Discovery of Anticancer Agents of Diverse Natural Origin.

Research progress from mainly over the last five years is described for a multidisciplinary collaborative program project directed toward the discovery of potential anticancer agents from a broad range of taxonomically defined organisms. Selected lead compounds with potential as new antitumor agents that are representative of considerable structural diversity have continued to be obtained from each of tropical plants, terrestrial and aquatic cyanobacteria, and filamentous fungi. Recently, a new focus has been on the investigation of the constituents of U.S. lichens and their fungal mycobionts. A medicinal chemistry and pharmacokinetics component of the project has optimized structurally selected lead natural products, leading to enhanced cytotoxic potencies against selected cancer cell lines. Biological testing has shown several compounds to have in vivo activity, and relevant preliminary structure-activity relationship and mechanism of action studies have been performed. Several promising lead compounds worthy of further investigation have been identified from the most recent collaborative work performed.

Aulosirazoles B and C from the Cyanobacterium Nostoc sp. UIC 10771: Analogues of an Isothiazolonaphthoquinone Scaffold that Activate Nuclear Transcription Factor FOXO3a in Ovarian Cancer Cells.

The known solid-tumor-selective cytotoxin aulosirazole (1) was identified from bioactive extracts from the culture medium of the cyanobacterium Nostoc sp. UIC 10771. Here, we demonstrate that 1 induces the nuclear accumulation of FOXO3a in OVCAR3 using both Western blot analysis and immunofluorescence confocal microscopy. We also report the discovery of two additional analogues, aulosirazoles B (2) and C (3). Structures for compounds 2 and 3 were determined using HR-ESI-LC-MS/MS and 1D and 2D NMR experiments. Aulosirazoles B (2) and C (3) represent the first natural analogues of the FOXO-activating compound aulosirazole (1) and are the second and third isothiazole-containing metabolites reported from this phylum.

Phormidepistatin from the Cyanobacterium UIC 10484: Assessing the Phylogenetic Distribution of the Statine Pharmacophore.

A new linear lipopeptide, phormidepistatin (1), containing an epi-statine amino acid was isolated from cf. Phormidium sp. strain UIC 10484. The planar structure was elucidated by 1D and 2D NMR experimentation. The relative configuration was determined by J-based configurational analysis and the absolute configuration by advanced Marfey's analysis. Given that the statine moiety is an established pharmacophore known to inhibit aspartic proteases, phormidepistatin was evaluated against cathepsin D and displayed limited activity. With 1 containing a statine-like moiety, we sought to assess the distribution of this γ-amino acid within the phylum Cyanobacteria. In-depth MS/MS analysis identified the presence of phormidepistatin in cf. Phormidium sp. UIC 10045 and cf. Trichormus sp. UIC 10039. A structure database search identified 33 known cyanobacterial metabolites containing a statine or statine-like amino acid and, along with phormidepistatin, were grouped into 10 distinct compound classes. A phylogenetic tree was built comprising all cyanobacteria with established 16S rRNA sequences known to produce statine or statine-like-containing compound classes. This analysis suggests the incorporation of the γ-amino acid into secondary metabolites is taxonomically widespread within the phylum. Overall, it is our assessment that cyanobacteria are a potential source for statine or statine-like-containing compounds.

A Family of Nonribosomal Peptides Modulate Collective Behavior in Pseudovibrio Bacteria Isolated from Marine Sponges*.

Although swarming motility and biofilms are opposed collective behaviors, both contribute to bacterial survival and host colonization. Pseudovibrio bacteria have attracted attention because they are part of the microbiome of healthy marine sponges. Two-thirds of Pseudovibrio genomes contain a member of a nonribosomal peptide synthetase-polyketide synthase gene cluster family, which is also found sporadically in Pseudomonas pathogens of insects and plants. After developing reverse genetics for Pseudovibrio, we isolated heptapeptides with an ureido linkage and related nonadepsipeptides we termed pseudovibriamides A and B, respectively. A combination of genetics and imaging mass spectrometry experiments showed heptapetides were excreted, promoting motility and reducing biofilm formation. In contrast to lipopeptides widely known to affect motility/biofilms, pseudovibriamides are not surfactants. Our results expand current knowledge on metabolites mediating bacterial collective behavior.

Laxaphycins B5 and B6 from the cultured cyanobacterium UIC 10484.

Two laxaphycin type-B cyclic dodecapeptides, laxaphycins B5 and B6, were obtained from UIC 10484, a freshwater cf. Phormidium sp. Analysis using the 16S rRNA sequence found UIC 10484 to clade with UIC 10045, a known laxaphycin type-A and -B producer, and MS/MS analysis revealed the presence of two novel laxaphycin type-B compounds. The structures of the metabolites were elucidated using 2D NMR and MS/MS. The absolute configurations of the amino acids were determined by advanced Marfey's analysis. Both metabolites were evaluated against the same three cancer cell lines. The IC50 of both laxaphycins B5 and B6 was near 1 µM against breast cancer MDA-MB-231, melanoma MDA-MB-435, and ovarian cancer OVCAR3 cell lines.

15N Stable Isotope Labeling and Comparative Metabolomics Facilitates Genome Mining in Cultured Cyanobacteria.

As genome mining becomes a more widely used approach to identify bacterial natural products, the challenge of matching biosynthetic gene clusters to their cognate secondary metabolites has become more apparent. Bioinformatic platforms such as AntiSMASH have made great progress in predicting chemical structures from genetic information, however the predicted structures are often incomplete. This complicates identifying the predicted compounds by mass spectrometry. Secondary metabolites produced by cyanobacteria represent a unique opportunity for bridging this gap. Cultured cyanobacteria incorporate inorganic nitrogen provided in chemically defined media into all nitrogen-containing secondary metabolites. Thus, stable isotope labeling with 15N labeled nitrate and subsequent comparative metabolomics can be used to match biosynthetic gene clusters to their cognate compounds in cell extracts. Analysis of the sequenced genome of Nostoc sp. UIC 10630 identified six biosynthetic gene clusters predicted to encode the production of a secondary metabolite with at least one nitrogen atom. Comparative metabolomic analysis of the 15N labeled and unlabeled cell extracts revealed four nitrogen containing compounds that contained the same number of nitrogen atoms as were predicted in the biosynthetic gene clusters. Two of the four compounds were new secondary metabolites, and their structures were elucidated by NMR, HRESIMS, and MS/MS.

Control of Stereoselectivity in Diverse Hapalindole Metabolites is Mediated by Cofactor-Induced Combinatorial Pairing of Stig Cyclases.

Stereospecific polycyclic core formation of hapalindoles and fischerindoles is controlled by Stig cyclases through a three-step cascade involving Cope rearrangement, 6-exo-trig cyclization, and a final electrophilic aromatic substitution. Reported here is a comprehensive study of all currently annotated Stig cyclases, revealing that these proteins can assemble into heteromeric complexes, induced by Ca2+ , to cooperatively control the stereochemistry of hapalindole natural products.

Scytodecamide from the Cultured Scytonema sp. UIC†10036 Expands the Chemical and Genetic Diversity of Cyanobactins.

Cyanobactins are a large family of cyanobacterial ribosomally synthesized and post-translationally modified peptides (RiPPs) often associated with biological activities, such as cytotoxicity, antiviral, and antimalarial activities. They are traditionally described as cyclic molecules containing heterocyclized amino acids. However, this definition has been recently challenged by the discovery of short, linear cyanobactins containing three to five amino acids as well as cyanobactins containing no heterocyclized residues. Herein we report the discovery of scytodecamide (1) from the freshwater cyanobacterium Scytonema sp. UIC 10036. Structural elucidation based on mass spectrometry, 1D and 2D NMR spectroscopy, and Marfey's method revealed 1 to be a linear decapeptide with an N-terminal N-methylation and a C-terminal amidation. The genome of Scytonema sp. UIC 10036 was sequenced, and bioinformatic analysis revealed a cyanobactin-like biosynthetic gene cluster consistent with the structure of 1. The discovery of 1 as a novel linear peptide containing an N-terminal N-methylation and a C-terminal amidation expands the chemical and genetic diversity of the cyanobactin family of compounds.

Commensal Gut Bacteria Convert the Immunosuppressant Tacrolimus to Less Potent Metabolites.

Tacrolimus exhibits low and variable drug exposure after oral dosing, but the contributing factors remain unclear. Based on our recent report showing a positive correlation between fecal abundance of Faecalibacterium prausnitzii and oral tacrolimus dose in kidney transplant patients, we tested whether F. prausnitzii and other gut abundant bacteria are capable of metabolizing tacrolimus. Incubation of F. prausnitzii with tacrolimus led to production of two compounds (the major one named M1), which was not observed upon tacrolimus incubation with hepatic microsomes. Isolation, purification, and structure elucidation using mass spectrometry and nuclear magnetic resonance spectroscopy indicated that M1 is a C-9 keto-reduction product of tacrolimus. Pharmacological activity testing using human peripheral blood mononuclear cells demonstrated that M1 is 15-fold less potent than tacrolimus as an immunosuppressant. Screening of 22 gut bacteria species revealed that most Clostridiales bacteria are extensive tacrolimus metabolizers. Tacrolimus conversion to M1 was verified in fresh stool samples from two healthy adults. M1 was also detected in the stool samples from kidney transplant recipients who had been taking tacrolimus orally. Together, this study presents gut bacteria metabolism as a previously unrecognized elimination route of tacrolimus, potentially contributing to the low and variable tacrolimus exposure after oral dosing.

The impact of culture conditions on growth and metabolomic profiles of freshwater cyanobacteria.

Cultured cyanobacteria produce secondary metabolites with a wide range of biological activities and are an important source of natural products. In the context of secondary metabolite discovery, microbial culture conditions are expected to support optimum growth, induce maximum chemical diversity, and be suitable for the majority of cyanobacterial strains. We investigated the effect of nitrate and phosphate on biomass production and metabolomic profiles of three filamentous freshwater cyanobacterial strains: cf. Oscillatoria sp. UIC 10045, Scytonema sp. UIC 10036, and Nostoc sp. UIC 10110. A standardized inoculation procedure allowed for the assessment of cell mass production. Dried cyanobacterial cell mass was extracted and analyzed by liquid chromatography coupled with high resolution mass spectrometry (UPLC-HRMS), followed by comparative metabolomics analysis using XCMS Online. Results showed that low nitrate media significantly reduced cell mass production for all three strains. Low nitrate also induced production of primary metabolites (heterocyst glycolipids) in strains UIC 10036 and UIC 10110. Changes in phosphate levels affected each strain differently. Strain UIC 10110 showed a significant increase in production of merocyclophane C when cultivated in low phosphate, while strain UIC 10036 displayed higher production of tolytoxin under high phosphate. Additionally, these experiments led to the identification of a potentially new peptide produced by strain UIC 10036.

Calothrixamides A and B from the Cultured Cyanobacterium Calothrix sp. UIC 10520.

Cyanobacteria are a source of chemically diverse metabolites with potential medicinal and biotechnological applications. Rapid identification of compounds is central to expedite the natural product discovery process. Mass spectrometry has been shown to be an important tool for dereplication of complex natural product samples. In addition, chromatographic separation and complementary spectroscopic analysis (e.g., UV) can enhance the confidence of the dereplication process. Here, we applied a droplet-liquid microjunction-surface sampling probe (droplet probe) coupled with UPLC-PDA-HRMS-MS/MS to identify two new natural products in situ from the freshwater strain Calothrix sp. UIC 10520. This allowed us to prioritize this strain for chemical investigation based on the presence of new metabolites very early in our discovery process, saving both time and resources. Subsequently, calothrixamides A (1) and B (2) were isolated from large-scale cultures, and the structures were elucidated by 1D and 2D NMR spectroscopy and mass spectrometry. The absolute configurations were determined by a combination of chemical degradation reactions, derivatization methods (Mosher's, Marfey's, and phenylglycine methyl ester), and J-based configurational analysis. Calothrixamides showed no cytotoxic activity against the MDA-MB-435, MDA-MB-231, and OVCAR3 cancer cell lines. They represent the first functionalized long-chain fatty acid amides reported from the Calothrix genus and from a freshwater cyanobacterium.

Complete Genome of Micromonospora sp. Strain B006 Reveals Biosynthetic Potential of a Lake Michigan Actinomycete.

Actinomycete bacteria isolated from freshwater environments are an unexplored source of natural products. Here we report the complete genome of the Great Lakes-derived Micromonospora sp. strain B006, revealing its potential for natural product biosynthesis. The 7-megabase pair chromosome of strain B006 was sequenced using Illumina and Oxford Nanopore technologies followed by Sanger sequencing to close remaining gaps. All identified biosynthetic gene clusters (BGCs) were manually curated. Five known BGCs were identified encoding desferrioxamine, alkyl- O-dihydrogeranylmethoxyhydroquinone, a spore pigment, sioxanthin, and diazepinomicin, which is currently in phase II clinical trials to treat Phelan-McDermid syndrome and co-morbid epilepsy. We report here that strain B006 is indeed a producer of diazepinomicin and at yields higher than previously reported. Moreover, 11 of the 16 identified BGCs are orphan, eight of which were transcriptionally active under the culture condition tested. Orphan BGCs include an enediyne polyketide synthase and an uncharacteristically large, 36-module polyketide synthase-nonribosomal peptide synthetase BGC. We developed a genetics system for Micromonospora sp. B006 that will contribute to deorphaning BGCs in the future. This study is one of the few attempts to report the biosynthetic capacity of a freshwater-derived actinomycete and highlights this resource as a potential reservoir for new natural products.

Hapalindole H Induces Apoptosis as an Inhibitor of NF-ĸB and Affects the Intrinsic Mitochondrial Pathway in PC-3 Androgen-insensitive Prostate Cancer Cells.

BACKGROUND: Prostate cancer presents the highest incidence rates among all cancers in men. Hapalindole H (Hap H), isolated from Fischerella muscicola (UTEX strain number LB1829) as part of our natural product anticancer drug discovery program, was found to be significantly active against prostate cancer cells. MATERIALS AND METHODS: In this study, Hap H was tested for nuclear factor-kappa B (NF-ĸB) inhibition and selective cytotoxic activity against different cancer cell lines. The apoptotic effect was assessed on PC-3 prostate cancer cells by fluorescence-activated cell sorting analysis. The underlying mechanism that induced apoptosis was studied and the effect of Hap H on mitochondria was evaluated and characterized using western blot and flow cytometric analysis. RESULTS: Hap H was identified as a potent NF-ĸB inhibitor (0.76 µM) with selective cytotoxicity against the PC-3 prostate cancer cell line (0.02 µM). The apoptotic effect was studied on PC-3 cells. The results showed that treatment of PC-3 cells with Hap H reduced the formation of NAD(P)H, suggesting that the function of the outer mitochondrial membrane was negatively affected. Thus, the mitochondrial transmembrane potential was assessed in Hap H treated cells. The results showed that the outer mitochondrial membrane was disrupted as an increased amount of JC-1 monomers were detected in treated cells (78.3%) when compared to untreated cells (10.1%), also suggesting that a large number of treated cells went into an apoptotic state. CONCLUSION: Hap H was found to have potent NF-ĸB p65-inhibitory activity and induced apoptosis through the intrinsic mitochondrial pathway in hormone-independent PC-3 prostate cancer cells.

Total Synthesis of Scytonemide A Employing Weinreb AM Solid-Phase Resin.

The human 20S proteasome inhibitor scytonemide A (1), a macrocyclic imine originally isolated from the cyanobacterium Scytonema hofmanni, was synthesized via a biomimetic solid-phase peptide synthesis (SPPS) approach employing the Weinreb AM resin. Utilizing this approach, cyclization of the protected heptapeptide via formation of the imine bond occurred spontaneously upon cleavage from the resin in the presence of a reducing agent and subsequent aqueous workup. The final deprotection step necessary to produce the natural product was accomplished under slightly basic conditions, facilitating cleavage of the silyl ether group while leaving the macrocycle intact. Purification of the synthetic scytonemide A was accomplished via normal-phase flash column chromatography, potentially facilitating larger scale preparation of the compound necessary for future mechanistic and SAR studies. The structure of the target compound was confirmed by NMR spectroscopy, which also shed light on differences in the spectroscopic data obtained for the synthetic and natural scytonemide A samples for some of the amide and alcohol signals in the 1H NMR spectrum.

Ribocyclophanes A-E, Glycosylated Cyclophanes with Antiproliferative Activity from Two Cultured Terrestrial Cyanobacteria.

The cell extracts of two cultured freshwater Nostoc spp., UIC 10279 and UIC 10366, both from the suburbs of Chicago, showed antiproliferative activity against MDA-MB-231 and MDA-MB-435 cancer cell lines. Bioassay-guided fractionation led to the isolation of five glycosylated cylindrocyclophanes, named ribocyclophanes A-E (1-5) and cylindrocyclophane D (6). The structure determination was carried out by HRESIMS and 1D and 2D NMR analyses and confirmed by single-crystal X-ray crystallography. The structures of ribocyclophanes A-E (1-5) contain a ß-d-ribopyranose glycone in the rare 1 C4 conformation. Among isolated compounds, ribocyclophane D (4) showed antiproliferative activity against MDA-MB-435 and MDA-MB-231 cancer cells with an IC50 value of less than 1 µM.

Merocyclophanes C and D from the Cultured Freshwater Cyanobacterium Nostoc sp. (UIC 10110).

Merocyclophanes C and D (1 and 2) were isolated from the cell extract of the cultured cyanobacterium UIC 10110. The structures were determined by one-dimensional nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry and confirmed by 2D NMR techniques. The absolute configurations were determined using electronic circular dichroism spectroscopy. Merocyclophanes C and D represent the first known analogues of the merocyclophane core structure, a recently discovered scaffold of [7,7] paracyclophanes characterized by an α-branched methyl at C-1/C-14; 1 and 2 showed antiproliferative activity against the MDA-MB-435 cell line with IC50 values of 1.6 and 0.9 µM, respectively. Partial 16S analysis determined UIC 10110 to be a Nostoc sp., and it was found to clade with UIC 10062 Nostoc sp., the only other strain known to produce merocyclophanes. The genome of UIC 10110 was sequenced, and a biosynthetic gene cluster was identified that is proposed to encode type I and type III polyketide synthases that are potentially responsible for production of the merocyclophanes; however, further experiments will be required to verify the true function of the gene cluster. The gene cluster provides a genetic basis for the observed structural differences of the [7,7] paracyclophane core structures.

Discovery of Anticancer Agents of Diverse Natural Origin.

Recent progress is described in an ongoing collaborative multidisciplinary research project directed towards the purification, structural characterization, chemical modification, and biological evaluation of new potential natural product anticancer agents obtained from a diverse group of organisms, comprising tropical plants, aquatic and terrestrial cyanobacteria, and filamentous fungi. Information is provided on how these organisms are collected and processed. The types of bioassays are indicated in which initial extracts, chromatographic fractions, and purified isolated compounds of these acquisitions are tested. Several promising biologically active lead compounds from each major organism class investigated are described, and these may be seen to be representative of a very wide chemical diversity.

Antimicrobial Activity of Croton macrostachyus Stem Bark Extracts against Several Human Pathogenic Bacteria.

In Kenya, leaves and roots from Croton macrostachyus are used as a traditional medicine for infectious diseases such as typhoid and measles, but reports on possible antimicrobial activity of stem bark do not exist. In this study, the antibacterial and antifungal effects of methanol, ethyl acetate and butanol extracts, and purified lupeol of C. macrostachyus stem bark were determined against important human gram-negative pathogens Escherichia coli, Salmonella typhi, Klebsiella pneumoniae, and Enterobacter aerogenes, gram-positive Listeria monocytogenes, and a fungus Candida albicans. The most promising broad scale antimicrobial activity against all the studied pathogens was shown by the ethyl acetate extract. The ethyl acetate extract induced the zone of inhibition between 10.1 ± 0.6 mm and 16.0 ± 1.2 mm against S. typhi, E. coli, K. pneumoniae, E. aerogenes, and L. monocytogenes with weaker antimicrobial activity against C. albicans (zone of inhibition: 5.6 ± 1.0 mm). The antibiotic controls (amoxicillin, ciprofloxacin, ampicillin, benzylpenicillin, clotrimazole, and cefotaxime) showed antimicrobial activity with zones of inhibition within 13.4 ± 0.7-22.1 ± 0.9 mm. The ethyl acetate extract had MIC in the range of 125-250 mg/mL against all the studied bacteria and against C. albicans MIC was 500 mg/mL. The present results give scientific evidence and support the traditional use of C. macrostachyus stem bark as a source for antimicrobials. We show that C. macrostachyus stem bark lupeol is a promising antimicrobial agent against several important human pathogens.

Application of high-field NMR spectroscopy for characterization and quantitation of submilligram quantities of isolated natural products.

We have investigated and compared a number of sample conditions on different NMR platforms in the search of maximum SNR and optimal experiment time efficiency for structure elucidation and quantitation of natural products. Using restricted volume 3 mm Shigemi microcell assembly in conjunction with a 900 MHz NMR spectrometer equipped with a 5 mm carbon-sensitive inverse cryoprobe, it was possible to achieve a substantial increase in SNR (46-fold) as compared with a conventional room temperature 400 MHz instrument. Switching from standard 5 mm NMR tube to 3 mm Shigemi microcell assembly typically improved SNR by threefold on either 600 or 900 MHz cryoplatform. A quantitation method that relies on a calibrated residual protonated NMR solvent signal as internal standard was developed using the same hardware setup and restricted sample volume tubes. Linearity of the method spans over 3 orders of magnitude, from low microgram to milligram quantities. We successfully applied this method to quantify a low micrgram sample of paclitaxel, verified by a UV/VIS quantitation measurement.

Microseiramide from the Freshwater Cyanobacterium Microseira sp. UIC 10445.

Microseiramide (1), a cyclic heptapeptide, was isolated from a sample of the freshwater cyanobacterium Microseira sp. UIC 10445 collected in a shallow lake in Northern Indiana. Taxonomic identification of UIC 10445 was performed by a combination of morphological and phylogenetic characterization. Phylogenetic analysis revealed that UIC 10445 was a member of the recently described genus Microseira, which is phylogenetically distinct from the morphologically similar genera. Moorea and Lyngbya. The planar structure of microseiramide (1) was determined by extensive 1D and 2D NMR experiments as well as HRESIMS analysis. The absolute configurations of amino acid residues were determined using acid hydrolysis followed by the advanced Marfey's analysis. microseiramide (1) is the first cyclic peptide reported from a Microseira sp., and the structure of microseiramide (1) is distinct from the previously known metabolites from cyanobacteria of the genera Moorea and Lyngbya.