ER translocon inhibitor ipomoeassin F inhibits triple-negative breast cancer growth via blocking ER molecular chaperones.

Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer where no effective therapy has been developed. Here, we report that the natural product ER translocon inhibitor ipomoeassin F is a selective inhibitor of TNBC cell growth. A proteomic analysis of TNBC cells revealed that ipomoeassin F significantly reduced the levels of ER molecular chaperones, including PDIA6 and PDIA4, and induced ER stress, unfolded protein response (UPR) and autophagy in TNBC cells. Mechanistically, ipomoeassin F, as an inhibitor of Sec61α-containing ER translocon, blocks ER translocation of PDIA6, inducing its proteasomal degradation. Silencing of PDIA6 or PDIA4 by RNA interferences or treatment with a small molecule inhibitor of the protein disulfide isomerases in TNBC cells successfully recapitulated the ipomoeassin F phenotypes, including the induction of ER stress, UPR and autophagy, suggesting that the reduction of PDIAs is the key mediator of the pharmacological effects of ipomoeassin F. Moreover, ipomoeassin F significantly suppressed TNBC growth in a mouse tumor xenograft model, with a marked reduction in PDIA6 and PDIA4 levels in the tumor samples. Our study demonstrates that Sec61α-containing ER translocon and PDIAs are potential drug targets for TNBC and suggests that ipomoeassin F could serve as a lead for developing ER translocon-targeted therapy for TNBC.

A common mechanism of Sec61 translocon inhibition by small molecules.

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

Mycolactone causes catastrophic Sec61-dependent loss of the endothelial glycocalyx and basement membrane: a new indirect mechanism driving tissue necrosis in Mycobacterium ulcerans infection.

The drivers of tissue necrosis in Mycobacterium ulcerans infection (Buruli ulcer disease) have historically been ascribed solely to the directly cytotoxic action of the diffusible exotoxin, mycolactone. However, its role in the clinically-evident vascular component of disease aetiology remains poorly explained. We have now dissected mycolactone's effects on primary vascular endothelial cells in vitro and in vivo. We show that mycolactone-induced changes in endothelial morphology, adhesion, migration, and permeability are dependent on its action at the Sec61 translocon. Unbiased quantitative proteomics identified a profound effect on proteoglycans, driven by rapid loss of type II transmembrane proteins of the Golgi, including enzymes required for glycosaminoglycan (GAG) synthesis, combined with a reduction in the core proteins themselves. Loss of the glycocalyx is likely to be of particular mechanistic importance, since knockdown of galactosyltransferase II (beta-1,3-galactotransferase 6; B3Galt6), the GAG linker-building enzyme, phenocopied the permeability and phenotypic changes induced by mycolactone. Additionally, mycolactone depleted many secreted basement membrane components and microvascular basement membranes were disrupted in vivo. Remarkably, exogenous addition of laminin-511 reduced endothelial cell rounding, restored cell attachment and reversed the defective migration caused by mycolactone. Hence supplementing mycolactone-depleted extracellular matrix may be a future therapeutic avenue, to improve wound healing rates.

Synthesis, Biological Evaluation and Docking Studies of Ring-Opened Analogues of Ipomoeassin F.

The plant-derived macrocyclic resin glycoside ipomoeassin F (Ipom-F) binds to Sec61α and significantly disrupts multiple aspects of Sec61-mediated protein biogenesis at the endoplasmic reticulum, ultimately leading to cell death. However, extensive assessment of Ipom-F as a molecular tool and a therapeutic lead is hampered by its limited production scale, largely caused by intramolecular assembly of the macrocyclic ring. Here, using in vitro and/or in cellula biological assays to explore the first series of ring-opened analogues for the ipomoeassins, and indeed all resin glycosides, we provide clear evidence that macrocyclic integrity is not required for the cytotoxic inhibition of Sec61-dependent protein translocation by Ipom-F. Furthermore, our modeling suggests that open-chain analogues of Ipom-F can interact with multiple sites on the Sec61α subunit, most likely located at a previously identified binding site for mycolactone and/or the so-called lateral gate. Subsequent in silico-aided design led to the discovery of the stereochemically simplified analogue 3 as a potent, alternative lead compound that could be synthesized much more efficiently than Ipom-F and will accelerate future ipomoeassin research in chemical biology and drug discovery. Our work may also inspire further exploration of ring-opened analogues of other resin glycosides.

Co-translational biogenesis of lipid droplet integral membrane proteins.

Membrane proteins destined for lipid droplets (LDs), a major intracellular storage site for neutral lipids, are inserted into the endoplasmic reticulum (ER) and then trafficked to LDs where they reside in a hairpin loop conformation. Here, we show that LD membrane proteins can be delivered to the ER either co- or post-translationally and that their membrane-embedded region specifies pathway selection. The co-translational route for LD membrane protein biogenesis is insensitive to a small molecule inhibitor of the Sec61 translocon, Ipomoeassin F, and instead relies on the ER membrane protein complex (EMC) for membrane insertion. This route may even result in a transient exposure of the short N termini of some LD membrane proteins to the ER lumen, followed by putative topological rearrangements that would enable their transmembrane segment to form a hairpin loop and N termini to face the cytosol. Our study reveals an unexpected complexity to LD membrane protein biogenesis and identifies a role for the EMC during their co-translational insertion into the ER.

An alternative pathway for membrane protein biogenesis at the endoplasmic reticulum.

The heterotrimeric Sec61 complex is a major site for the biogenesis of transmembrane proteins (TMPs), accepting nascent TMP precursors that are targeted to the endoplasmic reticulum (ER) by the signal recognition particle (SRP). Unlike most single-spanning membrane proteins, the integration of type III TMPs is completely resistant to small molecule inhibitors of the Sec61 translocon. Using siRNA-mediated depletion of specific ER components, in combination with the potent Sec61 inhibitor ipomoeassin F (Ipom-F), we show that type III TMPs utilise a distinct pathway for membrane integration at the ER. Hence, following SRP-mediated delivery to the ER, type III TMPs can uniquely access the membrane insertase activity of the ER membrane complex (EMC) via a mechanism that is facilitated by the Sec61 translocon. This alternative EMC-mediated insertion pathway allows type III TMPs to bypass the Ipom-F-mediated blockade of membrane integration that is seen with obligate Sec61 clients.

Ipomoeassin-F disrupts multiple aspects of secretory protein biogenesis.

The Sec61 complex translocates nascent polypeptides into and across the membrane of the endoplasmic reticulum (ER), providing access to the secretory pathway. In this study, we show that Ipomoeassin-F (Ipom-F), a selective inhibitor of protein entry into the ER lumen, blocks the in vitro translocation of certain secretory proteins and ER lumenal folding factors whilst barely affecting others such as albumin. The effects of Ipom-F on protein secretion from HepG2 cells are twofold: reduced ER translocation combined, in some cases, with defective ER lumenal folding. This latter issue is most likely a consequence of Ipom-F preventing the cell from replenishing its ER lumenal chaperones. Ipom-F treatment results in two cellular stress responses: firstly, an upregulation of stress-inducible cytosolic chaperones, Hsp70 and Hsp90; secondly, an atypical unfolded protein response (UPR) linked to the Ipom-F-mediated perturbation of ER function. Hence, although levels of spliced XBP1 and CHOP mRNA and ATF4 protein increase with Ipom-F, the accompanying increase in the levels of ER lumenal BiP and GRP94 seen with tunicamycin are not observed. In short, although Ipom-F reduces the biosynthetic load of newly synthesised secretory proteins entering the ER lumen, its effects on the UPR preclude the cell restoring ER homeostasis.

Ipomoeassin-F inhibits the in vitro biogenesis of the SARS-CoV-2 spike protein and its host cell membrane receptor.

In order to produce proteins essential for their propagation, many pathogenic human viruses, including SARS-CoV-2, the causative agent of COVID-19 respiratory disease, commandeer host biosynthetic machineries and mechanisms. Three major structural proteins, the spike, envelope and membrane proteins, are amongst several SARS-CoV-2 components synthesised at the endoplasmic reticulum (ER) of infected human cells prior to the assembly of new viral particles. Hence, the inhibition of membrane protein synthesis at the ER is an attractive strategy for reducing the pathogenicity of SARS-CoV-2 and other obligate viral pathogens. Using an in vitro system, we demonstrate that the small molecule inhibitor ipomoeassin F (Ipom-F) potently blocks the Sec61-mediated ER membrane translocation and/or insertion of three therapeutic protein targets for SARS-CoV-2 infection; the viral spike and ORF8 proteins together with angiotensin-converting enzyme 2, the host cell plasma membrane receptor. Our findings highlight the potential for using ER protein translocation inhibitors such as Ipom-F as host-targeting, broad-spectrum antiviral agents.This article has an associated First Person interview with the first author of the paper.

Ipomoeassin-F inhibits the in vitro biogenesis of the SARS-CoV-2 spike protein and its host cell membrane receptor.

In order to produce proteins essential for their propagation, many pathogenic human viruses, including SARS-CoV-2 the causative agent of COVID-19 respiratory disease, commandeer host biosynthetic machineries and mechanisms. Three major structural proteins, the spike, envelope and membrane proteins, are amongst several SARS-CoV-2 components synthesised at the endoplasmic reticulum (ER) of infected human cells prior to the assembly of new viral particles. Hence, the inhibition of membrane protein synthesis at the ER is an attractive strategy for reducing the pathogenicity of SARS-CoV-2 and other obligate viral pathogens. Using an in vitro system, we demonstrate that the small molecule inhibitor ipomoeassin F (Ipom-F) potently blocks the Sec61-mediated ER membrane translocation/insertion of three therapeutic protein targets for SARS-CoV-2 infection; the viral spike and ORF8 proteins together with angiotensin-converting enzyme 2, the host cell plasma membrane receptor. Our findings highlight the potential for using ER protein translocation inhibitors such as Ipom-F as host-targeting, broad-spectrum, antiviral agents.

Ring Expansion Leads to a More Potent Analogue of Ipomoeassin F.

Two new ring-size-varying analogues (2 and 3) of ipomoeassin F were synthesized and evaluated. Improved cytotoxicity (IC50: from 1.8 nM) and in vitro protein translocation inhibition (IC50: 35 nM) derived from ring expansion imply that the binding pocket of Sec61α (isoform 1) can accommodate further structural modifications, likely in the fatty acid portion. Streamlined preparation of the key diol intermediate 5 enabled gram-scale production, allowing us to establish that ipomoeassin F is biologically active in vivo (MTD: ∼3 mg/kg).

Posttranslational insertion of small membrane proteins by the bacterial signal recognition particle.

Small membrane proteins represent a largely unexplored yet abundant class of proteins in pro- and eukaryotes. They essentially consist of a single transmembrane domain and are associated with stress response mechanisms in bacteria. How these proteins are inserted into the bacterial membrane is unknown. Our study revealed that in Escherichia coli, the 27-amino-acid-long model protein YohP is recognized by the signal recognition particle (SRP), as indicated by in vivo and in vitro site-directed cross-linking. Cross-links to SRP were also observed for a second small membrane protein, the 33-amino-acid-long YkgR. However, in contrast to the canonical cotranslational recognition by SRP, SRP was found to bind to YohP posttranslationally. In vitro protein transport assays in the presence of a SecY inhibitor and proteoliposome studies demonstrated that SRP and its receptor FtsY are essential for the posttranslational membrane insertion of YohP by either the SecYEG translocon or by the YidC insertase. Furthermore, our data showed that the yohP mRNA localized preferentially and translation-independently to the bacterial membrane in vivo. In summary, our data revealed that YohP engages an unique SRP-dependent posttranslational insertion pathway that is likely preceded by an mRNA targeting step. This further highlights the enormous plasticity of bacterial protein transport machineries.

Design and Synthesis of Tetrazole- and Pyridine-Containing Itraconazole Analogs as Potent Angiogenesis Inhibitors.

Itraconazole, a widely used antifungal drug, was found to possess antiangiogenic activity and is currently undergoing multiple clinical trials for the treatment of different types of cancer. However, it suffers from extremely low solubility and strong interactions with many drugs through inhibition of CYP3A4, limiting its potential as a new antiangiogenic and anticancer drug. To address these issues, a series of analogs in which the phenyl group is replaced with pyridine or fluorine-substituted benzene was synthesized. Among them the pyridine- and tetrazole-containing compound 24 has significantly improved solubility and reduced CYP3A4 inhibition compared to itraconazole. Similar to itraconazole, compound 24 inhibited the AMPK/mTOR signaling axis and the glycosylation of VEGFR2. It also induced cholesterol accumulation in the endolysosome and demonstrated binding to the sterol-sensing domain of NPC1 in a simulation study. These results suggested that compound 24 may serve as an attractive candidate for the development of a new generation of antiangiogenic drug.

Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation.

Ipomoeassin F is a potent natural cytotoxin that inhibits growth of many tumor cell lines with single-digit nanomolar potency. However, its biological and pharmacological properties have remained largely unexplored. Building upon our earlier achievements in total synthesis and medicinal chemistry, we used chemical proteomics to identify Sec61α (protein transport protein Sec61 subunit alpha isoform 1), the pore-forming subunit of the Sec61 protein translocon, as a direct binding partner of ipomoeassin F in living cells. The interaction is specific and strong enough to survive lysis conditions, enabling a biotin analogue of ipomoeassin F to pull down Sec61α from live cells, yet it is also reversible, as judged by several experiments including fluorescent streptavidin staining, delayed competition in affinity pulldown, and inhibition of TNF biogenesis after washout. Sec61α forms the central subunit of the ER protein translocation complex, and the binding of ipomoeassin F results in a substantial, yet selective, inhibition of protein translocation in vitro and a broad ranging inhibition of protein secretion in live cells. Lastly, the unique resistance profile demonstrated by specific amino acid single-point mutations in Sec61α provides compelling evidence that Sec61α is the primary molecular target of ipomoeassin F and strongly suggests that the binding of this natural product to Sec61α is distinctive. Therefore, ipomoeassin F represents the first plant-derived, carbohydrate-based member of a novel structural class that offers new opportunities to explore Sec61α function and to further investigate its potential as a therapeutic target for drug discovery.

Novel Tetrazole-Containing Analogues of Itraconazole as Potent Antiangiogenic Agents with Reduced Cytochrome P450 3A4 Inhibition.

Itraconazole has been found to possess potent antiangiogenic activity, exhibiting promising antitumor activity in several human clinical studies. The wider use of itraconazole in the treatment of cancer, however, has been limited by its potent inhibition of the drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4). In an effort to eliminate the CYP3A4 inhibition while retaining its antiangiogenic activity, we designed and synthesized a series of derivatives in which the 1,2,4-triazole ring is replaced with various azoles and nonazoles. Among these analogues, 15n with tetrazole in place of 1,2,4-triazole exhibited optimal inhibition of human umbilical vein endothelial cell proliferation with an IC50 of 73 nM without a significant effect on CYP3A4 (EC50 > 20 µM). Similar to itraconazole, 15n induced Niemann-Pick C phenotype (NPC phenotype) and blocked AMPK/mechanistic target of rapamycin signaling. These results suggest that 15n is a promising angiogenesis inhibitor that can be used in combination with most other known anticancer drugs.

Structure-activity relationship study of itraconazole, a broad-range inhibitor of picornavirus replication that targets oxysterol-binding protein (OSBP).

Itraconazole (ITZ) is a well-known, FDA-approved antifungal drug that is also in clinical trials for its anticancer activity. ITZ exerts its anticancer activity through several disparate targets and pathways. ITZ inhibits angiogenesis by hampering the functioning of the vascular endothelial growth receptor 2 (VEGFR2) and by indirectly inhibiting mTOR signaling. Furthermore, ITZ directly inhibits the growth of several types of tumor cells by antagonizing Hedgehog signaling. Recently, we reported that ITZ also has broad-spectrum antiviral activity against enteroviruses, cardioviruses and hepatitis C virus, independent of established ITZ-activities but instead via a novel target, oxysterol-binding protein (OSBP), a cellular lipid shuttling protein. In this study, we analyzed which structural features of ITZ are important for the OSBP-mediated antiviral activity. The backbone structure, consisting of five rings, and the sec-butyl chain are important for antiviral activity, whereas the triazole moiety, which is critical for antifungal activity, is not. The features required for OSBP-mediated antiviral activity of ITZ overlap mostly with published features required for inhibition of VEGFR2 trafficking, but not Hh signaling. Furthermore, we use in silico studies to explore how ITZ could bind to OSBP. Our data show that several pharmacological activities of ITZ can be uncoupled, which is a critical step in the development of ITZ-based antiviral compounds with greater specificity and reduced off-target effects.

New insights into structure-activity relationship of ipomoeassin F from its bioisosteric 5-oxa/aza analogues.

Ipomoeassin F, a plant-derived macrolide, exhibited single-digit nanomolar growth inhibition activity against many cancer cell lines. In this report, a series of 5-oxa/aza analogues was prepared and screened for cytotoxicity. Replacement of 5-CH2 with O/NH simplified the synthesis and led to only a small activity loss. N-methylation almost completely restored the potency. Further studies with additional 5-oxa analogues suggested, for the first time, that size and flexibility of the ring also significantly influence the bioactivity of ipomoeassin F.

Design, synthesis and biological evaluation of fucose-truncated monosaccharide analogues of ipomoeassin F.

Ipomoeassin F is a plant-derived macrocyclic glycolipid with single-digit nanomolar IC50 values against cancer cell growth. In previous structure-activity relationship studies, we have demonstrated that certain modifications around the fucoside moiety did not cause significant cytotoxicity loss. To further elucidate the effect of the fucoside moiety on the biological activity, we describe here the design and synthesis of several fucose-truncated monosaccharide analogues of ipomoeassin F. Subsequent biological evaluation strongly suggests that the 6-membered ring of the fucoside moiety is essential to the overall conformation of the molecule, thereby influencing bioactivity.

Synergistic Contribution of Tiglate and Cinnamate to Cytotoxicity of Ipomoeassin F.

An efficient synthetic route for ipomoeassin F and its tiglate-modified analogues was developed. The route features late-stage conformation-controlled highly regioselective esterification of the glucose diol in the disaccharide core. The results from the NCI-60 cell line screens of ipomoeassin F were reported for the first time. Moreover, two new C-3-cinnamoyl-Glcp analogues (2 and 3) were prepared. Their in-house cytotoxicity data convey an important message that both identity and positioning of the two α,ß-unsaturated esters are crucial. They are not interchangeable.

Simultaneous Targeting of NPC1 and VDAC1 by Itraconazole Leads to Synergistic Inhibition of mTOR Signaling and Angiogenesis.

The antifungal drug itraconazole was recently found to exhibit potent antiangiogenic activity and has since been repurposed as an investigational anticancer agent. Itraconazole has been shown to exert its antiangiogenic activity through inhibition of the mTOR signaling pathway, but the molecular mechanism of action was unknown. We recently identified the mitochondrial protein VDAC1 as a target of itraconazole and a mediator of its activation of AMPK, an upstream regulator of mTOR. However, VDAC1 could not account for the previously reported inhibition of cholesterol trafficking by itraconazole, which was also demonstrated to lead to mTOR inhibition. In this study, we demonstrate that cholesterol trafficking inhibition by itraconazole is due to direct inhibition of the lysosomal protein NPC1. We further map the binding site of itraconazole to the sterol-sensing domain of NPC1 using mutagenesis, competition with U18666A, and molecular docking. Finally, we demonstrate that simultaneous AMPK activation and cholesterol trafficking inhibition leads to synergistic inhibition of mTOR, endothelial cell proliferation, and angiogenesis.

Estimation of the composition of intermetallic compounds in LiCl-KCl molten salt by cyclic voltammetry.

In this work, the compositions of Ce-Al, Er-Al and La-Bi intermetallic compounds were estimated by the cyclic voltammetry (CV) technique. At first, CV measurements were carried out at different reverse potentials to study the co-reduction processes of Ce-Al, Er-Al and La-Bi systems. The CV curves obtained were then re-plotted with the current as a function of time, and the coulomb number of each peak was calculated. By comparing the coulomb number of the related peaks, the compositions of the Ce-Al, Er-Al and La-Bi intermetallic compounds formed in the co-reduction process could be estimated. The results showed that Al11Ce3, Al3Ce, Al2Ce and AlCe could be formed by the co-reduction of Ce(iii) and Al(iii). For the co-reduction of Er(iii) and Al(iii), Al3Er2, Al2Er and AlEr were formed. In a La(iii) and Bi(iii) co-existing system in LiCl-KCl melts, LaBi2, LaBi and Li3Bi were the major products as a result of co-reduction.