Enzymatic Glucosylation Enhances the Solubility of Niclosamide but Abrogates Its Therapeutic Efficacy

High Resolution Image Download MS PowerPoint Slide Niclosamide (Nic) is a small molecule therapeutic listed as an essential medicine by the World Health Organization for the treatment of tapeworm infections. Despite its demonstrated potential for other therapeutic applications in vitro, including cancer cell proliferation and SARS-CoV-2 infection, any clinical use beyond as an anthelminthic agent has been precluded by its poor solubility and bioavailability. In this study, we explored the use of glucosylation as a strategy to modulate the properties of Nic. We determined that the O -glucoside of Nic (Nic-Glc) is 100 times more soluble than Nic at physiological conditions, outperforming all previous attempts at increasing Nic solubility. Motivated by the mandatory green transition, we identified five Nic-active glycosyltransferases (GTs) with the view to enable Nic-Glc bioproduction. Of these five plant GTs, TOGT1_1 from Nicotiana attenuata was identified as the best candidate and a rational engineering campaign was performed to improve its activity, with a total of 14 variants tested. Most of these mutants outperformed wildtype TOGT1_1 in both stability (64%) and relative activity (79%), highlighting the robustness and evolvability of this enzyme. However, when we tested Nic-Glc using in vitro cell-based assays, we found that glucosylation abrogated the antimicrobial and antiviral efficacy of Nic, with Nic-Glc having no impact on the growth of Staphylococcus aureus nor the infectivity of SARS-CoV-2 or hepatitis C virus. Therefore, the potential application of a Nic glycoside would likely need to function as a prodrug. This would preserve the solubility benefit afforded by glycosylation and release the therapeutically active Nic at the desired site. This work provides evidence that glycosylation is a viable strategy for improving the solubility of Nic, and that the development of an enzymatic glycosylation reaction is feasible as a means for the bioproduction of Nic-Glc.