**1. Introduction**

*Neosiphonia Superstes* is a species of marine sponge localized to the region of New Caledonia in the South Pacific [1]. Originally identified during the 1874 voyage of the HMS Challenger expedition, the sponge was resurrected in the early 1990s when D'auria et al. isolated novel macrolides they termed superstolides A and B, which were subsequently shown to display anticancer activity [2–4]. The unprecedented chemical structures of this group of marine natural products suggest they might have a unique cellular target(s) and a novel mechanism of action. However, the scarcity of natural products seriously hampered the biological investigation. In 2013, a truncated superstolide A was designed and synthesized (named ZJ-101) that maintains the potent anticancer activity of the original natural product, thereby solving the supply problem of superstolide A, albeit indirectly [5]. Subsequently, several analogs were produced with enhanced potency in the single-digit nanomolar range. However, the mechanism of action of ZJ-101 and other analogs has remained unknown.

Employing several phenotypic assays in cancer cell lines, we identified novel activities and phenotypes associated with ZJ-101 that have revealed a unique mechanism of action. Notably, an anti-adhesive effect was identified, which was approximately 30-fold more potent than the previously described anti-proliferative effect. Cell cycle and growth rate analysis revealed a strong cytostatic effect. Transcriptomic and lectin stain imaging analyses further converged on a dysregulation of glycosylation within the endomembrane system. Through this analysis, we identified a selectivity for dysregulation of *O*-linked glycosylation, which primarily occurs within the Golgi apparatus. The disruption of glycosylation by

**Citation:** Sanchez, P.R.; Head, S.A.; Qian, S.; Qiu, H.; Roy, A.; Jin, Z.; Zheng, W.; Liu, J.O. Modulation of the Endomembrane System by the Anticancer Natural Product Superstolide/ZJ-101. *Int. J. Mol. Sci.* **2023**, *24*, 9575. https://doi.org/ 10.3390/ijms24119575

Academic Editors: Barbara De Filippis, Alessandra Ammazzalorso and Marialuigia Fantacuzzi

Received: 8 May 2023 Revised: 24 May 2023 Accepted: 29 May 2023 Published: 31 May 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

ZJ-101 leads to pleiotropic effects on proliferation and adhesion, which synergize with etoposide in a 3D spheroid combination model. Taken together, these findings implicate a unique mechanism of action for the family of superstolides and other related analogs [3–6].
