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.

Synthesis and Pharmacological Characterization of 2-(2,6-Dichlorophenyl)-1-((1S,3R)-5-(3-hydroxy-3-methylbutyl)-3-(hydroxymethyl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one (LY3154207), a Potent, Subtype Selective, and Orally Available Positive Allosteric Modulator of the Human Dopamine D1 Receptor.

Clinical development of catechol-based orthosteric agonists of the dopamine D1 receptor has thus far been unsuccessful due to multiple challenges. To address these issues, we identified LY3154207 (3) as a novel, potent, and subtype selective human D1 positive allosteric modulator (PAM) with minimal allosteric agonist activity. Conformational studies showed LY3154207 adopts an unusual boat conformation, and a binding pose with the human D1 receptor was proposed based on this observation. In contrast to orthosteric agonists, LY3154207 showed a distinct pharmacological profile without a bell-shaped dose-response relationship or tachyphylaxis in preclinical models. Identification of a crystalline form of free LY3154207 from the discovery lots was not successful. Instead, a novel cocrystal form with superior solubility was discovered and determined to be suitable for development. This cocrystal form was advanced to clinical development as a potential first-in-class D1 PAM and is now in phase 2 studies for Lewy body dementia.

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.

Preclinical profile of a dopamine D1 potentiator suggests therapeutic utility in neurological and psychiatric disorders.

DETQ, an allosteric potentiator of the dopamine D1 receptor, was tested in therapeutic models that were known to respond to D1 agonists. Because of a species difference in affinity for DETQ, all rodent experiments used transgenic mice expressing the human D1 receptor (hD1 mice). When given alone, DETQ reversed the locomotor depression caused by a low dose of reserpine. DETQ also acted synergistically with L-DOPA to reverse the strong hypokinesia seen with a higher dose of reserpine. These results indicate potential as both monotherapy and adjunct treatment in Parkinson's disease. DETQ markedly increased release of both acetylcholine and histamine in the prefrontal cortex, and increased levels of histamine metabolites in the striatum. In the hippocampus, the combination of DETQ and the cholinesterase inhibitor rivastigmine increased ACh to a greater degree than either agent alone. DETQ also increased phosphorylation of the AMPA receptor (GluR1) and the transcription factor CREB in the striatum, consistent with enhanced synaptic plasticity. In the Y-maze, DETQ increased arm entries but (unlike a D1 agonist) did not reduce spontaneous alternation between arms at high doses. DETQ enhanced wakefulness in EEG studies in hD1 mice and decreased immobility in the forced-swim test, a model for antidepressant-like activity. In rhesus monkeys, DETQ increased spontaneous eye-blink rate, a measure that is known to be depressed in Parkinson's disease. Together, these results provide support for potential utility of D1 potentiators in the treatment of several neuropsychiatric disorders, including Parkinson's disease, Alzheimer's disease, cognitive impairment in schizophrenia, and major depressive disorder.

Diversity oriented biosynthesis via accelerated evolution of modular gene clusters.

Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes.

Electroencephalographic, cognitive, and neurochemical effects of LY3130481 (CERC-611), a selective antagonist of TARP-γ8-associated AMPA receptors.

6-[(1S)-1-[1-[5-(2-hydroxyethoxy)-2-pyridyl]pyrazol-3-yl]ethyl]-3H-1,3-benzothiazol-2-one (LY3130481 or CERC-611) is a selective antagonist of AMPA receptors containing transmembrane AMPA receptor regulatory protein (TARP) γ-8. This molecule has been characterized as a potent and efficacious anticonvulsant in an array of acute and chronic epilepsy models in rodents. The present set of experiments was designed to assess the effects of LY3130481 on the electroencephelogram (EEG), cognitive function, and neurochemical outflow. LY3130481 disrupted food-maintained responding in rats and spontaneous alternation in a Y-maze in mice. In rat fear conditioning, LY3130481 caused a deficit in trace (hippocampal-dependent), but not in delay fear conditioning. Although these effects on cognitive performances were observed, the known cognitive-impairing anticonvulsant, topiramate, did not always produce deficits under these assay conditions. LY3130481 produced modest increases in wake times in rats. In addition, LY3130481 was able to attenuate some impairing effects of standard antiepileptic drugs. The motor-impairing effects of the lacosamide were attenuated by LY3130481 as was the decrease in non-rapid-eye movement sleep induced by carbamazepine. Evaluation of the effect of LY3130481 on neurotransmitter and metabolite efflux in the rat medial prefrontal cortex, using in vivo microdialysis, revealed significant increases in the pro-cognitive and wake-promoting neurotransmitters, histamine and acetylcholine, as well as in serotonin, telemethylhistamine, 5-HIAA, HVA and MHPG. LY3130481 thus presents a novel behavioral profile that will have to be evaluated in patients to fully appreciate its implications for therapeutics. LY3130481 is currently under clinical development as CERC-611 as an antiepileptic.

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.

Fornix deep brain stimulation enhances acetylcholine levels in the hippocampus.

Deep brain stimulation (DBS) of the fornix has gained interest as a potential therapy for advanced treatment-resistant dementia, yet the mechanism of action remains widely unknown. Previously, we have reported beneficial memory effects of fornix DBS in a scopolamine-induced rat model of dementia, which is dependent on various brain structures including hippocampus. To elucidate mechanisms of action of fornix DBS with regard to memory restoration, we performed c-Fos immunohistochemistry in the hippocampus. We found that fornix DBS induced a selective activation of cells in the CA1 and CA3 subfields of the dorsal hippocampus. In addition, hippocampal neurotransmitter levels were measured using microdialysis before, during and after 60 min of fornix DBS in a next experiment. We observed a substantial increase in the levels of extracellular hippocampal acetylcholine, which peaked 20 min after stimulus onset. Interestingly, hippocampal glutamate levels did not change compared to baseline. Therefore, our findings provide first experimental evidence that fornix DBS activates the hippocampus and induces the release of acetylcholine in this region.

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.

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.

NP/MS since 1970: from the basement to the bench top.

This Highlight explores the evolution of applications of mass spectrometric technologies in the context of natural products research since the 1970's. The central themes are the analysis of mixtures, dereplication (identification) and structure determination. The ascension of HPLC as the method of choice for the analysis of pharmaceuticals was a driving force for the development of interfaces for coupling of HPLC and MS. An example of sequential analysis of fragment ions or MS/MS or MS(n) methods to provide detailed structural information on muraymycins, a family of uridyl-peptide antibiotics, is presented.

Recombinant strains for the enhanced production of bioengineered rapalogs.

The rapK gene required for biosynthesis of the DHCHC starter acid that initiates rapamycin biosynthesis was deleted from strain BIOT-3410, a derivative of Streptomyces rapamycinicus which had been subjected to classical strain and process development and capable of robust rapamycin production at titres up to 250mg/L. The resulting strain BIOT-4010 could no longer produce rapamycin, but when supplied exogenously with DHCHC produced rapamycin at titres equivalent to its parent strain. This strain enabled mutasynthetic access to new rapalogs that could not readily be isolated from lower titre strains when fed DHCHC analogs. Mutasynthesis of some rapalogs resulted predominantly in compounds lacking late post polyketide synthase biosynthetic modifications. To enhance the relative production of fully elaborated rapalogs, genes encoding late-acting biosynthetic pathway enzymes which failed to act efficiently on the novel compounds were expressed ectopically to give strain BIOT-4110. Strains BIOT-4010 and BIOT-4110 represent valuable tools for natural product lead optimization using biosynthetic medicinal chemistry and for the production of rapalogs for pre-clinical and early stage clinical trials.

Natural products and Pharma 2011: strategic changes spur new opportunities.

Although natural products have been marginalized by major pharmaceutical companies over the last 20-30 years, the changing landscape of drug discovery now favors a greatly enhanced role for Nature's privileged structures. Screening for drug leads in phenotypic screens provides the best opportunity to realize the value of natural products. Advances in total synthesis, especially function-oriented syntheses and biosynthetic technologies offer new avenues for the medicinal chemical optimization of biologically active secondary metabolites. Genomic research has given new insights into biosynthetic processes as well as providing evidence that a wealth of unrealized biosynthetic potential remains to be explored. As Pharma strives to develop innovative and highly effective new drugs, natural products will be increasingly valued as sources of novel leads whose further development will be expedited by emerging technologies.

Discovery and assembly-line biosynthesis of the lymphostin pyrroloquinoline alkaloid family of mTOR inhibitors in Salinispora bacteria.

The pyrroloquinoline alkaloid family of natural products, which includes the immunosuppressant lymphostin, has long been postulated to arise from tryptophan. We now report the molecular basis of lymphostin biosynthesis in three marine Salinispora species that maintain conserved biosynthetic gene clusters harboring a hybrid nonribosomal peptide synthetase-polyketide synthase that is central to lymphostin assembly. Through a series of experiments involving gene mutations, stable isotope profiling, and natural product discovery, we report the assembly-line biosynthesis of lymphostin and nine new analogues that exhibit potent mTOR inhibitory activity.

Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate.

The macrocyclic polyketides FK506, FK520, and rapamycin are potent immunosuppressants that prevent T-cell proliferation through initial binding to the immunophilin FKBP12. Analogs of these molecules are of considerable interest as therapeutics in both metastatic and inflammatory disease. For these polyketides the starter unit for chain assembly is (4R,5R)-4,5-dihydroxycyclohex-1-enecarboxylic acid derived from the shikimate pathway. We show here that the first committed step in its formation is hydrolysis of chorismate to form (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. This chorismatase activity is encoded by fkbO in the FK506 and FK520 biosynthetic gene clusters, and by rapK in the rapamycin gene cluster of Streptomyces hygroscopicus. Purified recombinant FkbO (from FK520) efficiently catalyzed the chorismatase reaction in vitro, as judged by HPLC-MS and NMR analysis. Complementation using fkbO from either the FK506 or the FK520 gene cluster of a strain of S. hygroscopicus specifically deleted in rapK (BIOT-4010) restored rapamycin production, as did supplementation with (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. Although BIOT-4010 produced no rapamycin, it did produce low levels of BC325, a rapamycin analog containing a 3-hydroxybenzoate starter unit. This led us to identify the rapK homolog hyg5 as encoding a chorismatase/3-hydroxybenzoate synthase. Similar enzymes in other bacteria include the product of the bra8 gene from the pathway to the terpenoid natural product brasilicardin. Expression of either hyg5 or bra8 in BIOT-4010 led to increased levels of BC325. Also, purified Hyg5 catalyzed the predicted conversion of chorismate into 3-hydroxybenzoate. FkbO, RapK, Hyg5, and Bra8 are thus founder members of a previously unrecognized family of enzymes acting on chorismate.

Biosynthetic potential of phylogenetically unique endophytic actinomycetes from tropical plants.

The culturable diversity of endophytic actinomycetes associated with tropical, native plants is essentially unexplored. In this study, 123 endophytic actinomycetes were isolated from tropical plants collected from several locations in Papua New Guinea and Mborokua Island, Solomon Islands. Isolates were found to be prevalent in roots but uncommon in leaves. Initially, isolates were dereplicated to the strain level by ribotyping. Subsequent characterization of 105 unique strains by 16S rRNA gene sequence analysis revealed that 17 different genera were represented, and rare genera, such as Sphaerisporangium and Planotetraspora, which have never been previously reported to be endophytic, were quite prevalent. Phylogenetic analyses grouped many of the strains into clades distinct from known genera within Thermomonosporaceae and Micromonosporaceae, indicating that they may be unique genera. Bioactivity testing and liquid chromatography-mass spectrometry (LC-MS) profiling of crude fermentation extracts were performed on 91 strains. About 60% of the extracts exhibited bioactivity or displayed LC-MS profiles with spectra indicative of secondary metabolites. The biosynthetic potential of 29 nonproductive strains was further investigated by the detection of putative polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes. Despite their lack of detectable secondary metabolite production in fermentation, most were positive for type I (66%) and type II (79%) PKS genes, and all were positive for NRPS genes. These results suggest that tropical plants from New Guinea and the adjacent archipelago are hosts to unique endophytic actinomycetes that possess significant biosynthetic potential.