Tolypocladamide H and the Proposed Tolypocladamide NRPS in Tolypocladium Species.

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.

The Marine-Derived Macrolactone Mandelalide A Is an Indirect Activator of AMPK.

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.

Synthetic Access to the Mandelalide Family of Macrolides: Development of an Anion Relay Chemistry Strategy.

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.

Jizanpeptins, Cyanobacterial Protease Inhibitors from a Symploca sp. Cyanobacterium Collected in the Red Sea.

Jizanpeptins A-E (1-5) are micropeptin depsipeptides isolated from a Red Sea specimen of a Symploca sp. cyanobacterium. The planar structures of the jizanpeptins were established using NMR spectroscopy and mass spectrometry and contain 3-amino-6-hydroxy-2-piperidone (Ahp) as one of eight residues in a typical micropeptin motif, as well as a side chain terminal glyceric acid sulfate moiety. The absolute configurations of the jizanpeptins were assigned using a combination of Marfey's methodology and chiral-phase HPLC analysis of hydrolysis products compared to commercial and synthesized standards. Jizanpeptins A-E showed specific inhibition of the serine protease trypsin (IC50 = 72 nM to 1 µM) compared to chymotrypsin (IC50 = 1.4 to >10 µM) in vitro and were not overtly cytotoxic to HeLa cervical or NCI-H460 lung cancer cell lines at micromolar concentrations.

ATG5 Promotes Death Signaling in Response to the Cyclic Depsipeptides Coibamide A and Apratoxin A.

Our understanding of autophagy and lysosomal function has been greatly enhanced by the discovery of natural product structures that can serve as chemical probes to reveal new patterns of signal transduction in cells. Coibamide A is a cytotoxic marine natural product that induces mTOR-independent autophagy as an adaptive stress response that precedes cell death. Autophagy-related (ATG) protein 5 (ATG5) is required for coibamide-induced autophagy but not required for coibamide-induced apoptosis. Using wild-type and autophagy-deficient mouse embryonic fibroblasts (MEFs) we demonstrate that coibamide-induced toxicity is delayed in ATG5-/- cells relative to ATG5+/+ cells. Time-dependent changes in annexin V staining, membrane integrity, metabolic capacity and caspase activation indicated that MEFs with a functional autophagy pathway are more sensitive to coibamide A. This pattern could be distinguished from autophagy modulators that induce acute ER stress (thapsigargin, tunicamycin), ATP depletion (oligomycin A) or mTORC1 inhibition (rapamycin), but was shared with the Sec61 inhibitor apratoxin A. Coibamide- or apratoxin-induced cell stress was further distinguished from the action of thapsigargin by a pattern of early LC3-II accumulation in the absence of CHOP or BiP expression. Time-dependent changes in ATG5-ATG12, PARP1 and caspase-3 expression patterns were consistent with the conversion of ATG5 to a pro-death signal in response to both compounds.

Enantioselective Total Synthesis of Mandelalide A and Isomandelalide A: Discovery of a Cytotoxic Ring-Expanded Isomer.

The total synthesis of mandelalide A and its ring-expanded macrolide isomer isomandelalide A has been achieved. Unexpected high levels of cytotoxicity were observed with the ring-expanded isomandelalide A with a rank order of potency: mandelalide A > isomandelalide A > mandelalide B. Key aspects of the synthesis include Ag-catalyzed cyclizations (AgCC's) to construct both the THF and THP rings present in the macrocycle, diastereoselective Sharpless dihydroylation of a cis-enyne, and lithium acetylide coupling with a chiral epoxide.

Interrogating the Tailoring Steps of Pactamycin Biosynthesis and Accessing New Pactamycin Analogues.

Pactamycin is a bacteria-derived aminocyclitol antibiotic with a wide-range of biological activity. Its chemical structure and potent biological activities have made it an interesting lead compound for drug discovery and development. Despite its unusual chemical structure, many aspects of its formation in nature remain elusive. Using a combination of genetic inactivation and metabolic analysis, we investigated the tailoring processes of pactamycin biosynthesis in Streptomyces pactum. The results provide insights into the sequence of events during the tailoring steps of pactamycin biosynthesis and explain the unusual production of various pactamycin analogues by S. pactum mutants. We also identified two new pactamycin analogues that have better selectivity indexes than pactamycin against malarial parasites.

Coibamide A, a natural lariat depsipeptide, inhibits VEGFA/VEGFR2 expression and suppresses tumor growth in glioblastoma xenografts.

Coibamide A is a cytotoxic lariat depsipeptide isolated from a rare cyanobacterium found within the marine reserve of Coiba National Park, Panama. Earlier testing of coibamide A in the National Cancer Institute in vitro 60 human tumor cell line panel (NCI-60) revealed potent anti-proliferative activity and a unique selectivity profile, potentially reflecting a new target or mechanism of action. In the present study we evaluated the antitumor activity of coibamide A in several functional cell-based assays and in vivo. U87-MG and SF-295 glioblastoma cells showed reduced migratory and invasive capacity and underwent G1 cell cycle arrest as, likely indirect, consequences of treatment. Coibamide A inhibited extracellular VEGFA secreted from U87-MG glioblastoma and MDA-MB-231 breast cancer cells with low nM potency, attenuated proliferation and migration of normal human umbilical vein endothelial cells (HUVECs) and selectively decreased expression of vascular endothelial growth factor receptor 2 (VEGFR2). We report that coibamide A retains potent antitumor properties in a nude mouse xenograft model of glioblastoma; established subcutaneous U87-MG tumors failed to grow for up to 28 days in response to 0.3 mg/Kg doses of coibamide A. However, the natural product was also associated with varied patterns of weight loss and thus targeted delivery and/or medicinal chemistry approaches will almost certainly be required to improve the toxicity profile of this unusual macrocycle. Finally, similarities between coibamide A- and apratoxin A-induced changes in cell morphology, decreases in VEGFR2 expression and macroautophagy signaling in HUVECs raise the possibility that both cyanobacterial natural products share a common mechanism of action.

Personnel training and patient education in medical marijuana dispensaries in Oregon.

OBJECTIVES: To determine the knowledge and training of Oregon Medical Marijuana Dispensary (OMMD) personnel and describe the information and type of advice provided to patients who use Oregon dispensaries. METHODS: Statewide cross-sectional email survey of OMMD personnel. RESULTS: Of the 141 surveys, 47 were initiated. The most frequently referenced types of training were on-the-job training and the Internet. Dispensary personnel most commonly used patients' preferences and symptoms as well as personal experiences to determine appropriate strains for patients. The majority of respondents advised patients about precautions and expected effects. Respondents were least likely to advise on drug interactions, or recommend a patient talk to a pharmacist or prescriber. CONCLUSION: Dispensary personnel in Oregon use a variety of resources to learn about medical marijuana. Although formal health or medical training was not indicated, personnel advise on marijuana's effects, use, and product selection. Further study is needed to assess the current training and advising on patients' ability to use medical marijuana safely and effectively.

Synthesis and biological evaluation of the [d-MeAla(11)]-epimer of coibamide A.

Coibamide A is a highly potent antiproliferative cyclic depsipeptide, which was originally isolated from a Panamanian marine cyanobacterium. In this study, the synthesis of coibamide A has been investigated using Fmoc-based solid-phase peptide synthesis followed by the cleavage of the resulting linear peptide from the resin and its subsequent macrolactonization. The peptide sequence of the linear coibamide A precursor was constructed on a solid-support following the optimization of the coupling conditions, where numerous coupling agents were evaluated. The macrocyclization of the resulting linear peptide provided the [d-MeAla(11)]-epimer of coibamide A, which exhibited nanomolar cytotoxic activity towards a number of human cancer cell lines.

Depsipeptide companeramides from a Panamanian marine cyanobacterium associated with the coibamide producer.

Two new cyclic depsipeptides, companeramides A (1) and B (2), have been isolated from the phylogenetically characterized cyanobacterial collection that yielded the previously reported cancer cell toxin coibamide A (collected from Coiba Island, Panama). The planar structures of the companeramides, which contain 3-amino-2-methyl-7-octynoic acid (Amoya), hydroxy isovaleric acid (Hiva), and eight α-amino acid units, were established by NMR spectroscopy and mass spectrometry. The absolute configuration of each companeramide was assigned using a combination of Marfey's methodology and chiral-phase HPLC analysis of complete and partial hydrolysis products compared to commercial and synthesized standards. Companeramides A (1) and B (2) showed high nanomolar in vitro antiplasmodial activity but were not overtly cytotoxic to four human cancer cell lines at the doses tested.

Identification of an atypical calcium-dependent calmodulin binding site on the C-terminal domain of GluN2A.

N-methyl-D-aspartate (NMDA) receptors are calcium-permeable ion channels assembled from four subunits that each have a common membrane topology. The intracellular carboxyl terminal domain (CTD) of each subunit varies in length, is least conserved between subunits, and binds multiple intracellular proteins. We defined a region of interest in the GluN2A CTD, downstream of well-characterized membrane-proximal motifs, that shares only 29% sequence similarity with the equivalent region of GluN2B. GluN2A (amino acids 875-1029) was fused to GST and used as a bait to identify proteins from mouse brain with the potential to bind GluN2A as a function of calcium. Using mass spectrometry we identified calmodulin as a calcium-dependent GluN2A binding partner. Equilibrium fluorescence spectroscopy experiments indicate that Ca(2+)/calmodulin binds GluN2A with high affinity (5.2±2.4 nM) in vitro. Direct interaction of Ca(2+)/calmodulin with GluN2A was not affected by disruption of classic sequence motifs associated with Ca(2+)/calmodulin target recognition, but was critically dependent upon Trp-1014. These findings provide new insight into the potential of Ca(2+)/calmodulin, previously considered a GluN1-binding partner, to influence NMDA receptors by direct association.

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

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.

Apratoxin H and apratoxin A sulfoxide from the Red Sea cyanobacterium Moorea producens.

Cultivation of the marine cyanobacterium Moorea producens, collected from the Nabq Mangroves in the Gulf of Aqaba (Red Sea), led to the isolation of new apratoxin analogues apratoxin H (1) and apratoxin A sulfoxide (2), together with the known apratoxins A-C, lyngbyabellin B, and hectochlorin. The absolute configuration of these new potent cytotoxins was determined by chemical degradation, MS, NMR, and CD spectroscopy. Apratoxin H (1) contains pipecolic acid in place of the proline residue present in apratoxin A, expanding the known suite of naturally occurring analogues that display amino acid substitutions within the final module of the apratoxin biosynthetic pathway. The oxidation site of apratoxin A sulfoxide (2) was deduced from MS fragmentation patterns and IR data, and 2 could not be generated experimentally by oxidation of apratoxin A. The cytotoxicity of 1 and 2 to human NCI-H460 lung cancer cells (IC50 = 3.4 and 89.9 nM, respectively) provides further insight into the structure-activity relationships in the apratoxin series. Phylogenetic analysis of the apratoxin-producing cyanobacterial strains belonging to the genus Moorea, coupled with the recently annotated apratoxin biosynthetic pathway, supports the notion that apratoxin production and structural diversity may be specific to their geographical niche.

Design of Synthetic Surrogates for the Macrolactone Linker Motif in Coibamide A.

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 ß- 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 ß-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.

Mandelalides A-D, cytotoxic macrolides from a new Lissoclinum species of South African tunicate.

Mandelalides A-D are variously glycosylated, unusual polyketide macrolides isolated from a new species of Lissoclinum ascidian collected from South Africa, Algoa Bay near Port Elizabeth and the surrounding Nelson Mandela Metropole. Their planar structures were elucidated on submilligram samples by comprehensive analysis of 1D and 2D NMR data, supported by mass spectrometry. The assignment of relative configuration was accomplished by consideration of homonuclear and heteronuclear coupling constants in tandem with ROESY data. The absolute configuration was assigned for mandelalide A after chiral GC-MS analysis of the hydrolyzed monosaccharide (2-O-methyl-α-L-rhamnose) and consideration of ROESY correlations between the monosaccharide and aglycone in the intact natural product. The resultant absolute configuration of the mandelalide A macrolide was extrapolated to propose the absolute configurations of mandelalides B-D. Remarkably, mandelalide B contained the C-4' epimeric 2-O-methyl-6-dehydro-α-L-talose. Mandelalides A and B showed potent cytotoxicity to human NCI-H460 lung cancer cells (IC(50), 12 and 44 nM, respectively) and mouse Neuro-2A neuroblastoma cells (IC(50), 29 and 84 nM, respectively).

Canine osteosarcoma cells exhibit basal accumulation of multiple chaperone proteins and are sensitive to small molecule inhibitors of GRP78 and heat shock protein function.

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.

Cyclic depsipeptides, grassypeptolides D and E and Ibu-epidemethoxylyngbyastatin 3, from a Red Sea Leptolyngbya cyanobacterium.

Two new grassypeptolides and a lyngbyastatin analogue, together with the known dolastatin 12, have been isolated from field collections and laboratory cultures of the marine cyanobacterium Leptolyngbya sp. collected from the SS Thistlegorm shipwreck in the Red Sea. The overall stereostructures of grassypeptolides D (1) and E (2) and Ibu-epidemethoxylyngbyastatin 3 (3) were determined by a combination of 1D and 2D NMR experiments, MS analysis, Marfey's methodology, and HPLC-MS. Compounds 1 and 2 contain 2-methyl-3-aminobutyric acid and 2-aminobutyric acid, while biosynthetically distinct 3 contains 3-amino-2-methylhexanoic acid and the ß-keto amino acid 4-amino-2,2-dimethyl-3-oxopentanoic acid (Ibu). Grassypeptolides D (1) and E (2) showed significant cytotoxicity to HeLa (IC50 = 335 and 192 nM, respectively) and mouse neuro-2a blastoma cells (IC50 = 599 and 407 nM, respectively), in contrast to Ibu-epidemethoxylyngbyastatin 3 (neuro-2a cells, IC50 > 10 µM) and dolastatin 12 (neuro-2a cells, IC50 > 1 µM).

Targeting of HER/ErbB family proteins using broad spectrum Sec61 inhibitors coibamide A and apratoxin A.

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 A- or 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. Time- and 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.

MDR1 function is sensitive to the phosphorylation state of myosin regulatory light chain.

Multiple drug resistance protein 1 (MDR1) is composed of two homologous halves separated by an intracellular linker region. The linker has been reported to bind myosin regulatory light chain (RLC), but it is not clear how this can occur in the context of a myosin II complex. We characterized MDR1-RLC interactions and determined that binding occurs via the amino terminal of the RLC, a domain that typically binds myosin heavy chain. MDR1-RLC interactions were sensitive to the phosphorylation state of the light chain in that phosphorylation by myosin light chain kinase (MLCK) resulted in a loss of binding in vitro. We used ML-7, a specific inhibitor of MLCK, to study the functional consequences of disrupting RLC phosphorylation in intact cells. Pretreatment of polarized Madin-Darby canine kidney cells stably expressing MDR1 with ML-7 produced a significant increase in apical to basal permeability and a corresponding decrease in the efflux ratio (threefold; p<0.01) of [(3)H]-digoxin, a classic MDR1 substrate. Together these data show that MDR1-mediated transport of [(3)H]-digoxin can be modulated by pharmacological manipulation of myosin RLC, but direct MDR1-RLC interactions are atypical and not explained by the structure of the myosin II holoenzyme.