Tamoxifen Derivatives Alter Retromer-Dependent Endosomal Tubulation and Sorting to Block Retrograde Trafficking of Shiga Toxins

Abstract

Shiga toxin 1 and 2 (STx1 and STx2) undergo retrograde trafficking to reach the cytosol of cells where they target ribosomes. As retrograde trafficking is essential for disease, inhibiting STx1/STx2 trafficking is therapeutically promising. Recently, we discovered that the chemotherapeutic drug tamoxifen potently inhibits the trafficking of STx1/STx2 at the critical early endosome-to-Golgi step. We further reported that the activity of tamoxifen against STx1/STx2 is independent of its selective estrogen receptor modulator (SERM) property and instead depends on its weakly basic chemical nature, which allows tamoxifen to increase endolysosomal pH and alter the recruitment of retromer to endosomes. The goal of the current work was to obtain a better understanding of the mechanism of action of tamoxifen against the more disease-relevant toxin STx2, and to differentiate between the roles of changes in endolysosomal pH and retromer function. Structure activity relationship (SAR) analyses revealed that a weakly basic amine group was essential for anti-STx2 activity. However, ability to deacidify endolysosomes was not obligatorily necessary because a tamoxifen derivative that did not increase endolysosomal pH exerted reduced, but measurable, activity. Additional assays demonstrated that protective derivatives inhibited the formation of retromer-dependent, Golgi-directed, endosomal tubules, which mediate endosome-to-Golgi transport, and the sorting of STx2 into these tubules. These results identify retromer-mediated endosomal tubulation and sorting to be fundamental processes impacted by tamoxifen; provide an explanation for the inhibitory effect of tamoxifen on STx2; and have important implications for the therapeutic use of tamoxifen, including its development for treating Shiga toxicosis.

C910 chemical compound inhibits the traffiking of several bacterial AB toxins with cross-protection against influenza virus

“…The importance of these chemical modulators of vesicular trafficking in deciphering the key steps of these pathways and toxin mechanisms of action perfectly illustrates the cross-feeding between the disciplines of cell biology and infectiology ( Forrester et al., 2020 ; Sandvig et al., 1991 ; Selyunin et al., 2021 ). Notably, these different compounds have been used as tools to discern the different physicochemical requirements of AB toxins.…”

“…For example, CNFy and CNF3 showed distinctive sensitivity to EGA but similar dose-response profiles to ABMA, indicating differential endosomal escape of the two highly homologous toxins ( Haywood et al., 2021 ). Similarly, tamoxifen was identified in a drug repurposing screen to block the fusion of late endosomes with lysosomes with consequences on the dynamics of retromer cycling on early endosomes, leading to the impaired sorting of Stx1 and Stx2 toxins from early endosomes to the Golgi network ( Selyunin et al., 2019 , 2021 ).…”

Out of the ESCPE room: Emerging roles of endosomal SNX‐BARs in receptor transport and host–pathogen interaction

“…Furthermore, the role of ESCPE‐1 function in bacterial and viral infections is becoming clearer and could be of pivotal importance in uncovering novel pathways to target for innovative therapeutic approaches. For example, small molecules that inhibit SNX1‐dependent endosomal tubulation and retrograde trafficking of Shiga toxins were recently identified 152 . We hope that this work could encourage the exploration of ESCPE‐1 role in host–pathogen interaction and promote the identification of molecular interventions to target such interactions for reduced infection.…”

“…For example, small molecules that inhibit SNX1-dependent endosomal tubulation and retrograde trafficking of Shiga toxins were recently identified. 152 We hope that this work could encourage the exploration of ESCPE-1 role in host-pathogen interaction and promote the identification of molecular interventions to target such interactions for reduced infection.…”

Application of Protein Toxins as Cell Biological and Pharmacological Tools

“…Selyunin and colleagues have observed that the chemotherapeutic agent tamoxifen protects cells against Shiga toxins 1 and 2 by inhibiting their intracellular trafficking from early endosomes to the Golgi apparatus [3]. Initially, the authors thought that this effect was due to a modification of endosomal pH by tamoxifen.…”