We report the engineering and selection of two synthetic proteins—FSR16m and FSR22—for the possible treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. FSR16m and FSR22 are trimeric proteins composed of DARPin SR16m or SR22 fused with a T4 foldon. Despite selection by a spike protein from a now historical SARS-CoV-2 strain, FSR16m and FSR22 exhibit broad-spectrum neutralization of SARS-CoV-2 strains, inhibiting authentic B.1.351, B.1.617.2 and BA.1.1 viruses, with respective IC₅₀ values of 3.4, 2.2 and 7.4 ng ml⁻¹ for FSR16m. Cryo-EM structures revealed that these DARPins recognize a region of the receptor-binding domain (residues 456, 475, 486, 487 and 489) overlapping a critical portion of the angiotensin-converting enzyme 2 (ACE2)-binding surface. K18-hACE2 transgenic mice inoculated with B.1.617.2 and receiving intranasally administered FSR16m showed less weight loss and 10–100-fold lower viral burden in upper and lower respiratory tracts. The strong and broad neutralization potency makes FSR16m and FSR22 promising candidates for the prevention and treatment of infection by SARS-CoV-2.

Shiga toxin-producing E. coli (STEC) is a common cause of bloody diarrhea. The pathology of STEC infection derives from two exotoxins—Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2)—that are secreted by STEC in the gut, from where they are systemically absorbed, causing severe kidney damage leading to hemolytic uremic syndrome (HUS). Currently, there is no effective treatment for HUS, and only supportive care is recommended. We report the engineering of a panel of designed ankyrin repeat proteins (DARPin) with potent neutralization activity against Stx2a, the major subtype associated with HUS. The best dimeric DARPin, SD5, created via a combination of directed evolution and rational design, neutralizes Stx2a with a half maximal effective concentration (EC₅₀) of 0.61 nM in vitro. The two monomeric DARPin constituents of SD5 exhibit complementary functions—SHT targets the enzymatic A subunit of Stx2a and inhibits the toxin’s catalytic activity, while DARPin #3 binds the B subunit, based on the cryo-EM study, and induces a novel conformational change in the B subunit that distorts its five-fold symmetry and presumably interferes with toxin attachment to target cells. SD5 was fused to an albumin-binding DARPin, and the resulting trimeric DARPin DA1-SD5 efficiently protects mice in a toxin challenge model, pointing to a high potential of this DARPin as a therapeutic for STEC infection. Finally, the unprecedented toxin conformational change induced by DARPin #3 represents a novel mode of action for neutralizing Stx2 toxicity and reveals new targets for future drug development.

Clostridioides difficile is an enteric bacterium whose exotoxins, TcdA and TcdB, inactivate small GTPases within the host cells, leading to bloody diarrhea. In prior work, our group engineered a panel of potent TcdB-neutralizing designed ankyrin repeat proteins (DARPin) as oral therapeutics against C. difficile infection. However, all these DARPins are highly susceptible to digestion by gut-resident proteases, i.e. trypsin and chymotrypsin. Close evaluation of the protein sequence revealed a large abundance of positively charged and aromatic residues in the DARPin scaffold. In this study, we significantly improved the protease stability of one of the DARPins, 1.4E, via protein engineering. Unlike 1.4E, whose anti-TcdB EC₅₀ increased >83-fold after 1-hour incubation with trypsin (1 mg/ml) or chymotrypsin (0.5 mg/ml), the best progenies—T10-2 and T10b—exhibit similar anti-TcdB potency as their parent in PBS regardless of protease treatment. The superior protease stability of T10-2 and T10b is attributed to the removal of nearly all positively charged and aromatic residues except those directly engaged in target binding. Furthermore, T10-2 was found to retain significant toxin-neutralization ability in ex vivo cecum fluid and can be easily detected in mouse fecal samples upon oral administration. Both T10-2 and T10b enjoy a high thermo- and chemo-stability and can be expressed very efficiently in Escherichia coli (>100 mg/l in shaker flasks). We believe that, in additional to their potential as oral therapeutics against C. difficile infection, T10-2 and T10b can also serve as a new generation DARPin scaffold with superior protease stability. Directed evolution and rational design have led to the creation of two new DARPin proteins with superior trypsin stability. These DARPins can serve as attractive new generation protease-stable protein scaffolds for oral therapeutic applications.

In the originally published version of this manuscript, the wrong file was provided as supplementary data. The correct supplementary data, which contains five figures, is now attached to the online paper.

Proximity ligation assay (PLA), in which the ligation of two DNA probes is greatly accelerated by the associating target molecules, has emerged as a highly sensitive technique for protein detection. The detection of the ligated DNA typically relies on PCR, which requires temperature cycling. In this study, we report on a novel disco ntinuous (DISCO)-LAMP assay that enables the wash-free detection of PLA products via loop-mediated isothermal amplification (LAMP). Due to the exponential amplification nature of LAMP, a careful balance between efficient amplification of the ligated full-length DNA and minimal background amplification from the individual constituent probes is essential but often challenging to achieve. After extensive template/primer design and assay optimization, DISCO-LAMP assay achieved a detection limit of 1 fM for the ligated DNA probe while maintaining undetectable background amplification at 1 nM of each individual probe. DISCO-LAMP detected Shiga toxin 2 (Stx2) with a limit of detection (LoD) of 100 fM when functionalized with Stx2-binders, as well as both Wuhan-1 and Omicron spike protein when functionalized with D _S16 , a newly engineered DARPin targeting a conserved epitope on the SARS-CoV-2 Spike protein. We believe DISCO-LAMP represents a versatile and efficient LAMP-based PLA technology that is readily adaptable for sensing diverse targets.