New Micropeptins with Anti-Neuroinflammatory Activity Isolated from a Cyanobacterial Bloom.

Metabolite mining of environmentally collected aquatic and marine microbiomes offers a platform for the discovery of new therapeutic lead molecules. Combining a prefractionated chromatography library with liquid chromatography tandem mass spectrometry (LC-MS/MS)-based molecular networking and biological assays, we isolated and characterized two new micropeptins ( and ) along with the previously characterized micropeptin 996. These metabolites showed potency in anti-neuroinflammatory assays using BV-2 mouse microglial cells, showing a 50% reduction in inflammation in a range from 1 to 10 μM.

Novel PIKK inhibitor antibody-drug conjugates: Synthesis and anti-tumor activity.

Novel neolymphostin-based antibody-drug conjugate (ADC) precursors were synthesized either through amide couplings between both cleavable and non-cleavable linkers and neolymphostin derivatives, or through Cu(I)-catalyzed acetylene-azide click cycloadditon between non-cleavable linkers and neolymphostin acetal derivatives. These precursors were site-specifically conjugated to cysteine mutant trastuzumab-A114C to provide neolymphostin-based ADCs. Preliminary in vitro data indicated that the corresponding ADCs were active against HER2-expressing tumor cell lines, thus providing a proof-of-concept for using neolymphostin as ADC-based anticancer agents.

Cyanobufalins: Cardioactive Toxins from Cyanobacterial Blooms.

Cyanobufalins A-C (1-3), a new series of cardiotoxic steroids, have been discovered from cyanobacterial blooms in Buckeye Lake and Grand Lake St. Marys in Ohio. Compounds 1-3 contain distinctive structural features, including geminal methyl groups at C-4, a 7,8 double bond, and a C-16 chlorine substituent that distinguish them from plant- or animal-derived congeners.

Microcystins Containing Doubly Homologated Tyrosine Residues from a Microcystis aeruginosa Bloom: Structures and Cytotoxicity.

Four new microcystin congeners are described including the first three examples of microcystins containing the rare doubly homologated tyrosine residue 2-amino-5-(4-hydroxyphenyl)pentanoic acid (Ahppa) (1-4). Large-scale harvesting and biomass processing allowed the isolation of substantial quantities of these compounds, thus enabling complete structure determination by NMR as well as cytotoxicity evaluation against selected cancer cell lines. The new Ahppa-toxins all incorporate Ahppa residues at the 2-position, and one of these also has a second Ahppa at position 4.

Multivalent peptidic linker enables identification of preferred sites of conjugation for a potent thialanstatin antibody drug conjugate.

Antibody drug conjugates (ADCs) are no longer an unknown entity in the field of cancer therapy with the success of marketed ADCs like ADCETRIS and KADCYLA and numerous others advancing through clinical trials. The pursuit of novel cytotoxic payloads beyond the mictotubule inhibitors and DNA damaging agents has led us to the recent discovery of an mRNA splicing inhibitor, thailanstatin, as a potent ADC payload. In our previous work, we observed that the potency of this payload was uniquely tied to the method of conjugation, with lysine conjugates showing much superior potency as compared to cysteine conjugates.

Trichophycin A, a Cytotoxic Linear Polyketide Isolated from a Trichodesmium thiebautii Bloom.

In an effort to isolate and characterize bioactive secondary metabolites from blooms, collected cyanobacteria biomass was subjected to bioassay-guided extraction and fractionation using the human colon cancer cell line HCT-116, resulting in the isolation and subsequent structure characterization of a linear polyketide trichophycin A (). The planar structure of was completed using 1D and 2D NMR spectroscopy and high-resolution electrospray ionization mass spectrometry (HRESIMS). Trichophycin A was moderately toxic against the murine neuroblastoma cell line Neuro-2A (EC: 6.

Structure revision of trichotoxin, a chlorinated polyketide isolated from a bloom.

NMR-guided fractionation of the lipophilic extract of filaments led to the isolation of a phenyl-containing chlorinated polyketide () and an alkyne-containing analogue (). Comparison of spectroscopic and spectrometric data of with the data of the previously reported trichotoxin, strongly suggested that these metabolites were identical and supports a structural revision of trichotoxin and its designation as trichotoxin A. In addition, we report the isolation and characterization of the alkyne-containing analogue trichotoxin B ().

Discovery of anabaenopeptin 679 from freshwater algal bloom material: Insights into the structure-activity relationship of anabaenopeptin protease inhibitors.

Cyanobacteria possess a unique capacity for the production of structurally novel secondary metabolites compared to the biosynthetic abilities of other environmental prokaryotes such as bacteria of the genus Streptomyces. Two different strategies to explore cyanobacteria-derived natural products have been explored previously: (1) cultivation of single cyanobacterial strains, in bioreactors for example; (2) bulk collections from the environment of so called 'algal blooms' that are dominated by cyanobacteria. In this study a new environmentally friendly collection technique for obtaining large quantities of algal bloom biomass was utilized.

Natural Product Splicing Inhibitors: A New Class of Antibody-Drug Conjugate (ADC) Payloads.

There is a considerable ongoing work to identify new cytotoxic payloads that are appropriate for antibody-based delivery, acting via mechanisms beyond DNA damage and microtubule disruption, highlighting their importance to the field of cancer therapeutics. New modes of action will allow a more diverse set of tumor types to be targeted and will allow for possible mechanisms to evade the drug resistance that will invariably develop to existing payloads. Spliceosome inhibitors are known to be potent antiproliferative agents capable of targeting both actively dividing and quiescent cells.

Isolation of pyrrolocins A-C: cis- and trans-decalin tetramic acid antibiotics from an endophytic fungal-derived pathway.

Three new decalin-type tetramic acid analogues, pyrrolocins A (1), B (2), and C (3), were defined as products of a metabolic pathway from a fern endophyte, NRRL 50135, from Papua New Guinea. NRRL 50135 initially produced 1 but ceased its production before chemical or biological evaluation could be completed. Upon transfer of the biosynthetic pathway to a model host, 1-3 were produced.

Cytotoxic Spliceostatins from Burkholderia sp. and Their Semisynthetic Analogues.

The spliceostatin class of natural products was reported to be potent cytotoxic agents via inhibition of the spliceosome, a key protein complex in the biosynthesis of mature mRNA. As part of an effort to discover novel leads for cancer chemotherapy, we re-examined this class of compounds from several angles, including fermentation of the producing strains, isolation and structure determination of new analogues, and semisynthetic modification. Accordingly, a group of spliceostatins were isolated from a culture broth of Burkholderia sp.

Total synthesis and stereochemistry revision of mannopeptimycin aglycone.

Development of efficient methods for preparation of bioactive nonribosomal peptides, containing densely functionalized nonproteinogenic amino acids, is an important task in organic synthesis. We have employed a concise synthesis for such amino acids by asymmetric aldol addition coupled with an isomeric resolution via diastereoselective cyclization. This approach is successfully applied to the first total synthesis of the cyclic hexapeptide aglycone of the mannopeptimycins, a group of glycopeptides known for potent activity against drug-resistant bacteria.

Discovery of the lomaiviticin biosynthetic gene cluster in .

The lomaiviticins are a family of cytotoxic marine natural products that have captured the attention of both synthetic and biological chemists due to their intricate molecular scaffolds and potent biological activities. Here we describe the identification of the gene cluster responsible for lomaiviticin biosynthesis in strains DPJ-0016 and DPJ-0019 using a combination of molecular approaches and genome sequencing. The link between the gene cluster and lomaiviticin production was confirmed using bacterial genetics, and subsequent analysis and annotation of this cluster revealed the biosynthetic basis for the core polyketide scaffold.

Probing natural product biosynthetic pathways using Fourier transform ion cyclotron resonance mass spectrometry.

Two natural products, diazepinomicin (1) and dioxapyrrolomycin (2), containing stable isotopic labels of (15)N or deuterium, were used to demonstrate the utility of Fourier transform ion cyclotron resonance mass spectrometry for probing natural product biosynthetic pathways. The isotopic fine structures of significant ions were resolved and subsequently assigned elemental compositions on the basis of highly accurate mass measurements. In most instances the mass measurement accuracy is less than one part per million (ppm), which typically makes the identification of stable-isotope labeling unambiguous.

Investigating the biosynthetic origin of the nitro group in pyrrolomycins.

Feasible modes of introducing the nitro group into pyrrolomycin antibiotics were investigated based on incorporation of (15)N-labeled arginine and proline into dioxapyrrolomycin, produced by the actinomycete culture LL-F42248. Biosynthesis of nitrated pyrrolomycins was unaffected by the presence of nitric oxide synthase (NOS) inhibitors. The culture was able to grow in nitrogen-free (minimal) media and produce nitrated secondary metabolites.

Reassessing the structure of pyranonigrin.

Fermentation extracts of the marine fungus Aspergillus niger LL-LV3020 were found to have relevant activity in a number of assays. Chemical screening of the extracts revealed that this organism produced numerous secondary metabolites in addition to its principal metabolite, citric acid. The compound with the most significant UV peak was isolated and its structure elucidated.

Culicinin D, an antitumor peptaibol produced by the fungus Culicinomyces clavisporus, strain LL-12I252.

A group of new 10mer linear peptides, designated culicinins A-D (1-4), was isolated from the fermentation broth of the entomopathogenic fungus Culicinomyces clavisporus, strain LL-12I252. The structures of the culicinins were determined by a combination of 2D NMR and MS analysis. The major compound, culicinin D (4), exhibited selective inhibitory activity against PTEN-negative MDA468 tumor cells.

Production of fungal antibiotics using polymeric solid supports in solid-state and liquid fermentation.

The use of inert absorbent polymeric supports for cellular attachment in solid-state fungal fermentation influenced growth, morphology, and production of bioactive secondary metabolites. Two filamentous fungi exemplified the utility of this approach to facilitate the discovery of new antimicrobial compounds. Cylindrocarpon sp.

Lichenicolins A and B, new bisnaphthopyrones from an unidentified lichenicolous fungus, strain LL-RB0668.

Lichenicolous fungus LL-RB0668 was isolated from a processed lichen thallus on a modified Lilly-Barnett solid medium. Two new bisnaphthopyrone compounds, lichenicolins A (1) and B (2), were isolated from the culture broth of this organism fermented on a rice-based solid medium. These results demonstrate that lichen-associated fungi potentially are a good resource for new bioactive natural products for current screening programs.

Mannopeptimycins, a novel class of glycopeptide antibiotics active against gram-positive bacteria.

Mannopeptimycins alpha-epsilon, novel glycopeptides with activity against methicillin-resistant staphylococci and vancomycin-resistant enterococci, are purified from the fermentation broth of a strain of Streptomyces hygroscopicus, LL-AC98, and their structures characterized using spectroscopic analyses and chemical methods. The SAR data of the natural and synthetic esters demonstrate that the presence of hydrophobic groups near the terminal mannosyl moiety is critical for antibacterial potency. Scalable syntheses of 4,6-cyclic acetals and ketals on this moiety are used to produce significant quantities of the respective mannopeptimycin derivatives.

Hydrophobic acetal and ketal derivatives of mannopeptimycin-alpha and desmethylhexahydromannopeptimycin-alpha: semisynthetic glycopeptides with potent activity against Gram-positive bacteria.

The effect of introducing hydrophobic groups onto the disaccharide portion of the mannopeptimycins has been examined. Under acid-catalyzed conditions dimethyl acetals and ketals react on the terminal mannose of the disaccharide moiety of mannopeptimycin-alpha and the cyclohexylalanyl analogue 2. The preferentially formed monofunctionalized 4,6-acetals and -ketals display potent antibacterial activities against Gram-positive microorganisms, including MRSA, PRSP, and VRE pathogens.

Acremonidins, new polyketide-derived antibiotics produced by Acremonium sp., LL-Cyan 416.

Acremonidins A to approximately E (1 to approximately 5) were produced by fermentation of Acremonium sp., LL-Cyan 416, in heterogeneous phases. The structures of these compounds, containing a bridging keto group, were determined by spectroscopic analysis.

Mannopeptimycin esters and carbonates, potent antibiotic agents against drug-resistant bacteria.

A series of ester and carbonate derivatives of the glycopeptide mannopeptimycin alpha (1) with potent activity against G+ bacteria, including the methicillin-resistant staphylococci and vancomycin-resistant enterococci, was synthesized. The SAR data obtained from natural and semisynthetic compounds demonstrated the importance of a hydrophobic group in the terminal mannosyl moiety for antibacterial activity.

Cytosporacin, a highly unsaturated polyketide: application of the ACCORD-ADEQUATE experiment to the structural determination of natural products.

Cytosporacin (1), a novel antibacterial polyketide containing naphthopyranone and isochromandione moieties, was isolated from the fermentation broth of the fungus Cytospora rhizophorae. A (1)H-detected ACCORD-ADEQUATE pulse sequence that distinguished (2)J(CH) from (3)J(CH) correlations provided critical information for structural determination. NOE studies established the relative configuration and revealed the presence of two rotamers.