A common mechanism of Sec61 translocon inhibition by small molecules

Abstract

The Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum (ER) membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural and synthetic small molecules specifically inhibit the Sec61 channel, generating cellular effects that are potentially useful for therapeutic purposes, but their inhibitory mechanisms remain unclear. Here we present near-atomic-resolution structures of the human Sec61 channel inhibited by a comprehensive panel of structurally distinct small moleculescotransin, decatransin, apratoxin F, ipomoeassin F, mycolactone, cyclotriazadisulfonamide (CADA) and eeyarestatin I (ESI). Remarkably, all inhibitors bind to a common lipid-exposed pocket formed by the partially open lateral gate and plug domain of the channel. Mutations conferring resistance to the inhibitors are clustered at this binding pocket. The structures indicate that Sec61 inhibitors stabilize the plug domain of Sec61 in a closed state, thereby preventing the protein-translocation pore from opening. Our study reveals molecular interactions between Sec61 and its inhibitors in atomic detail and offers the structural framework for further pharmacological studies and drug design.

Lipid scrambling is a general feature of protein insertases

“…4e). Finally, for the specific case of Sec61, we tested its ability to scramble lipids in its “closed” and “open” states (Gemmer et al, 2023; Gérard et al, 2020; Itskanov et al, 2023), and our results indicate that when the Sec61 lateral gate is partially closed or closed, lipid scrambling is strongly reduced or abolished, respectively (Fig. 4f).…”

Diastereomers of Coibamide A Show Altered Sec61 Client Selectivity and Ligand-Dependent Activity against Patient-Derived Glioma Stem-like Cells

“…Further, mutant Sec61(R66I) HEK293 cells were resistant to 1 and AprA yet remained highly sensitive to marizomib (Figure S3A−J). These data strongly suggest that diastereomers 2 and 3 retain structural conformations that are sufficient for target engagement with Sec61 and that R66I, located in the mobile plug region of the Sec61α subunit, 18 is either in contact with, or modulated by, 2 and 3. In the presence of a relatively high (1 μM) concentration of each diastereomer, fixed at 50-fold higher than the IC 50 for GLuc inhibition by 3, BiP was suppressed by 2 in normal HUVECs and U87-MG GBM cells, whereas 3 induced no statistically significant change in BiP (Figure 6A,B).…”

“…Further, the striking differential sensitivity of secreted proteins to 2 and 3 (Figure 7C) suggested that these diastereomers have distinct pharmacological signatures, presumably due to subtle differences in the spatial arrangement of 1, 2, and 3 within the Sec61 inhibitorbinding pocket. 3,18 Human Glioma Stem-like Cells are Differentially Sensitive to Coibamide A, [L-Hiv 2 ]-CbA, and [L-Hiv 2 , L-MeAla 11 ]-CbA. Six different GSCs isolated from patients with primary or recurrent GBM, collectively representing a range of distinct molecular features with clinical significance (Figure S1), were used to compare the bioactivity profiles of 2 and 3.…”

“…3,14 Recent structural models of ligandbound human Sec61 complexes obtained using cryogenic electron microscopy (cryo-EM) continue to support and extend a critical role for the SP as the basis for Sec61 substrate selectivity. 12,18 For example, promising selectivity has recently been achieved for a nontoxic cotransin-like molecule, KZR-8445, that discriminates between pro-inflammatory cytokines in a SP-dependent manner. 12 Cytokines, such as IL-2, IL-7 and TNFα, are sensitive to low nanomolar concentrations of KZR-8445, while the SPs of other Sec61 substrates are insensitive with IC 50 values > 25 μM.…”

“…12 Besides HUN-7293, most natural Sec61 inhibitors have apparently evolved to inhibit ER translocation of a broad range of Sec61 substrates and are eventually cytotoxic. 2,12,18,29 This general mechanism underlies the pharmacological properties of several natural and synthetic Sec61 inhibitors against solid and hematological cancer cell types. 21,30−35 Human cancer cells are sensitive to low nanomolar concentrations of CbA (1), which can be attributed, at least initially, to the functional loss of a broad-range of Sec61 client proteins that are critical for cell proliferation and survival.…”