Inhibition of cotranslational translocation by apratoxin S4: Effects on oncogenic receptor tyrosine kinases and the fate of transmembrane proteins produced in the cytoplasm

Abstract

Receptor tyrosine kinases (RTKs) have become major targets for anticancer therapy. However, resistance and signaling pathway redundancy has been problematic. The marine-derived apratoxins act complementary to direct kinase inhibitors by downregulating the levels of multiple of these receptors and additionally prevent the secretion of growth factors that act on these receptors by targeting Sec61α, therefore interfering with cotranslational translocation. We have profiled the synthetic, natural product-inspired apratoxin S4 against panels of cancer cells characterized by differential sensitivity to RTK inhibitors due to receptor mutations, oncogenic KRAS mutations, or activation of compensatory pathways. Apratoxin S4 was active at low-nanomolar to sub-nanomolar concentrations against panels of lung, head and neck, bladder, and pancreatic cancer cells, concomitant with the downregulation of levels of several RTKs, including EGFR, MET and others. However, the requisite concentration to inhibit certain receptors varied, suggesting some differential substrate selectivity in cellular settings. This selectivity was most pronounced in breast cancer cells, where apratoxin S4 selectively targeted HER3 over HER2 and showed greater activity against ER+ and triple negative breast cancer cells than HER2+ cancer cells. Depending on the breast cancer subtype, apratoxin S4 differentially downregulated transmembrane protein CDCP1, which is linked to metastasis and invasion in breast cancer and modulates EGFR activity. We followed the fate of CDCP1 through proteomics and found that nonglycosylated CDCP1 associates with chaperone HSP70 and HUWE1 that functions as an E3 ubiquitin ligase and presumably targets CDCP1, as well as potentially other substrates inhibited by apratoxins, for proteasomal degradation. By preventing cotranslational translocation of VEGF and other proangiogenic factors as well as VEGFR2 and other receptors, apratoxins also possess antiangiogenic activity, which was validated in endothelial cells where downregulation of VEGFR2 was observed, extending the therapeutic scope to angiogenic diseases.

Sec61 Inhibitor Apratoxin S4 Potently Inhibits SARS-CoV-2 and Exhibits Broad-Spectrum Antiviral Activity

“…A different type of Sec61 inhibitor, ipomoeassin F, was recently shown to inhibit the in vitro biogenesis of both spike protein and the SARS-CoV receptor ACE2; however, this observation remains to be confirmed in cell culture models . Consistent with the known effects of Apra S4 on host membrane proteins, Apra S4 additionally downregulates ACE2, although not completely. Further studies are required to determine if this indirect effect would additionally interfere with virus entry upon longer exposure to Apra S4 (Figure ).…”

“…Apratoxin S4 (Apra S4) is a synthetic hybrid analogue of apratoxins A/E, which prevents protein translocation by directly targeting Sec61α, the central subunit of the protein translocation channel on the endoplasmic reticulum (ER) membrane. , Among known Sec61 inhibitors, apratoxins possess unique resistant profiles in cancer cells, indicating a unique mode of target interaction. , It prevents protein ER translocation at an earlier stage than cotransin and appears to impact ER import of Sec61 substrate proteins in a substrate-nonselective fashion based on biochemical data . However, the substrate selectivity for host proteins is tunable and highly context-dependent, as we recently reported . Since viral glycoproteins are dependent on Sec61-mediated secretion, we observed blockage of viral proteostasis.…”

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

“…3 Moreover, it is unclear why some secretory and Type-I membrane proteins are more sensitive to CbA (1), or AprA, than closely related Sec61 clients with similar primary sequence homology. 37,43,44 Given the structural complexity and physiochemical properties of lariat depsipetide scaffolds, 45 the present study aimed to assess the potential impact of stereoselectivity on the action of 1. Several research groups have previously shown that single alterations in the stereochemistry of chiral centers in the macrocycle, or tail, of 1, results in significant decreases in cytotoxic potency relative to the authentic natural product.…”

“…Although all Sec61 inhibitors are predicted to bind and compete for a common region within the Sec61α subunit, extensive resistance mapping in cells and advanced structural models have confirmed that these ligands form nonidentical interactions with Sec61. ,,,,,, Point mutations in Sec61α conferring strong resistance to the broad-spectrum cyanobacterial Sec61 inhibitor apratoxin A (AprA), confer only mild resistance to 1 , which suggests that the two molecules bind distinct interfaces or possibly different conformational states of the native channel . Moreover, it is unclear why some secretory and Type-I membrane proteins are more sensitive to CbA ( 1 ), or AprA, than closely related Sec61 clients with similar primary sequence homology. ,, Given the structural complexity and physiochemical properties of lariat depsipetide scaffolds, the present study aimed to assess the potential impact of stereoselectivity on the action of 1 . Several research groups have previously shown that single alterations in the stereochemistry of chiral centers in the macrocycle, or tail, of 1 , results in significant decreases in cytotoxic potency relative to the authentic natural product. ,,,− We compared the pharmacological properties of 1 , and two major diastereoisomers, against a panel of patient-derived glioblastoma (GBM) stem-like cells (GSCs) with clinically relevant features of GBM including the amplification of known Sec61 substrates such as EGFR and PDGFR (Figure S1).…”