Nanonine, a Synthetic Nanobody Targeting Protein Disulfide Isomerase, Is a New Weapon in Our Antithrombotic Arsenal

Background: Protein disulfide isomerase (PDI) is a potential target for developing treatments that can prevent blood clotting without increasing the risk of excessive bleeding. Small-molecule inhibitors of PDI have been under investigation for about ten years, and a compound called isoquercetin is currently being tested in clinical trials for treating blood clotting associated with cancer. However, developing drugs that specifically target PDI has proven to be challenging, as PDI is structurally similar to other thiol-isomerases. Nanobodies, a new type of biotherapeutics, stand out due to their small size, ease of production, and specificity for their targets, offering significant advantages over traditional monoclonal antibodies and small molecules. Goal: This study aimed to discover, functionally characterize, and structurally characterize synthetic nanobodies (Synabodies) that target PDI and have antithrombotic properties. Methods: Yeast-display technology combined with next-generation sequencing was conducted to identify a library of Synabodies that specifically bind to PDI. Surface plasmon resonance (SPR) and kinetic experiments were used to evaluate the affinity, specificity, and impact on PDI reductase activity. Cryogenic electron microscopy (cryo-EM) was employed to determine the mechanism of action and binding mode of a selected inhibitory Synabody called NanoNine. Platelet aggregometry was performed to assess the functional profile of NanoNine in human platelets. Results: Ten Synabodies were selected from the yeast display experiments, out of which six were successfully expressed in E.coli. Three of these Synabodies bound to PDI in the nanomolar range, as determined by SPR. None of the Synabodies cross-reacted with ERp57 and ERp72, two thiol-isomerases structurally similar to PDI that are known to play key roles in thrombosis. One of the six available Synabodies, NanoNine, inhibited PDI's reductase activity against multiple substrates, including insulin and Bodipy-L-cystine, with greater potency than most small molecule compounds discovered thus far (i.e., IC50∼100 nM). To determine NanoNine's mechanism of action, the complex was isolated using size-exclusion chromatography, and its structure was solved using cryo-EM at 5.0 Å resolution. The cryo-EM structure of the complex revealed the spatial arrangement of all four PDI domains a-b-b'-a' in the typical flexible U-shaped conformation. It also provided insights into the binding mode of NanoNine, showing that it interacts more stably with the b' and a' domains but also has transient interactions with the a domain due to the multiple conformations of PDI visible by cryo-EM. The binding energy mostly comes from the particularly long complementarity-determining region 3 (CDR3) of NanoNine, which deeply inserts into the hydrophobic binding pocket of the b' domain, making multiple hydrophilic and hydrophobic contacts with surrounding residues. Ala mutagenesis experiments confirmed the key role of H256 in the b' domain, and competition experiments with small molecules bepristat 2a and rutin established shared binding sites and reversibility. Finally, NanoNine displayed inhibition of platelet aggregation using platelet-rich plasma and washed platelets when stimulated with collagen or thrombin. Conclusions: NanoNine, a synthetic nanobody targeting PDI, shows promise as a new candidate for preclinical development. The cryo-EM structure of the PDI-NanoNine complex will facilitate further drug discovery and structure-based optimization of PDI-targeted antithrombotics.