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

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 <i>in vitro</i>, 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 <i>O</i>-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 <i>Nicotiana attenuata</i> 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 <i>in vitro</i> 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 <i>Staphylococcus aureus</i> 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.