The endoplasmic reticulum (ER) to Golgi secretory compartment of eukaryotic cells is highly sensitive to changes in intracellular homeostasis. Using a primary screening assay that monitors the function of this pathway, we prioritized a cyanobacterial extract from the Red Sea that decreased secretion of a bioluminescent reporter, Gaussia luciferase (GLuc), in living cells. A comparison of LCMS2 data against the GNPS database revealed a match for macrocyclic depsipeptide hoiamide A (1). Biological testing confirmed the ability of 1 to induce a mixed, non-lethal stress response in human U87-MG glioblastoma cells; analysis of stress markers by qRT-PCR revealed early upregulation of superoxide dismutase 1 (SOD1) and C/EBP-homologous protein (CHOP) relative to the control. Co-treatment of cells with 1 (100 nM to 3 μM) and antioxidant N-acetylcysteine afforded full protection from 1-induced decreases in GLuc secretion. We report that terminally differentiated human SH-SY5Y neuroblastoma cells, with a neuron-like phenotype, are highly sensitive to nanomolar concentrations of 1, whereas undifferentiated cells remained viable at 3 μM. These results expand the known biology of hoiamides and suggest that neurotoxic potential of 1 is likely due to an inherent failure of neurons to adapt to the loss of redox homeostasis and sustained ER stress.

Paraisariamides (Psds) are a family of fungal N-methylated cyclic heptapeptides with structural similarity to the translation eukaryotic elongation factor 1A inhibitor ternatin. Initial characterization of the eight known Psds A-H revealed a complex biological activity profile; Psd A and Psds E-H showed differential cytotoxicity to HCT116 colon cancer cells, whereas Psds B-D were inactive. Here, we report that Psd A is a weak inhibitor of total protein synthesis and that three synthetic diastereomers of Psd A retain comparable pharmacological activity. Psd A and all structural variants decreased the level of expression of extracellular Gaussia luciferase in a phenotypic assay that senses endoplasmic reticulum stress in living cells. Inhibition of cellular secretion was fully reversible; however, the three Psd A diastereomers were all eventually cytotoxic to HCT116 colon cancer cells. The stereochemistry of Psd A, a weak cytotoxin relative to Psds E-H, is anticipated to inform future structure-activity relationship studies of these ternatin-like molecules.

Paraisariamides (Psds) are a family of fungal N-methylated cyclic heptapeptides with structural similarity to the translation eukaryotic elongation factor 1A inhibitor ternatin. Initial characterization of the eight known Psds A-H revealed a complex biological activity profile; Psd A and Psds E-H showed differential cytotoxicity to HCT116 colon cancer cells, whereas Psds B-D were inactive. Here, we report that Psd A is a weak inhibitor of total protein synthesis and that three synthetic diastereomers of Psd A retain comparable pharmacological activity. Psd A and all structural variants decreased the level of expression of extracellular luciferase in a phenotypic assay that senses endoplasmic reticulum stress in living cells. Inhibition of cellular secretion was fully reversible; however, the three Psd A diastereomers were all eventually cytotoxic to HCT116 colon cancer cells. The stereochemistry of Psd A, a weak cytotoxin relative to Psds E-H, is anticipated to inform future structure-activity relationship studies of these ternatin-like molecules.

Insect-associated fungi are prolific producers of peptidic natural products with potent biological activities. Paraisaria is a genus of insect-pathogenic fungi (Family Ophiocordycipitaceae) in the Order Hypocreales, which also includes other Cordyceps-like fungi. Such fungi are increasingly used as functional foods and nutraceuticals, but Paraisaria may also be unintentionally consumed through infected, potentially toxic, edible insects. Here we report the discovery, isolation, and structure elucidation of paraisariamides A-D (1-4) from Paraisaria cascadensis and paraisariamides E-H (5-8) from P. insignis. Total synthesis of 1 and 5-8 was instrumental for comprehensive structure assignment and provided pure compounds for advanced biological testing. The paraisariamide family of N-methylated cyclic heptapeptides has been detected in all Paraisaria specimens analyzed to date from a variety of ecological niches. Paraisariamides A-H (1-8) display differential cell-type specific toxicity to human cancer cells, and we demonstrate that the most cytotoxic paraisariamides E-H (5-8) potently and rapidly inhibit mammalian protein synthesis. Molecular cartography was used to visualize the spatial distribution of paraisariamides in a lyophilized specimen of a beetle larva parasitized by P. insignis. Localization of paraisariamides to the fungal endosclerotium within the host larva is consistent with a role in localized disruption of host protein synthesis.

An investigation of living phosphatic stromatolites from Schoenmakerskop barrage pool near Gqeberha (Port Elizabeth), South Africa, yielded new cyclic octadepsipeptides, amatyemides A (1) and B (2), named using the Xhosa word 'amatye' for 'rock'. The amatyemides were isolated from methanol extracts of a targeted stromatolite sample collection, following an initial metabolomic survey of the Schoenmakerskop pool. Planar structure elucidation of 1 and 2 relied on NMR and LCMS2 data, which delineated the same six amino acids and one 2-hydroxy-3-methylpentanoic acid (Hmpa) residues in each compound. The two octadepsipeptides differed only in the presence of a 2-hydroxydodecanoic acid (Hdda) residue in 1 and a 2-hydroxydecanoic acid (Hda) residue in 2. The absolute configurations of most amino acid residues in 1 were determined using an enhanced Marfey's reagent. The configurations of the 2-hydroxy acids and O-methylthreonine were assigned, and the absolute structures of amatyemides A (1) and B (2) were confirmed, by total solid-phase peptide synthesis of two possible diastereomers for each natural product. Biological testing of natural and synthetic amatyemides against human U87-MG glioblastoma, HCT116 colon, and SH-SY5Y neuroblastoma cells revealed weak, cell-type specific, cytotoxic potential where 2 > 1, and this was attributed to induction of oxidative stress by 2.

Coibamide A (CbA) is a cyanobacterial lariat depsipeptide that selectively inhibits multiple secreted and integral membrane proteins from entering the endoplasmic reticulum secretory pathway through binding the alpha subunit of the Sec61 translocon. As a complex peptide-based macrocycle with 13 stereogenic centers, CbA is presumed to adopt a conformationally restricted orientation in the ligand-bound state, resulting in potent antitumor and antiangiogenic bioactivity. A stereochemical structure-activity relationship for CbA was previously defined based on cytotoxicity against established cancer cell lines. However, the ability of synthetic isomers to inhibit the biosynthesis of specific Sec61 substrates was unknown. Here, we report that two less toxic diastereomers of CbA, [L-Hiv2]-CbA and [L-Hiv2, L-MeAla11]-CbA, are pharmacologically active Sec61 inhibitors. Both compounds inhibited the expression of a secreted reporter (Gaussia luciferase), VEGF-A, and a Type 1 membrane protein (VCAM1), while [L-Hiv2]-CbA also decreased the expression of ICAM1 and BiP/GRP78. Analysis of 43 different chemokines in the secretome of SF-268 glioblastoma cells revealed different inhibitory profiles for the two diastereomers. When the cytotoxic potential of CbA compounds was compared against a panel of patient-derived glioblastoma stem-like cells (GSCs), Sec61 inhibitors were remarkably toxic to five of the six GSCs tested. Each ligand showed a distinct cytotoxic potency and selectivity pattern for CbA-sensitive GSCs, with IC50 values ranging from subnanomolar to low micromolar concentrations. Together, these findings highlight the extreme sensitivity of GSCs to Sec61 modulation and the importance of ligand stereochemistry in determining the spectrum of inhibited Sec61 client proteins.

A marine cyanobacterial cyclic depsipeptide, coibamide A (CbA), inhibits the mammalian protein secretory pathway by blocking the Sec61 translocon, which is an emerging drug target for cancer and other chronic diseases. In our previous structure–activity relationship study of CbA, the macrolactone ester linker was replaced with alkyl/alkenyl surrogates to provide synthetically accessible macrocyclic scaffolds. To optimize the cellular bioactivity profile of CbA analogues, novel lysine mimetics having β- and ε-methyl groups have now been designed and synthesized by a stereoselective route. A significant increase in cytotoxicity was observed upon introduction of these two methyl groups, corresponding to the d-MeAla α-methyl and MeThr β-methyl of CbA. All synthetic products retained the ability to inhibit secretion of a model Sec61 substrate. Tandem evaluation of secretory function inhibition in living cells and cytotoxicity was an effective strategy to assess the impact of structural modifications to the linker for ring closure.

Coibamide A is a cyclopeptide natural product with potent antiproliferative activity against cultured human cancer cells and subcutaneous U87-MG glioblastoma tumors in mice. We have recently determined that coibamide A binds to the Sec61 translocon channel at a site that is close, but not identical, to the binding site of the peptide/polyketide natural product apratoxin A. As the majority of secreted proteins in mammalian cells are trafficked through the conventional secretory pathway via the Sec61 translocon, this raised the possibility that coibamide A and apratoxin A block import and secretion of different Sec61 client proteins. In the present study, we analyzed and compared the secretome of human U87-MG glioblastoma cells in the presence and absence of both marine natural products. U87-MG glioblastoma cells were treated with coibamide A (5 nM), apratoxin A (5 nM), or vehicle for 24 h. The growth media was then replaced with serum-free media for a further 24 h. Tryptic proteomic samples were generated from cell-free conditioned media and analyzed on a LC-coupled Orbitrap Fusion mass spectrometer. The high-resolution Orbitrap mass analyzer was used to both identify precursor ions for tandem mass (MS2) analysis in the ion trap mass analyzer and to quantitate those ions (MS1). Secreted proteins were identified from peptide–spectrum matches to the human protein database using a decoy database to assign an FDR and quantified from their MS1 ion intensities. Comparison of mass spectrometric data against the human secretome database revealed 175 proteins in the U87-MG cell secretome that were identified with high confidence. Differences between coibamide A and apratoxin A treatment were not significant, however both Sec61 inhibitors induced significant suppression of the U87-MG secretome relative to vehicle-treated cells. Treatment with coibamide A or apratoxin A resulted in the reduction of 75% of quantifiable secretome (131 proteins). Of the remaining 44 proteins, 34 are directed to intracellular organelles or contain a motif associated with retention in the endoplasmic reticulum and 10 were not suppressed with treatment but were hypothesized to be Sec61 clients. Malignant glioblastoma cells secrete proteins into the extracellular space and use complex intercellular signaling mechanisms for a survival advantage. Our results indicate that broad-acting inhibitors of Sec61-dependent co-translational translocation block the progression of a significant percentage of the U87-MG glioblastoma cell secretome including several proteins that have previously been detected as biomarkers in the cerebrospinal fluid, blood, or urine of brain tumor patients. Taken together, our work illustrates the feasibility of inducing a reversible suppression of numerous (undrugged) extracellular signals by targeting Sec61-dependent protein biogenesis in the secretory pathway with drug-like molecules. Citation Format: Daphne R. Mattos, Jeffrey D. Serrill, Philip R. Gafken, Kerry L. McPhail, Walter K. Vogel, Jane E. Ishmael. Cyanobacterial natural product Sec61α inhibitors induce broad suppression of the glioma cell secretome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3916.

Introduction: The 2018 Farm Bill changed the legal status of cannabis plants that meet the definition of industrial hemp and allowed for the rapid expansion of hemp-based products into commercial spaces. With an emphasis on industrial hemp as the source of naturally-occurring cannabinoid compounds, a niche market for cannabidiol (CBD)-containing products was quickly established in pharmacies and grocery stores. Although the U.S. Food and Drug Administration (FDA) has retained oversight of all cannabis-based products, labelling of hemp-derived products for retail markets remains largely unregulated. Under federal law, CBD cannot be added to foods, beverages, sold as a dietary supplement or marketed for a therapeutic benefit, however the perceived health benefits of CBD as an acceptable and safe ingredient contribute to the growing market for these health products. Objective: The objective of this study was to evaluate the range of over-the-counter (OTC) hemp- and CBD-based products available to consumers and determine the prevalence of other pharmacologically active agents identified as ingredients in these products. Labels were scored for the presence of: active and inactive ingredients, percent CBD, full-spectrum hemp, full-spectrum CBD or CBD isolate. Methods: Two large pharmacy chains and one medium-sized grocery store located in the Pacific Northwest were surveyed between May 2020 and February 2021 and OTC hemp-derived products on display were recorded. Identification of pharmacologically active ingredients on the product label was validated using the National Medicines Comprehensive Database. Products that were noted to have CBD or hemp ingredients were included in the study, while any products that did not accurately report the amount of CBD in the product were excluded. Products that did not list the total weight of the product were excluded from the analysis. Results: Thirty-three unique products were recorded from 19 different manufacturers. 39% of product labels indicated the presence of Full-Spectrum Hemp as part of the base product, while 66% of products listed Hemp Extract as the base product. Text labels on CBD-containing products, on average, indicated more than 3 additional pharmacologically active ingredients were contained in each product. Topical CBD products were more likely to have other ingredients such as arnica montana, menthol and camphor, whereas products for oral ingestion were more likely to have only CBD as the primary active ingredient. Text labels on 52% of topical CBD products listed the presence of 10% menthol. Average concentrations of CBD in OTC products was found to be 1.12% ± 1.48 %, based on dry weight. Conclusion: Product text labels on OTC hemp-and CBD-containing preparations is varied and presented in a non-standardized format. Topical CBD products were more likely to contain other pharmacologically active natural products that can be used for the treatment of pain symptoms. Concentrations of arnica montana, menthol and camphor were as much as 10-fold higher than the proportion of CBD contained in these products. The absence of a standard format for labelling of OTC hemp- and CBD-derived products and the frequent presence of other active ingredients has the potential to create confusion and risk for the consumer.

The mandelalides are complex macrolactone natural products with distinct macrocycle motifs and a bioactivity profile that is heavily influenced by compound glycosylation. Mandelalides A and B are direct inhibitors of mitochondrial ATP synthase (complex V) and therefore more toxic to mammalian cells with an oxidative metabolic phenotype. To provide further insight into the pharmacology of the mandelalides, we studied the AMP-activated protein kinase (AMPK) energy stress pathway and report that mandelalide A is an indirect activator of AMPK. Wild-type mouse embryonic fibroblasts (MEFs) and representative human non-small cell lung cancer (NSCLC) cells showed statistically significant increases in phospho-AMPK (Thr172) and phospho-ACC (Ser79) in response to mandelalide A. Mandelalide L, which also harbors an A-type macrocycle, induced similar increases in phospho-AMPK (Thr172) and phospho-ACC (Ser79) in U87-MG glioblastoma cells. In contrast, MEFs co-treated with an AMPK inhibitor (dorsomorphin), AMPKα-null MEFs, or NSCLC cells lacking liver kinase B1 (LKB1) lacked this activity. Mandelalide A was significantly more cytotoxic to AMPKα-null MEFs than wild-type cells, suggesting that AMPK activation serves as a protective response to mandelalide-induced depletion of cellular ATP. However, LKB1 status alone was not predictive of the antiproliferative effects of mandelalide A against NSCLC cells. When EGFR status was considered, erlotinib and mandelalide A showed strong cytotoxic synergy in combination against erlotinib-resistant 11-18 NSCLC cells but not against erlotinib-sensitive PC-9 cells. Finally, prolonged exposures rendered mandelalide A, a potent and efficacious cytotoxin, against a panel of human glioblastoma cell types regardless of the underlying metabolic phenotype of the cell. These results add biological relevance to the mandelalide series and provide the basis for their further pre-clinical evaluation as ATP synthase inhibitors and secondary activators of AMPK.

The genome of entomopathogenic fungus Tolypocladium inflatum Gams encodes 43 putative biosynthetic gene clusters for specialized metabolites, although genotype-phenotype linkages have been reported only for the cyclosporins and fumonisins. T. inflatum was cultured in defined minimal media, supplemented with or without one of nine different amino acids. Acquisition of LC-MS/MS data for molecular networking and manual analysis facilitated annotation of putative known and unknown metabolites. These data led us to target a family of peptaibols and guided the isolation and purification of tolypocladamide H (1), which showed modest antibacterial activity and toxicity to mammalian cells at micromolar concentrations. HRMS/MS, NMR, and advanced Marfey's analysis were used to assign the structure of 1 as a peptaibol containing 4-[(E)-2-butenyl]-4-methyl-l-threonine (Bmt), a hallmark structural motif of the cyclosporins. LC-MS detection of homologous tolypocladamide metabolites and phylogenomic analyses of peptaibol biosynthetic genes in other cultured Tolypocladium species allowed assignment of a putative tolypocladamide nonribosomal peptide synthetase gene.

Osteosarcoma is the most common type of bone cancer in dogs and humans, with significant numbers of patients experiencing treatment failure and disease progression. In our search for new approaches to treat osteosarcoma, we previously detected multiple chaperone proteins in the surface-exposed proteome of canine osteosarcoma cells. In the present study, we characterized expression of representative chaperones and find evidence for stress adaptation in canine osteosarcoma cells relative to osteogenic progenitors from normal bone. We compared the cytotoxic potential of direct (HA15) and putative (OSU-03012) inhibitors of Grp78 function and found canine POS and HMPOS osteosarcoma cells to be more sensitive to both compounds than normal cells. HA15 and OSU-03012 increased the thermal stability of Grp78 in intact POS cells at low micromolar concentrations, but each induced distinct patterns in Grp78 expression without significant change in Grp94. Both inhibitors were as effective alone as carboplatin and showed little evidence of synergy in combination treatment. However, HMPOS cells with acquired resistance to carboplatin were sensitive to inhibition of Grp78 (by HA15; OSU-03012), Hsp70 (by VER-155008), and Hsp90 (by 17-AAG) function. These results suggest that multiple nodes within the osteosarcoma chaperome may be relevant chemotherapeutic targets against platinum resistance.

Coibamide A is a potent cancer cell toxin and one of a select group of natural products that inhibit protein entry into the secretory pathway via a direct inhibition of the Sec61 protein translocon. Many Sec61 client proteins are clinically relevant drug targets once trafficked to their final destination in or outside the cell, however the use of Sec61 inhibitors to block early biosynthesis of specific proteins is at a pre-clinical stage. In the present study we evaluated the action of coibamide A against human epidermal growth factor receptor (HER, ErbB) proteins in representative breast and lung cancer cell types. HERs were selected for this study as they represent a family of Sec61 clients that is frequently dysregulated in human cancers, including coibamide-sensitive cell types. Although coibamide A inhibits biogenesis of a broad range of Sec61 substrate proteins in a presumed substrate nonselective manner, endogenous HER3 (ErbB-3) and EGFR (ErbB-1) proteins were more sensitive to coibamide A, and the related Sec61 inhibitor apratoxin A, than HER2 (ErbB-2). Despite this rank order of sensitivity (HER3 > EGFR > HER2), Sec61-dependent inhibition by coibamide A was sufficient to decrease cell surface expression of HER2. We report that coibamide Aor apratoxin A-mediated block of HER3 entry into the secretory pathway is unlikely to be mediated by the HER3 signal peptide alone. HER3 (G11L/S15L), that is fully resistant to the highly substrate-selective cotransin analogue CT8, was more resistant than wild-type HER3 but only at low coibamide A (3 nM) concentrations; HER3 (G11L/S15L) expression was inhibited by higher concentrations of either natural product. Timeand concentration-dependent decreases in HER protein expression induced a commensurate reduction in AKT/MAPK signaling in breast and lung cancer cell types and loss in cell viability. Coibamide A potentiated the cytotoxic efficacy of small molecule kinase inhibitors lapatinib and erlotinib in breast and lung cancer cell types, respectively. These data indicate that natural product modulators of Sec61 function have value as chemical probes to interrogate HER/ErbB signaling in treatment-resistant human cancers.

Coibamide A (CbA) is a marine natural product with potent antiproliferative activity against human cancer cells and a unique selectivity profile. Despite promising antitumor activity, the mechanism of cytotoxicity and specific cellular target remain unknown. Here, we develop an optimized synthetic CbA photoaffinity probe (photo-CbA) and use it to demonstrate that CbA directly targets the Sec61α subunit of the trimeric Sec61 translocon. CbA binding to Sec61 results in broad substrate-nonselective inhibition of ER protein import and potent cytotoxicity against specific cancer cell lines. CbA targets a lumenal cavity of Sec61α that is partially shared with known Sec61 inhibitors, yet profiling against resistance conferring Sec61α mutations identified from human HCT116 cells suggests a distinct binding mode for CbA. Specifically, despite conferring strong resistance to all previously known Sec61 inhibitors, the Sec61α mutant R66I remains sensitive to CbA. A further unbiased screen for Sec61α resistance mutations identified the CbA-resistant mutation S71P, which confirms non-identical binding sites for CbA and apratoxin A and supports the susceptibility of the Sec61 plug region for channel inhibition. Remarkably, CbA, apratoxin A andipomoeassin F do not display comparable patterns of potency and selectivity in the NCI60 panel of human cancer cell lines.Our work connecting CbA activity with selective prevention of secretory and membrane protein biogenesis by inhibition of Sec61 opens up possibilities for developing new Sec61 inhibitors with improved drug-like properties that are based on the coibamide pharmacophore.

Coibamide A, is a rare cytotoxic N-methyl-stabilized cyclopeptide originally isolated from a cyanobacterium growing within the marine reserve of Coiba National Park, Panama. We have recently determined that this natural product targets the co-translational translocation machinery and potently inhibits expression of secreted, resident endoplasmic reticulum (ER) and membrane-bound proteins. This mechanism of coibamide A action leads to a pattern of cellular consequences including inhibition of glucose regulated protein 78 (GRP78) expression and a specific pattern of cell stress and death signaling in cultured glioblastoma cells. Exposure to nanomolar concentrations of coibamide A, or the related cyclic depsipeptide natural product apratoxin A, promotes proteasomal degradation of GRP78 and a compensatory upregulation of cytosolic heat shock proteins 40 and 70 that precedes cell death. Co-translational translocation is mediated by the Sec61 translocation channel, which comprises a conserved hetero-oligomeric protein composed of a main pore-forming Sec61alpha subunit plus beta and gamma subunits. Sec61 also signals directly with the protein folding machinery of the ER lumen to maintain proteostasis. The Sec61 translocon channel is not a direct target of any currently approved or experimental drug, however, several natural product structures are now known to target the Sec61 channel and inhibit the co-translational translocation process. Pharmacological inhibitors of Sec61 may be valuable tools to probe GBM biology as SEC61gamma has previously been identified as a proto-oncogene, and Sec61gamma overexpression reported in high versus low grade glioma or normal astrocytes. Natural products have historically been important sources of new chemical structures, particularly for cancer and infectious disease, rather than providing the final drug entity. Although preliminary, the discovery of natural product ligands to target the ER translocation machinery reveals a distinct mechanism to perturb proteostasis in aggressive CNS cancers characterized by high expression of GRP78 and therapeutic resistance.

The mandelalides comprise a family of structurally complex marine macrolides that display significant cytotoxicity against several human cancer cell lines. Presented here is a full account on the development of an Anion Relay Chemistry (ARC) strategy for the total synthesis of (-)-mandelalides A and L, the two most potent members of the mandelalide family. The design and implementation of a three-component type II ARC/cross-coupling protocol and a four-component type I ARC union permits rapid access respectively to the key tetrahydrofuran and tetrahydropyran structural motifs of these natural products. Other highlights of the synthesis include an osmium-catalyzed oxidative cyclization of an allylic 1,3-diol, a mild Yamaguchi esterification to unite the northern and southern hemispheres, and a late-stage Heck macrocyclization. Synthetic mandelalides A and L displayed potent cytotoxicity against human HeLa cervical cancer cells (EC50, 1.3 and 3.1 nM, respectively). This synthetic approach also provides access to several highly potent non-natural mandelalide analogs, including a biotin-tagged mandelalide probe for future biological investigation.