during EHEC infection, which complicate pathogenesis. Significantly Gb3 is upregulated in the neovasculature of many tumours, irrespective of tumour Gb3 status. Gb3 is markedly increased in pancreatic, ovarian, breast, testicular, renal, astrocytic, gastric, colorectal, cervical, sarcoma and meningeal cancer relative to the normal tissue. VT has been shown to be effective in mouse xenograft models of renal, astrocytoma, ovarian, colorectal, meningioma, and breast cancer. These studies are herein reviewed. Both CT and VT (and several other bacterial toxins) access the cell cytosol via cell surface ->ER transport. Once in the ER they interface with the protein folding homeostatic quality control pathway of the cell -ERAD, (ER associated degradation), which ensures that only correctly folded nascent proteins are allowed to progress to their cellular destinations. Misfolded proteins are translocated through the ER membrane and degraded by cytosolic proteosome. VT and CT A subunits have a C terminal misfolded protein mimic sequence to hijack this transporter to enter the cytosol. This interface between exogenous toxin and genetically encoded endogenous mutant misfolded proteins, provides a new therapeutic basis for the treatment of such genetic diseases, e.g., Cystic fibrosis, Gaucher disease, Krabbe disease, Fabry disease, Tay-Sachs disease and many more. Studies showing the efficacy of this approach in animal models of such diseases are presented.

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Development of a novel tetravalent peptide that absorbs subtilase cytotoxin by targeting the receptor-binding B-subunit

Hama

Nakahara

Watanabe-Takahashi

et al. 2022

Biochemical and Biophysical Research Communications

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Identification of a peptide motif that potently inhibits two functionally distinct subunits of Shiga toxin

Cited by 14 publications

References 29 publications

A unique peptide-based pharmacophore identifies an inhibitory compound against the A-subunit of Shiga toxin

A unique peptide-based pharmacophore identifies an inhibitory compound against the A-subunit of Shiga toxin

Therapeutic Uses of Bacterial Subunit Toxins

Development of a novel tetravalent peptide that absorbs subtilase cytotoxin by targeting the receptor-binding B-subunit

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