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Updated: Aug 23, 2025

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A Multi-Specific DARPin Potently Neutralizes Shiga Toxin 2 via Simultaneous Modulation of Both Toxin Subunits.

Yu Zeng1, Mengqiu Jiang2, Sally Robinson3

  • 1Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, 8847 Riverside Pkwy, Bryan, TX 77807, USA.

Bioengineering (Basel, Switzerland)
|October 27, 2022
PubMed
Summary

Researchers developed a novel protein therapy to neutralize Shiga toxin 2a (Stx2a), a key cause of hemolytic uremic syndrome (HUS). This engineered protein, DARPin SD5, shows potent neutralization and protects mice, offering a promising therapeutic for STEC infections.

Keywords:
antibioticbiologicdysenterykidneytherapy

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Area of Science:

  • Microbiology
  • Protein Engineering
  • Immunology

Background:

  • Shiga toxin-producing Escherichia coli (STEC) causes bloody diarrhea and severe kidney damage, leading to hemolytic uremic syndrome (HUS).
  • Current treatments for HUS are limited to supportive care, highlighting the need for effective therapeutic interventions.
  • Shiga toxins (Stx1 and Stx2) are the primary drivers of STEC-induced pathology, with Stx2a being the major HUS-associated subtype.

Purpose of the Study:

  • To engineer novel designed ankyrin repeat proteins (DARPins) with potent neutralization activity against Stx2a.
  • To characterize the mechanism of action of the developed DARPins against Stx2a.
  • To evaluate the therapeutic potential of engineered DARPins in a preclinical model of STEC infection.

Main Methods:

  • Directed evolution and rational design were employed to create a panel of DARPins targeting Stx2a.
  • In vitro neutralization assays were performed to determine the half-maximal effective concentration (EC50) of DARPins.
  • Cryo-electron microscopy (cryo-EM) was utilized to elucidate the structural basis of DARPin-toxin interactions.
  • A mouse model of toxin challenge was used to assess the in vivo efficacy of the lead DARPin candidate.

Main Results:

  • The dimeric DARPin SD5 demonstrated potent in vitro neutralization of Stx2a with an EC50 of 0.61 nM.
  • SD5's monomeric components, SHT and DARPin #3, act via distinct mechanisms: SHT inhibits catalytic activity, while DARPin #3 induces a novel conformational change in the Stx2a B subunit.
  • The trimeric DARPin DA1-SD5, a fusion of SD5 with an albumin-binding DARPin, provided efficient protection in a mouse toxin challenge model.
  • DARPin #3's induction of a conformational change in the Stx2a B subunit represents a novel neutralization mechanism.

Conclusions:

  • Engineered DARPins, particularly SD5 and its derivatives, exhibit potent Stx2a neutralization capabilities.
  • The dual-action mechanism of SD5, involving inhibition of catalytic activity and disruption of toxin structure, offers a robust strategy against Stx2a.
  • The demonstrated in vivo efficacy of DA1-SD5 highlights its potential as a therapeutic candidate for STEC infections and HUS.
  • The novel conformational change induced by DARPin #3 opens new avenues for drug development targeting Shiga toxin toxicity.