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 (1 and 2) 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. These results show promise for cyanobacterial peptides in the therapeutic realm apart from their impact on environmental health and provide another example of the utility of large prefractionated natural product libraries for therapeutic hit and lead identification.

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. Despite these structural differences, the compounds are qualitatively identical to bufalin in their cytotoxic profiles versus cell lines in tissue culture and cardiac activity, as demonstrated in an impedance-based cellular assay conducted with IPSC-derived cardiomyocytes. Cyanobufalins are nonselectively toxic to human cells in the single-digit nanomolar range and show stimulation of contractility in cardiomyocytes at sub-nanomolar concentrations. The estimated combined concentration of 1-3 in the environment is in the same nanomolar range, and consequently more precise quantitative analyses are recommended along with more detailed cardiotoxicity studies. This is the first time that cardioactive steroid toxins have been found associated with microorganisms in an aquatic environment. Several factors point to a microbial biosynthetic origin for the cyanobufalins.

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. The two most lipophilic Ahppa-containing microcystins showed 10-fold greater cytotoxic potency against human tumor cell lines (A549 and HCT-116) compared to microcystin-LR (5). The presence of an Ahppa residue in microcystin congeners is difficult to ascertain by MS methods alone, due to the lack of characteristic fragment ions derived from the doubly homologated side chain. Owing to their unexpected cytotoxic potency, the potential impact of the compounds on human health should be further evaluated.

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. However, the ADC field is rapidly shifting towards site-specific ADCs due to their advantages in manufacturability, characterization and safety. In this work we report the identification of a highly efficacious site-specific thailanstatin ADC. The site of conjugation played a critical role on both the in vitro and in vivo potency of these ADCs. During the course of this study, we developed a novel methodology of loading a single site with multiple payloads using an in situ generated multi-drug carrying peptidic linker that allowed us to rapidly screen for optimal conjugation sites. Using this methodology, we were able to identify a double-cysteine mutant ADC delivering four-loaded thailanstatin that was very efficacious in a gastric cancer xenograft model at 3mg/kg and was also shown to be efficacious against T-DM1 resistant and MDR1 overexpressing tumor cell lines.

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

In an effort to isolate and characterize bioactive secondary metabolites from Trichodesmium thiebautii 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 (1). The planar structure of 1 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 (EC50: 6.5 µM) and HCT-116 cells (EC50: 11.7 µM). Trichophycin A was significantly more cytotoxic than the previously isolated polyketides trichotoxin A and trichotoxin B. These cytotoxicity observations suggest that toxicity may be related to the polyol character of these polyketide compounds.

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. Algal biomass derived from eight million liters of lake water was concentrated using a novel continuous countercurrent filtration system. Analysis of this freshwater algal bloom from Grand Lake-Saint Marys, Ohio led to the discovery of anabaenopeptin 679 (1), as well as the known anabaenopeptins B, F, H and 908. Anabaenopeptin 679 is unusual in that it possesses the classical anabaenopeptin-like cyclic pentapeptide core, but lacks the typical sidechain attached to the constitutive ureido group. Screening of all anabaenopeptin derivatives in an enzymatic assay for inhibitory activity toward carboxypeptidase A identified anabaenopeptin 679 as a strong inhibitor of carboxypeptidase A with an IC50 value of 4.6µg/mL. This result defines a new minimal core structure for carboxypeptidase activity among the anabaenopeptin class, and provides further insight into the structure-activity relationship of anabaenopeptin-like carboxypeptidase A inhibitors.

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. A series of thailanstatin-antibody conjugates were prepared in order to evaluate their potential utility in the treatment of cancer. After exploring a variety of linkers, we found that the most potent antibody-drug conjugates (ADCs) were derived from direct conjugation of the carboxylic acid-containing payload to surface lysines of the antibody (a "linker-less" conjugate). Activity of these lysine conjugates was correlated to drug-loading, a feature not typically observed for other payload classes. The thailanstatin-conjugates were potent in high target expressing cells, including multidrug-resistant lines, and inactive in nontarget expressing cells. Moreover, these ADCs were shown to promote altered splicing products in N87 cells in vitro, consistent with their putative mechanism of action. In addition, the exposure of the ADCs was sufficient to result in excellent potency in a gastric cancer xenograft model at doses as low as 1.5 mg/kg that was superior to the clinically approved ADC T-DM1. The results presented herein therefore open the door to further exploring splicing inhibition as a potential new mode-of-action for novel ADCs.

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

NMR-guided fractionation of the lipophilic extract of Trichodesmium thiebautii filaments led to the isolation of a phenyl-containing chlorinated polyketide (1) and an alkyne-containing analogue (2). Comparison of spectroscopic and spectrometric data of 1 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 (2). Absolute configuration of 1 and 2 is proposed based on spectroscopic comparison to a close structural analog.

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. All three compounds are structurally related to equisetin-type compounds, with 1 and 3 having a trans-decalin ring system, while 2 has a cis-fused decalin. All were active against Mycobacterium tuberculosis, with the trans-decalin analogues 1 and 3 exhibiting lower MICs than the cis-decalin analogue 2. Here we report the isolation, structure elucidation, and antimycobacterial activities of 1-3 from the recombinant expression as well as the isolation of 1 from the wild-type fungus NRRL 50135.

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. FERM BP-3421, and their structures identified by analysis of spectroscopic data. Semisynthesis was performed on the major components 4 and 5 to generate ester and amide derivatives with improved in vitro potency. With their potent activity against tumor cells and unique mode of action, spliceostatins can be considered potential leads for development of cancer drugs.

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. The facile preparation of the key amino acids and the synthesis of the aglycone pave the way for further studies on this class of antibiotics and the development of new lead compounds with therapeutic potential. In addition, our studies have led to the revision of the stereochemistry of the ß-methylphenylalanine residue in the mannopeptimycin aglycone.

Discovery of the lomaiviticin biosynthetic gene cluster in Salinispora pacifica.

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 Salinispora pacifica strains DPJ-0016 and DPJ-0019 using a combination of molecular approaches and genome sequencing. The link between the lom 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. Additionally, we have used comparative genomics to identify candidate enzymes for several unusual tailoring events, including diazo formation and oxidative dimerization. These findings will allow further elucidation of the biosynthetic logic of lomaiviticin assembly and provide useful molecular tools for application in biocatalysis and synthetic biology.

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. In the case of the mono-(15)N-labeled diazepinomicin (1) derived from labeled tryptophan, tandem mass spectrometry located this (15)N label at the non-amide nitrogen. Through the use of exceptionally high mass resolving power of over 125,000, the isotopic fine structure of the molecular ion cluster of 1 was revealed. Separation of the (15)N(2) peak from the isobaric (13)C(15)N peak, both having similar abundances, demonstrated the presence of a minor amount of doubly (15)N-labeled diazepinomicin (1). Tandem mass spectrometry amplified this isotopic fine structure (Deltam=6.32 mDa) from mDa to 1 Da scale thereby allowing more detailed scrutiny of labeling content and location. Tandem mass spectrometry was also used to assign the location of deuterium labeling in two deuterium-labeled diazepinomicin (1) samples. In one case three deuterium atoms were incorporated into the dibenzodiazepine core; while in the other a mono-D label was mainly incorporated into the farnesyl side chain. The specificity of (15)N-labeling in dioxapyrrolomycin (2) and the proportion of the (15)N-label contained in the nitro group were determined from the measurement of the relative abundance of the (14)NO(2)(1-) and (15)NO(2)(1-) fragment ions.

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. These results indicate that LL-F42248 is capable of fixing nitrogen.

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. Physical data suggested that this compound is identical with pyranonigrin A (1); however, our structure elucidation led to a different assignment than previously reported. On the basis of analysis of all data, we propose a correction to the structure of pyranonigrin A. Its absolute configuration was determined by electronic circular dichroism measurements in comparison with theoretical values calculated via ab initio time-dependent density functional theory and assigned as (7R)-3,7-dihydroxy-2-[(1E)-prop-1-enyl]-6,7-dihydropyrano[2,3-c]pyrrole-4,5-dione.

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. LL-Cyan426 produced pyrrocidines A and B and Acremonium sp. LL-Cyan416 produced acremonidins A-E when grown on agar bearing moist polyester-cellulose paper and generated distinctly different metabolite profiles than the conventional shaken or stationary liquid fermentations. Differences were also apparent when tenfold concentrated methanol extracts from these fermentations were tested against antibiotic-susceptible and antibiotic-resistant Gram-positive bacteria, and zones of inhibition were compared. Shaken broth cultures of Acremonium sp. or Cylindrocarpon sp. showed complex HPLC patterns, lower levels of target compounds, and high levels of unwanted compounds and medium components, while agar/solid support cultures showed significantly increased yields of pyrrocidines A and B and acremonidins A-E, respectively. This method, mixed-phase fermentation (fermentation with an inert solid support bearing liquid medium), exploited the increase in surface area available for fungal growth on the supports and the tendency of some microorganisms to adhere to solid surfaces, possibly mimicking their natural growth habits. The production of dimeric anthraquinones by Penicillium sp. LL-WF159 was investigated in liquid fermentation using various inert polymeric immobilization supports composed of polypropylene, polypropylene cellulose, polyester-cellulose, or polyurethane. This culture produced rugulosin, skyrin, flavomannin, and a new bisanthracene, WF159-A, after fermentation in the presence and absence of polymeric supports for mycelial attachment. The physical nature of the different support systems influenced culture morphology and relative metabolite yields, as determined by HPLC analysis and measurement of antimicrobial activity. The application of such immobilized-cell fermentation methods under solid and liquid conditions facilitated the discovery of new antibiotic compounds, and offers new approaches to fungal fermentation for natural product discovery.

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. Studies on the 3D structure of 4 using NOE data and computer modeling revealed a dominant conformation of the right-handed helix.

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. These acetal and ketal derivatives exhibit potent activities against susceptible and resistant Gram-positive bacteria in both in vitro and in vivo experiments, comparable with or exceeding the activity of vancomycin. Studies on the mechanism of action suggest that the mannopeptimycins interfere with the late stages of bacterial cell wall biosynthesis. It is believed that these antibiotics inhibit the transglycosylation by binding to the transglycosylase substrate, lipid II.

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.