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Maritime Natural Products Conference August 16 – 18, 2017
University of Prince Edward Island Location: School of Sustainable Design Engineering, Rm 128B
Sponsorship:
Synapse Nautilus Biosciences Canada
Bruker Canada DSM (Ocean Nutrition Inc.)
PEI BioAlliance University of Prince Edward Island
BioVectra Thermo Fisher Scientific
Fisher Scientific Delivra Waters Tecan
Natural Products Canada Canadian Journal of Chemistry
VWR
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Program Schedule
Wednesday, August 16th – The Pourhouse (above The Old Triangle) 18:00 – 20:00 Welcome mixer (drinks and snacks) Thursday, August 17th – UPEI School of Sustainable Design Engineering Rm 128B 08:10 – 08:20 Welcome – Russ Kerr Session 1 Chair – Russ Kerr 08:20 – 09:20 David Newman, “Converting Natural Product Discoveries to Drugs,
with an Emphasis on Marine Sources” 09:20 – 09:40 Kelsie Armstrong (Gray/Johnson, UNB) 09:40 – 10:00 Logan MacIntyre (Kerr, UPEI) 10:00 – 10:20 Madison Carroll (Jakeman, Dal) 10:20 – 10:40 Break (SSDE Foyer) Session 2 Chair – Chris Gray 10:40 – 11:00 Sarah Greening (Thompson, Dal) 11:00 – 11:20 Jennifer Kolwich (Sit, SMU) 11:20 – 11:40 Beth Buchanan (Delivra, PEI) 11:40 – 12:00 Leon Liang (Kerr, UPEI) 12:00 – 12:20 Michael Beh (Thompson, Dal) 12:20 – 13:20 Lunch (SSDE Foyer) Session 3 Chair – David Jakeman 13:20 – 13:40 John Riley (BioVectra, PE) 13:40 – 14:00 Stephanie Forget (Jakeman, Dal) 14:00 – 14:20 Aleya Quilty (Kirby, UPEI) 14:20 – 14:40 Trevor Clark (Gray/Johnson, UNB) 14:40 – 15:00 Andrew Robertson (Kerr, UPEI) 15:00 – 15:20 Break (SSDE Foyer) 15:20 –17:00 Poster session (SSDE Foyer) 18:00 Dinner (Peake’s Quay Restaurant)
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Friday, August 18th – UPEI School of Sustainable Design Engineering Rm 128B Session 4 Chair – Clarissa Sit 08:20 – 8:40 Connor Lamont (Thompson, Dal) 08:40 – 9:00 Riley Jackson (Kerr, UPEI) 09:00 – 09:20 Bradley Davis (Gray/Johnson, UNB) 09:20 – 09:40 Hope Igboeli (Kerr, UPEI) 09:40 – 10:00 Jeanna MacLeod (Jakeman, Dal) 10:00 – 10:20 Jennifer Vacon (Kirby, UPEI) 10:20 – 10:40 Break (SSDE Foyer) Session 5 Chair – Justin Moores 10:40 – 11:00 Marie Charles (Kerr, UPEI) 11:00 – 11:20 James Tweel and Tiffany Cameron (Kerr, UPEI) 11:20 – 11:40 Sabrena MacKenzie (NRC-Halifax) 11:40 – 12:00 Morgan Crosby (Sit, SMU) 12:00 – 12:20 Andrew Flewelling (Gray/Johnson, UNB) 12:20 – 13:20 Lunch (SSDE Foyer) Session 6 Chair – Alison Thompson 13:20 – 13:40 Qingfeg Meng (Delivra, PE) 13:40 – 14:00 Jian-she Zhu (Jakeman, Dal) 14:00 – 14:20 Stacey Goldberg (Kerr, UPEI) 14:20 – 14:40 Lauren Forgrave (Gray/Johnson, UNB) 14:40 Final Remarks – Russ Kerr
Thanks for attending and safe travels.
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Abstracts for Oral Presentations Converting Natural Product Discoveries to Drugs, with an Emphasis on Marine Sources Dr. David Newman Newman Consulting llc Retired Chief, Natural Products Branch at National Cancer Institute
Although there have been around 25,000 reported bioactive materials isolated from marine sources over the last 40 or so years, the number of “Marine-sourced drugs” approved world-wide is less than 20, though there are a number of agents in clinical trials where a significant number may well “make the cut” in the relatively near future. This presentation will discuss the problems, both foreseen and unforeseen, that have allowed researchers from all areas of science to convert an initial finding of bioactivity into a viable drug candidate. A common thread that will run through the presentation is that the “source” of a particular compound, may well not be the actual one, and will demonstrate how those “beautiful marine invertebrates” have utilized microbes to aid in their defence, and how man has now been able to exploit these findings, albeit many years later.
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Antimycobacterial natural products from the Canadian medicinal plant Pinus strobus Kelsie Armstrong,1 Trevor N. Clark,1 John A. Johnson1 and Christopher A. Gray1,2 1 Department of Biological Sciences, University of New Brunswick, Saint John 2 Department of Chemistry, University of New Brunswick, Fredericton Presenting author: Undergraduate student Pinus strobus, the eastern white pine, is a plant used medicinally by some Canadian First Nations to treat symptoms associated with tuberculosis. Bioassay screening revealed that the methanolic extract of P. strobus branches significantly inhibited the growth of Mycobacterium tuberculosis H37Ra in vitro. Fractionation of the extract resulted in the isolation of two diterpene acids, isopimaric acid and copalic acid, that were responsible for the observed antimycobacterial activity of the extract (IC50 values of 16 µM and 120 µM respectively). This study provides an additional example in which the bioactivities of natural products isolated from a plant extract correlate well with the documented ethnopharmacological uses of the plant.
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Fungal co-culture: mimicking nature to discover new antimicrobials Logan MacIntyre, Hebelin Correa, Bradley Haltli, Douglas Marchbank and Russell Kerr Department of Biomedical Sciences, Atlantic Veterinary College, Charlottetown, PE Nautilus Biosciences Inc., Charlottetown, PE Many fungal taxa have been shown to possess natural product gene clusters that remain unexpressed under typical laboratory conditions and the corresponding natural products are termed ‘cryptic’. Cryptic natural products comprise an attractive new chemical resource for drug discovery and it is the objective of this work to access such compounds in an effort to uncover new antimicrobials. As a pilot study, a small collection of seven fungi collected from the same marine habitat (the Caribbean octocoral Eunicea fusca) were grown in every pairwise combination in order to induce the production of cryptic natural products by the constituent strains. Each pair was compared to its respective pure cultures in a UPLC-HRMS based metabolomic experiment and metabolites present only in co-culture were identified as putatively cryptic. To complement this experiment, all culture extracts were tested for antimicrobial activity against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, Pseudomonas aeruginosa and Candida albicans. The results of this pilot study validate the biochemometric approach taken to identify potentially new antimicrobials from fungal co-cultures and substantiate its application to larger fungal collections.
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Expression of 19F-Labeled β-Phosphoglucomutase and the Study of its TSA Complexes Madison Carroll, Anna Ampaw, Debabrata Bhattasali, David L. Jakeman. β-phosphoglucomutase (βPGM), a phosphate transfer mutase, catalyzes the transfer of a phosphate group from β-glucose 1-phosphate (G1P) to produce glucose 6-phosphate (G6P), proceeding though a β-glucose 1,6-bisphosphate (βG16P) intermediate as well as two transition states (Scheme 1).1 To study the two transition states of the phosphoryl transfer, transition state analogue (TSA) complexes were made using metal fluorides (MgF3- or AlF4-) and phospho or phosphono-glucosides.2 To easily monitor the complexation, 19F was incorporated into the protein as a spectroscopic probe. A βPGM containing two 5-fluorotryptophan (5-FW) residues was produced, as well as two mutants (W24F and W216F), each containing only one of the 5-FW residues, with the other replaced by a phenylalanine residue. 19F NMR spectroscopy was used first to assign the resonances belonging to each 5-FW residue, and then to monitor the formation of TSA complexes. The W216F 5-FW-βPGM formed transition state analogue 2 (TSA2) complexes with G6P and transition state analogue 1 (TSA1) complexes with β-glucose 1-C-phosphonate (G1CP) and (S)-1-β-phosphonofluoromethylene-1-deoxy-D-glucopyranose (G1CFsP), with the protein forming the AlF4-G1CFsP complex most readily. The formation of the complex was most easily observed for the W216F mutant, where a new signal at -121.7 ppm corresponded to the complexed protein. The intensity of this resonance was comparable to the intensity of the resonance for the bound G1CFsP and for the metal fluoride complexes. This is the first time 19F NMR spectroscopy has been used to probe the enzyme, ligand, and metal fluoride complex, demonstrating that the molar ratios of each species exist and indicating that the metal fluorides are representative of the transition state.
Scheme 1. βPGM catalyzed conversion of β-glucose 1-phosphate to β-glucose 6-phosphate.
1. Jin Y, Bhattasali D, Pellegrini E, et al. PNAS. 2014;; 111(34):12384-12389. 2. Baxter NJ, Olguin LF, Goličnik M, et al. PNAS. 2006;; 103(40):14732-14737.
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Synthesis and Characterization of Prodigiosene F-BODIPYs
Sarah Greening and Alison Thompson Dalhousie University
PET
Fluorescence OFFin CH2Cl2
PET
Fluorescence ONin hexanes
Prodigiosin is a natural product isolated from select bacterial strains. This tripyrrolic natural product and synthetic analogs termed prodigiosenes, are reported to have an impressive array of biological activities including immunosuppressive, antimalarial, anticancer, and antimicrobial effects, among others. Additionally, prodigiosin has been shown to have potential as a natural colorant in the food industry as well as use in sunscreen. Boron dipyrrin (BODIPY) dyes have attracted much attention due to their chemical robustness, large molar absorption coefficients, high fluorescent quantum yields and high photophysical stability. These criteria are critical when developing dyes for fluorescent imaging and other biomedical applications. As such, a series of synthetic analogs of the natural product prodigiosin have been transformed into the respective F-BODIPYs and the photophysical properties observed. Variation of substituents on the B- and C-ring results in a red-shift in absorption and emission maximum wavelengths approaching 600 nm. Additionally, the Stokes’ shift of prodigiosene F-BODIPYs are controlled by the electronic effects of B-ring substituents. The prodigiosene F-BODIPY bearing a dimethylaniline substituent exhibited a photo-induced electron transfer (PET) phenomenon which was effectively controlled depending on the polarity of the solvent, and the protonation state.
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Utilizing micro-ecological activity against bat pathogen Psuedogymnoascus destructans as a means of discovering novel antimicrobial compounds. Jennifer Kolwich, Clarissa S. Sit Saint Mary’s University, Halifax NS White nose syndrome (WNS) is an emergent disease in North American bat populations. Since the first documented case in New York one decade ago, WNS has spread to 31 states and 5 provinces, leading to population decreases of 90-100% in some sites. The causative agent of WNS was determined to be the psychrophilic fungal pathogen Pseudogymnoascus destructans, a species native to the Palearctic Region, which was anthropogenically spread to North America. Previous investigations have revealed that despite the presence of P. destructans and subsequent WNS in Europe and Asia, these regions are not experiencing similar widespread mortality in their bat populations. Recent studies which have had success at treating WNS revolve around the status of P. destructans as an invasive species in the the skin microbiome of North American bats and how micro-ecological defenses of pre-existing cutaneous specimens could be the key in reducing the effect of the fungus.1-3 Most of these studies focus on determining which strains of microbes can most effectively inhibit the P. destructans fungus, but little has been done in terms of separating and testing the metabolites of effective strains to determine their identity and mechanism of inhibition. To investigate these interactions, cutaneous samplings from various unaffected Canadian bat species will be pairwise tested against P. destructans and the closely related Pseudogymnoascus bhatii. Strains which show effective inhibition will be cultured on a larger scale and identified using rRNA sequencing. Metabolites will be extracted and fractionated, then tested directly against the Pseudogymnoascus species. Active compounds will be compared to previously reported natural products and unreported compounds will undergo complete characterisation. Cornelison, C. T., et al. BMC Microbiology, (2014), 14: 246. Hoyt JR, et al. PLoS ONE (2015) 10(4): e0121329. Cheng, T. L., et al. J Appl Ecol (2017), 54: 701–708.
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Natural Product for Early Indications of Metabolic Disorder Buchanan, Beth., Meng, Qingfang., Poulin, Mathieu-Marc., Zuccolo, Jon., Zuccolo, Amir., Azike, Chike., Gabriele, Joseph., Baranowski, David. Metabolic disorders encompass pathologies in which general energy metabolism and circulation are dysfunctional and are the second leading cause of death in Canada. Individuals with metabolic syndrome typically have irregular levels of glucose (diabetes), cholesterol, and/or triglyceride circulation and storage (dyslipidemia) and are at higher risk for heart attack and stroke. Delivra Inc. has begun development of a new product that focuses on early indications of cardiovascular disease using berberine (BBr), a naturally occurring isoquinoline in plants known for its lipid lowering properties. To date, berberines potential has not been fully realized due to its poor oral bioavailability (~1%) and gastrointestinal upset.
Delivra Inc. has formulated berberine for transdermal administration with promising results, however, due to its intense yellow colour it is not ideal for a topical application. To overcome this challenge, Delivra Inc. synthesized several berberine congeners with reduced spectroscopic properties to screen for bioactivity analogous to berberine. One berberine derivative (DHB) was observed to mirror berberines PCSK9 inhibition in vitro.
Acute pharmacokinetics in Sprague-Dawley rats comparing oral BBr and transdermal-DHB (patent pending formulation) demonstrated an increase in bioavailability with AUC0-8 of 26.5 and 202.3 ng·hr/mL, respectively. Importantly, the metabolite berberine-glucuronide is observed with similarly improved kinetics demonstrating that transdermal application achieves hepatic targeting. This same trend is observed during chronic administration (14 days, once daily) with oral BBr, Delivra-BBr, and Delivra-DHB yielding 1.0, 0.5, and 20 ng/mL, respectively. In a pilot study using Zucker fa/fa rodents as a model of metabolic disorder, Delivra-DHB treated animals achieved a reduction in cholesterol by 34% and reduction in triglycerides by 85%, compared to control. Thus far, experimentation has supported the development of a novel transdermal-DHB formulation and subsequent testing is designed to further solidify the products efficacy and safety in pre-clinical and clinical studies.
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Co-culture Mimic as a Tool for Natural Product Discovery Leon Libang Liang and Rusell Kerr Department of Chemistry, University of Prince Edward Island Co-cultivation of microbial organisms has led to the discovery of novel natural products. Co-culture mimic modifies the conventional co-culture methodology aiming to improve reproducibility. By supplementing autoclaved “inducer” cultures to the “producer” fermentations, co-culture mimic has resulted in various alterations in the “producer” metabolome. Fermentations were analyzed by a high-throughput LC-HRMS platform. Non-targeted metabolomics studies are carried out to monitor various stimulation effects from addition of “inducer” cultures. These effects include strong induction of biosynthesis of a novel natural product, biotransformation of quorum sensing molecules, and stimulation of natural product production.
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The Hunt for Air-Stable Tetrapyrroles to Mimic those Found in Heme Biosynthesis Michael H. R. Beh and Alison Thompson Dalhousie University
Tetrapyrroles are a common substrate for enzymes in the biosynthesis of heme. Their inherent instability, often oxidizing spontaneously in air, makes studying the enzymatic pathway a challenging endeavour. Puckered tetrapyrrolic compounds linked by C-CH2-C bridges (bold, Figure 1), e.g. uroporphyrinogen III (1), are the most susceptible to this oxidation, thus leading to C=CH-C bridges and planar structures. Replacing the C-CH2-C bridges with functional groups of similar size and tetrahedral geometry has the potential to produce synthetic inhibitors for the biosynthesis of heme. Replacement of the C-CH2-C moiety is a long-standing project in the Thompson group and the current goal is substitution with sulfur-based groups, thus producing a sulfur-bridged tetrapyrrole (3, Figure 1B). This presentation will focus on recent work developing a method to prepare sulfide-bridged bispyrroles.
Oxidation
1 2
R1 = CH2CO2HR2 = CH2CH2CO2H
R1 = CH2CO2HR2 = CH2CH2CO2H
X = S, sulfideX = SO, sulfoxideX = SO2, sulfone
NHH2C
CH2
HNCH2
HN
H2C
NHR1
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X
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A) B)
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Figure 1
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Culturing a cure: A case study on the manufacturing of Doxorubicin John Riley BioVectra Inc., Charlottetown, PEI Doxorubicin is a cytotoxic anthracycline based compound that is isolated from cultures of Streptomyces peucetius. It is employed as a chemotherapy medication to treat various forms of cancer including;; breast, bladder, Kaposi's sarcoma, lymphoma, and acute lymphocytic leukemia. It is also often used together with other chemotherapy agents and in the development of novel, complex formulations for targeting various forms of cancer. Due to its wide use, access to large quantities of Doxorubicin is needed to ensure that patients receive medicine when needed. The manufacturing of Doxorubicin will be presented as a case study to demonstrate key concepts for scaling up microbial fermentation processes.
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HO
O
CH3
O
OOH
HOHO
OHO
O
CH3
O
+ UDP-glcSv0189
Mg2+, pH 8.0+ UDP
Characterization of a Promiscuous Glycosyltransferase from S. venezuelae ISP5230 Stephanie M. Forget,a David L. Jakemana,b aDepartment of Chemistry and bCollege of Pharmacy, Dalhousie University A 2015 analysis of bacterial secondary metabolites found that one fifth were glycosylated. 1 The glycosylation of secondary metabolites alters physiochemical properties with implications on bioactivity. In some instances, glycosylation is essential for bioactivity, as is the case for the sugar moiety of the chemotherapeutic doxorubicin,2 while in other cases glycosylation deactivates bioactivity, for instance glucuronidation during phase II metabolism. This potential to tune bioactivity has resulted in interest in the discovery of expedient and reliable methods for glycodiversification. S. venezuelae ISP5230 (ATCC 10712) is a prolific producer of secondary metabolites with more than 30 putative secondary metabolite gene clusters of which seven have associated characterized natural products, including chloramphenicol and jadomycin. Of five GT-1 family glycosyltransferases encoded within the S. venezuelae genome, one protein, SVEN_0189, stood out for its potential application as a glycosylation tool, given its high sequence identity (74 %) to a well-studied glycosyltransferase, OleD. 3 SVEN_0189 was cloned into a pET28a E. coli expression vector;; purification of the N-His6 tagged protein after IPTG induction was accomplished using Nickel affinity chromatography. While OleD is known to physiologically catalyse transfer of glucose to the macrolide oleandomycin,4 the physiological acceptor of SVEN_0189 is unknown. Glycosyltransferase activity has been demonstrated using UDP-glucose as the sugar donor and 4-methylumbelliferone as the acceptor, as shown in the scheme. In this talk progress towards the characterisation of SVEN_0189 will be discussed, including kinetic assays and screening assays with a diverse set of acceptor molecules.
1. Elshahawi, S. I.;; Shaaban, K. A.;; Kharel, M. K.;; Thorson, J. S. Chem. Soc. Rev. 2015, 44, 7591-7697. 2. Di Marco, A.;; Casazza, A. M.;; Gambetta, R.;; Supino, R.;; Zunino, F. Cancer Res. 1976, 36, 1962-1966. 3. Zhou, M.;; Hamza, A.;; Zhan, C.;; Thorson, J. S. J. Nat. Prod. 2013, 76, 279-286. 4. Quiros, L. M.;; Aguirrezabalaga, I.;; Olano, C.;; Mendez, C.;; Salas, J. A. Mol. Microbiol. 1998, 28, 1177-1185.
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Anthocyanin Derived Pigments for Use in Food Dyes Aleya T. Quiltya,c, Jason L. McCallumb,c, Brian D. Wagnera and Christopher W. Kirbya,c Departments of aChemistry and bBiology, University of Prince Edward Island, Charlottetown, PE, C1A 4P3 cCharlottetown Research and Development Centre, Agriculture and Agri-Food Canada, Charlottetown, PE, C1A 4N6 A library of Anthocyanin Derived Pigments (ADPs) is being developed by the reaction of anthocyanins (the compounds responsible for the red, blue and purple colours in most fruits and vegetables) with hydroxycinnamic acids. These abundant pigmented compounds are water-soluble, non-toxic, anti-oxidant agents and are commonly used as food colourants;; however they are sensitive to industrial processing. Pyranoanthocyanins are ADPs which are more stable than simple anthocyanins in acid-base chemistry which prevents aromatic ring fission reactions during cooking/pasteurization, storage and fermentation processes. Along with the benefits of an anthocyanin, ADPs are also anti-inflammatory and are promising compounds for development of novel food dyes. Our ongoing efforts towards creation and characterization of ADPs will be presented. The ultimate goal of this research is to create novel ADP food dyes from Canadian agricultural industry waste streams.
Figure 1. Proposed combinatory reaction for the production of ADPs.
Pyranoanthocyanin
Hydroxycinnamic Acid Anthocyanin
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Bioactive natural products from endophytes of Canadian medicinal plants Trevor N. Clark,1 John A. Johnson1 and Christopher A. Gray1,2 1 Department of Biological Sciences, University of New Brunswick, Saint John 2 Department of Chemistry, University of New Brunswick, Fredericton Presenting author: Graduate student Natural products are an excellent source of diverse and complex chemical structures and endophytic fungi have proven to be a useful source of natural products. The principal objective of the UNB Natural Products Research Group is the discovery of bioactive natural products from fungal endophytes. This presentation will provide an overview of our progress and highlight the natural products isolated thus far from endophytes of Canadian medicinal plants using bioassay and NMR metabolomics prioritization methods.
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Isolation of Imaqobactin, an Amphiphilic Siderophore from the Arctic Marine Bacterium Variovorax paradoxus RKJM285 Andrew W. Robertson and Russell G. Kerr Iron is an essential micronutrient used by almost all organisms for cellular processes requiring iron dependent heme-enzymes. Despite the biological importance of iron, and its high natural abundance on earth, the predominant oxidation state in aerobic environments is Fe(III), often existing as insoluble ferric oxides. Due to this poor solubility, iron bioavailability within these environments is often limited. To counter this, many microorganisms release high affinity small molecule chelators known as siderophores. These natural products are excreted into the surrounding environment to scavenge Fe(III) which is then actively transported back into the organism where it can then be further processed for use in cellular operations. These compounds are important for environmental iron cycling, and are directly involved in shaping microbial biodiversity within microbiomes. The Canadian arctic represents a relatively unexploited microbiome for natural products discovery. The arctic marine environment has been typically dismissed as a rich source of natural products due to belief that low biodiversity within the area will yield low chemical diversity. Conversely, recent literature suggests for microorganisms to tolerate this extreme climate and low nutrient availability, unique chemical diversity must have developed. Herein we describe the isolation and structural characterization of an amphiphilic siderophore from the bacterium Variovorax paradoxus RKJM285 isolated from sediment sample near Clyde River, Nunavut. Absolute stereochemistry of a depsipeptide core was determined by Marfey’s analysis, and the relative stereochemistry of a 2-methyl-3-hydroxy-4,7-diaminoheptanoic acid moiety was determined by NOESY and selective nOe experiments. Metal binding and photo-reductive properties are discussed. Antimicrobial and cytotoxic screening identified the compound as a moderate antimicrobial agent. To the best of our knowledge this represents the first siderophore structurally characterized from a marine arctic source, and highlights the importance of continued efforts towards screening for chemical diversity within this extreme environment.
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Complexation of Boron to Functionalized Pyrrole Rings Connor J. Lamont and Alison Thompson Dalhousie University
Pyrroles are well documented as key structural components of a diverse number of natural products. Notable examples of pyrroles and their roles in a biological context include the antimicrobial and anticancer properties of prodigiosin, the antibiotic effects of clorobiocin and coumermycin A1, and the chelating ability of porphyrins such as heme b, chlorophyll α, vitamin B12, and factor 430. The chelating ability of pyrrole-containing compounds is highlighted by boron dipyrrin (BODIPY) complexes, a class of dyes whose high fluorescent quantum yields has led to their use in medical imaging procedures and other biomedical applications. Successful synthesis of boron iminopyrrole (BOIMPY) complexes has led to the further investigation of functionalized pyrroles and their ability to form novel boron complexes. This presentation will focus on the variety of synthetic routes that were taken in attempts to form novel boron complexes with pyrroles containing formyl, keto, thioester, and thione moieties.
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Synthesis of Cystargolide Analogues, 20S Proteasome Inhibitors with Potent Anticancer Activity Riley Jackson, Andrew Robertson, Douglas Marchbank, Russell Kerr Cystargolides A and B, two β-lactone-containing natural products previously isolated from Kitasatospora cystarginea by the Kerr Lab, exhibit anticancer activity through inhibition of the 20S proteasome.1 A total synthesis has been established by Tello-Aburto et al. which showed that benzylation of the cystargolides improved their activity against the 20S proteasome by two orders of magnitude.2 The Kerr lab recently investigated structure-activity relationships (SAR) around the β-lactone region, revealing that the naturally occurring isopropyl group resulted in more potent activity than other substituents in the 20S proteasome assay.3 The purpose of this research is to assess the SAR around the dipeptide region by synthesizing cystargolide analogues containing different dipeptides appended to the isopropyl β-lactone core in an effort to identify analogues with improved anticancer activity. In order to achieve this objective, the isopropyl β-lactone core must be synthesized in sufficient quantities to generate the library of cystargolide analogues. The molecular docking program AutoDock 4 with AutoDock Tools will then be utilized to predict SAR in silico before selecting individual amino acid building blocks for the dipeptide synthesis. Progress to date shall be discussed.
References (1) Gill, K. A., Berrué, F., Arens, J. C., Carr, G., and Kerr, R. G. (2015) Cystargolides, 20S Proteasome Inhibitors Isolated from Kitasatospora cystarginea. J. Nat. Prod. 78, 822–826. (2) Tello-Aburto, R., Hallada, L. P., Niroula, D., and Rogelj, S. (2015) Total synthesis and absolute stereochemistry of the proteasome inhibitors cystargolides A and B. Org. Biomol. Chem. 13, 10127–10130. (3) Robertson, A.W.;; Marchbank, D.H.;; Kerr, R.G. Manuscript in preparation.”
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Isolation of endophytes from the roots of Canadian medicinal plants Bradley R. Davis,1 John A. Johnson1 and Christopher A. Gray.1,2 1 Department of Biological Sciences, University of New Brunswick, Saint John 2 Department of Chemistry, University of New Brunswick, Fredericton Presenting author: Graduate student Endophytic fungi have been identified as a relatively unexplored source of new chemical diversity. We are investigating the assemblage of endophytic fungi in the root systems of four plants with a history of medicinal use (Chimaphila umbellata, Heracleum maximum, Polypodium virginianum, and Rubus allegheniensis) using both culture-dependent and culture-independent techniques. To date, 91 distinct endophytic fungi have been isolated overall, with greater diversities being obtained on 2.4% potato dextrose agar (52 distinct isolates) than on 2% malt extract agar (39 distinct isolates). However, based on recent estimates, it is likely that the vast majority of fungal endophytes are not being isolated using traditional approaches on solid media. We will therefore be undertaking a culture-independent estimate of the fungal endophyte species present in the root tissues of C. umbellata, H. maximum, P. virginianum, and R. allegheniensis to obtain a more representative estimate of the endophytes resident in these plants.
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Discovery of a new polyketide via co-treatment with an epigenetic modifier and an osmotic stress
Hope Igboeli, Douglas Marchbank, Hebelin Correa, David Overy, Russell Kerr
Department of Chemistry, University of Prince Edward Island
Genomic analysis of filamentous fungi has revealed the presence of “silent” biosynthetic pathways which encode for unknown natural products. Silent biosynthetic genes are ones that are present in an organism’s genome but are not expressed in detectable amounts during routine cultivations. The number of silent biosynthetic gene clusters often greatly outnumbers the number of commonly expressed natural products. This insight has fuelled the development of new strategies for natural products discovery in fungi to optimize their biosynthetic potential. Herein we describe a new method of biosynthetic gene activation in fungi involving the initiation of both stress and epigenetic pathways to simultaneously up-regulate cryptic or low abundant natural products. This method has lead to the discovery of a new polyketide, which was isolated from the fermentation extract of Asteromyces cruciatus treated with suberoylanilide hydroxamic acid and salt. The polyketide was purified by reversed-phase flash column chromatography followed by preparative high performance liquid chromatography. The structure of this compound was established using one-and-two dimensional NMR experiments.
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Targeted natural product discovery by activation of silent Streptomyces venezuelae gene cluster through overexpression of pathway-specific regulator Jeanna M. MacLeod (1), David L. Jakeman (1,2) 1 College of Pharmacy, Dalhousie University 2 Department of Chemistry, Dalhousie University Natural products from bacteria and plants are a valuable source of drug candidates, as they are designed through evolution to interact with biological systems. Streptomyces venezuelae ISP5230 has been studied for its ability to produce natural products with biological activity, chloramphenicol and the jadomycin family of compounds. An analysis of the S. venezuelae genome sequence using antiSMASH 3.0 software indicated the presence of more than thirty uncharacterized biosynthetic gene clusters (BGCs). Many of the identified BGCs bear limited homology to known gene clusters or sub-clusters, suggesting they may produce natural products with novel chemistry. These “silent” BGCs which are un- or under-expressed in laboratory conditions can be overexpressed by inserting promoter sequences upstream of activator or structural genes or derepressed by deleting repressor genes. Cluster R6 contains a sub-cluster homologous to the indolocarbazole scaffold biosynthetic genes downstream from a putative large ATP-binding LuxR (LAL) family activator. Indolocarbazoles are generally effective inhibitors of protein kinases and are candidates for anti-cancer drug therapy. A vector was constructed to insert the strong constitutive ermE* promoter and a ribosomal binding site ahead of the LuxR gene by homologous recombination. The vector was transformed into wild-type S. venezuelae ISP5230 by conjugation, and selection of a mutant strain with the ermE* promoted LuxR gene was successful. Multiple culture conditions favorable for indolocarbazole production are being screened to assess the production of novel natural products. Any new compounds detected through screening will be isolated and characterized. Novel structures will be assessed for biological activity in anti-proliferative assays.
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Bio-Protectant Development to Control Potato Pathogens
ND Vacona,b <[email protected]>, SM Boyetchkoc and CW Kirbya,b aDepartment of Chemistry, University of Prince Edward Island, Charlottetown, PE, C1A 4P3, bCharlottetown Research and Development Centre, Agriculture and Agri-Food Canada, Charlottetown, PE, C1A 4N6, cSaskatoon Research and Development Centre, Agriculture and Agri-Food Canada, Saskatoon, SK, S7N 0X2
Bioautography methods are being used to screen for bio-protectant candidates produced from an established soil bacterial collection to control potato post-harvest pathogens: Phytophthora erythroseptica (Pink Rot), Colletotrichum coccodes (Black Dot), Helminthosporium solani (Silver Scurf), and Streptomyces scabies (Common Scab). Direct bioautography methods require large amounts of spores to conduct the assay in a reasonable time-frame;; however determining growth conditions to induce sporulation of our target pathogens has proved to be taxing. To overcome this problem, we are using a modified method where the culture can be taken directly from its standard grown conditions on a petri plate to create the microbial suspension for the assay to allow rapid screening for antimicrobial compounds. These ongoing efforts to improve potato seed storage and viability through the screening and isolation of potential bio-protectant candidates for environmentally-friendly post-harvest treatment methods against these pathogens will be presented.
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Natural Product Discovery and Development of a Novel Method to Quantify Bacteria from Marine Habitats in Remote Locations
Marie Charles, Brad Haltli, and Russell G. Kerr Department of Chemistry, University of Prince Edward Island Many approaches in natural product discovery have been focused on inducing the expression of silent biosynthetic genes in previously isolated microbes. However, the microbial diversity of earth’s biosphere is much larger than the microbial libraries from which these organisms are sourced. To reveal the missing microbial diversity, and to possibly discover untapped resources of novel natural products, new isolation methods are required. In 2010, a research team from Northeastern University developed the ‘isolation chip’ (iCHIP). We have modified this device for use in sponges in a preliminary attempt to isolate new strains of bacteria. Preliminary field collections of sponges were conducted in The Bahamas. Following collection, isolated microbes were fermented in different marine media to source natural products. Additionally, the field research revealed that to effectively use this device it is necessary to quantify the bacterial densities of the environmental samples. In the laboratory, this can be achieved via fluorescence microscopy;; however, this technique is not applicable to field studies. Alternatively, adenosine triphosphate (ATP) can be used as a measure of living cells. Due to the availability of relatively low cost portable luminometers and the commercial availability of ATP test kits, this approach is an appealing method for estimating bacterial abundance for iCHIP studies conducted in the field. Preliminary data will be discussed.
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Miniaturized Growth Chambers for the Isolation of “Unculturable” Bacteria James Tweel, Tiffany Cameron, Bradley Haltli, Ali Ahmadi, Russell Kerr University of Prince Edward Island, Charlottetown, Canada Large scale bacteria cultivation is commonly used to discover the biological potential of micro-organisms and to produce novel bioactive compounds. One major difficulty experienced in microbiology is known as “The Great Plate Count Anomaly” which refers to the fact that in any given sample, only 1% of the various bacteria strains present may be cultivated under standard laboratory conditions. The other 99% of “unculturable” bacteria are unable to grow through in vitro techniques as they require unique environmental factors and physical conditions for successful and significant growth. The resolution of this issue is of great importance as it leads to the discovery of novel natural products that can lead to the development of new products in a wide range of industries including healthcare. To address the great plate count anomaly problem and to highlight the importance of environmental elements and signaling molecules, microbiologists have attempted in situ cultivation of bacteria, particularly, through development of the isolation chip (iChip). In short, the iChip allows in situ cultivation of bacteria by allowing exchange of environmental nutrients through miniature diffusion chambers found within the device. Although iChips have shown great potential in discovery of novel compounds, they are not suitable for high throughput assays. To address the limitations of the current iChip design, we propose a new design of a growth chamber and a novel microfluidic-based method of encapsulating bacteria in agarose beads. Samples of bacteria are retrieved from sponges or sediment and encapsulated in agarose micro-beads. These micro-beads are in turn placed inside the growth chamber that is inserted back into the sediment or sponge from which the bacteria originated. The device includes membranes to allow diffusion of exterior nutrients from the natural environment all the while preventing migration of bacteria cells. Using the proposed in situ approach, bacteria develop monospecific cultures within the beads while exchanging crucial signaling molecules and other environmental factors. Overall, the proposed new miniaturized growth chambers is expected to facilitate the isolation and culture of a range of new bacteria leading to discovery of novel bioactive natural products.
Figure 1: (a) The two latest 3D printed prototypes of the growth chamber (b) Preliminary micro-bead formation using water and oil taken with high speed camera
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Building an Algal Repository at the National Research Council
Sabrena MacKenzie, Pat McGinn, Joseph Hui, Fabrice Berrué Aquatic and Crop Resource Development, National Research Council, 1411 Oxford St., Halifax, Nova Scotia, Canada B3H 3Z1 The objective of the NRC’s Algal Carbon Conversion (ACC) flagship program is to develop commercially viable and environmentally sustainable solutions to transform CO2 emissions into value-added products. Microalgae are photosynthetic organisms of choice as they are capable of converting carbon dioxide into valuable biomass and natural products with applications including biofuels, food & animal feed, and natural health products. Over the past five years, the Algal Carbon Conversion Program has been collecting algal isolates from both marine and freshwater habitats and has built a library of more than 400 algal strains. Consequently, the Natural Product Chemistry team in NRC-Halifax has recently initiated a screening campaign in order to characterize the chemical composition of many of these microalgae strains grown under various conditions. Molecules of interest include amino acids, pigments, carotenoids, lipids (triacylglycerol (TAG), glycolipids, phospholipids), small natural products, and polysaccharides. These classes of compounds exhibit a wide range of polarity and molecular weight and have required the development and validation of complementary analytical techniques using both LC-HRMS and NMR. This presentation will introduce the overall analytical strategy to build an integrated and searchable algal repository and assist researchers and collaborators in the prioritization of lead microalgae strains with potential industrial applications.
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Pairwise Assay Investigation of Microbial Isolates from Nova Scotian Honey Bees as Potential Biocontrols for AFB Causing Bacterium Paenibacillus larvae Morgan C. Crosby, Dr. Clarissa S. Sit* Saint Mary’s University Honey bees are known to be one of the most important pollinators in the largely profitable world of agriculture. Yet, there has been a global decline of honey bees in recent years, with Colony Collapse Disorder (CCD) and American Foulbrood disease (AFB) as potential contributors to this decline, with no current well-established solutions. Previous studies suggest that there is potential for microbes isolated from the gut of honey bees to possess the capability to combat the bacterium at the root of AFB, Paenibacillus larvae.1 External microbes and internal gut microbes isolated from honey bees from several apiaries across Nova Scotia were sampled and are currently being analyzed for microbial content for pairwise assay testing against a commercially available strain of P. larvae. Future plans for genomic sequencing and chemical component analysis of metabolites, as well as potential natural products isolation are discussed. 1. Forsgren, E.;; Olofsson, T. C.;; Vásquez, A.;; Fries, I. Apidologie, 2010, 41, 99-108.
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Application of a simple bioactivity profiling strategy to natural product discovery from endophytes of marine macroalgae Andrew J. Flewelling,1 John A. Johnson,1 Christopher A. Gray1,2 1 Department of Biological Sciences, University of New Brunswick, Saint John 2 Department of Chemistry, University of New Brunswick, Fredericton Presenting author: Graduate student The natural product chemistry of marine macroalgal endophytes is relatively unexplored despite these fungi being recognized as a promising source of new bioactive molecules. As redundancy in natural products discovery increases, new techniques are needed to prioritise extracts for fractionation. The use of bioactivity profiling provides an excellent, albeit labour intensive screening approach that facilitates the discovery of antibiotics with novel modes of action or cellular targets. Here we present a simplified method for bioactivity profiling that we have applied to a library of one hundred and forty extracts of endophytic fungi isolated from 20 species of marine macroalgae from the Bay of Fundy, Canada. Extracts were screened for antimicrobial activity against a suite of Gram positive and Gram negative bacteria, mycobacteria and fungi. These data were used to compile bioactivity profiles of each extract that were compared to each other and the profiles of known antibiotics representing a range of modes of action. Principle component analysis revealed that 37 extracts exhibited unique profiles within the extract library, and hierarchical cluster analysis indicated 26 of these extracts possessed profiles different from those of the antibiotics. Bioassay-guided fractionation has led to the isolation of antimicrobial natural products from the extracts of a Penicillium sp., a Tolypocladium sp., an unidentified sterile filamentous isolate, and Aspergillus fumigatus. We have demonstrated that a simple and efficient bioactivity profiling technique is effective for prioritising fungal extract libraries. We are confident that this technique will be a valuable tool for identifying natural products with unique antimicrobial modes of action.
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Cannabis sativa in the Canadian Marketplace and its Anti-inflammatory Potential Meng, Qingfang., Zuccolo, Jon., Poulin, Mathieu-Marc, Buchanan, Beth., Gabriele, Joseph., Baranowski, David. Cannabis sativa has been popular for over a millennium for both its medicinal and psychoactive properties. The medicinal and recreational properties of this plant are largely attributed to phytocannabinoids, a family of prenylated polyketides with >100 members. The major phytocannabinoids of Cannabis sativa are tetrahydrocannabinol (THC) and cannabidiol (CBD) along with their acidic forms THCA and CBDA. While legal recreational marijuana is forthcoming in Canada, the non-THC variety of Cannabis sativa - hemp - has been cultivated at an industrial agricultural level for several decades. Hemp (particularly seed oils) must demonstrate <0.03% (w/w) THC with no other limitations on cannabinoid content. Undoubtedly hemp-derived products illicit no psychoactive effect, however non-THC cannabinoids may have additional biological consequences, particularly anti-inflammatory properties. Using a series of commercially available hemp products, we investigated the concentrations of these major cannabinoids and the overall anti-inflammatory effects of each using HPLC/MS/MS and an in vitro assay monitoring cyclooxygenase activity, respectively. A method for the absolute quantification of THC, THCA, CBD, and CBDA in complex finished products was developed using appropriate deuterated standards in combination with high performance liquid chromatography and multiple reaction monitoring mass spectrometry. This method is amenable to finished products of various formats (e.g. oils, cosmetic creams, solids, etc.) with a lower limit of quantification of 0.195 ng/mL and a linear range from 0.195-50.0 ng/mL. The observed concentration of CBD, THC, CBDA and THCA in 40 commercial hemp products varied from 0-0.635% (w/w), 0-0.019% (w/w), 0-0.0052% (w/w), and 0-0.000497% (w/w). Based upon this data, hemp products adhere to the federally regulated limitation on THC content and may contain biologically significant levels of other cannabinoids, including CBD. In tandem to cannabinoid quantification a commercially available assay for human cyclooxygenase-2 (COX2) activity was modified to investigate the potential anti-inflammatory effects of these products along with the compound-specific COX2 inhibitory effects of pure cannabinoids. In contrast to published data our results indicate CBDA and THCA have no effect, whereas the IC50 values of CBD and THC are ~130 µM and ~66 µM, respectively. Screening all 40 commercial hemp products generated a variety of results and this COX-2 inhibitory activity did not correlate with cannabinoid content, suggesting hemp oils may contain other naturally occurring anti-inflammatory compounds. Given the plants origin and the legal restrictions on hemp cultivation it is not surprising that THC content meets regulatory criterion, albeit curious that a singular compound represents the barrier to market given the known effects of other cannabinoid family members. Furthermore, cannabinoid and non-cannabinoid content should be taken into account when evaluating the medicinal properties of this genus, particularly when the goal is to evaluate disease-modifying properties.
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Synthesis of C-1 branched α-D-glucopyranose 1- and 6-phosphate analogues: inhibitory evaluation of phosphoglucomutases and thymidylyltransferase
Jian-She Zhu1, David L. Jakeman*1
1College of Pharmacy, Dalhousie University, Halifax, Nova Scotia, Canada B3H 3J5
Phosphoglucomutase (PGM) exists in various living organism from bacteria to human.1 It catalyzes reversible intramolecular conversion of glucopyranose 1-phosphate (G1P) to glucopyranose 6-phosphate (G6P). It involves in the early steps of many biosynthetic pathways.2 Given its function, PGM has been viewed as an important connection between catabolic and anabolic processes. Cps2L,3 cloned from S. pneumoniae is a class of thymidylyltransferase, which converts G1P to dTDP-glucose, a precursor of many deoxy-sugar donor for production of various oligo- and polysaccharides.4 Both enzymes (PGM and Cps2L) can use G1P as a starting material. It was thus hypothesized that G1P or G6P analogues derivatized at their anomeric positions (C-1) will be potential inhibitors of these enzymes, due to the disfavor of steric and/or electronic factors resulting from these extra substitutions (CH3, CH2F, CHF2, CF3). As a result, a series of G1P and G6P analogues were synthesized. All of the G1P analogues were prepared by the conventional chemical synthesis starting from lactone, featuring a bypass route to install phosphate group through phosphitylation of benzylated ketose, followed by oxidation of the resultant phosphite.5 While G6P analogues were readily obtained in an enzymatic way using E. coli glucokinase (GlK).6
As expected, none of these analogues are substrates of Cps2L or PGM. Instead, they exhibit inhibition against these enzymes. Measuring their inhibition profiles is in progress. 1. Whitehouse, D. B., McMillan, W. O. Mol. Biol. Evol. 1998, 15, 456 2. O. C. Goldberg JB, S. R, Soberon-Chavez G, FEMS Microbiol Lett., 1999, 179, 85 3. Timmons, S. C., Jakeman, D. L. Org. Lett. 2007, 9, 857. 4. Thibodeaux, C. J., Liu, H.-w. Angew. Chem. Int. Ed. 2008, 47, 9814. 5. Mui Mui Sim, H. Kondo, Chi-Huey Wong, J. Am. Chem. Soc., 1993, 115, 2260 6. D. H. Kwan, Stephen G. Withers, FEBS Letters, 2016, 590, 461
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Investigation and Classification of Cultivable Bacteria Associated with Marine Sponges, Sediment and Seawater S. Goldberg1, B. Haltli1,2, H. Correa2, R. Kerr1,2
1 Department of Biomedical Sciences, Atlantic Veterinary College, PEI 2 Department of Chemistry, University of Prince Edward Island, PEI Marine sponges are increasingly becoming more relevant and essential to the advancement of microbiology research because of the abundance and diversity of their microbial communities. Sponge-associated microbial communities are ubiquitous, abundant, and diverse. Despite the fact that microorganisms can constitute up to 60% of sponge biomass, sponges remain as rich sources of potential for obtaining novel ‘yet to be cultured’ bacteria. The microbiomes of marine sponges are often considerably different from those in surrounding benthic habitats. For this reason, we investigated the composition of bacteria cultivated from four sponge species (Aplysina fistularis, Verongula gigantea, Xestospongia muta, and Ectoplasia ferox), collected from The Bahamas, in comparison to that of surrounding seawater and sediment. Close to 1000 bacterial colonies were cultured, collected and purified. Phylogenetic analysis revealed that the overall bacterial library encompassed 4 phyla, 19 families, and 30 genera, whereby 1 family, 4 genera, and 6 species were unclassified (<97% rRNA sequence similarity to described strains). Upon assessment of the high percentage of cultivated novel bacteria, we directed our efforts to describing and classifying these new strains. Here, we present the characterization of six novel strains of bacteria, five of them being sponge-associated and one derived from sediment. This research upholds the unceasing appeal of marine sources, sponges in particular, as ecologically rich niches for novel microbial discoveries.
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Natural products from Canadian medicinal plants of the genus Pyrola Lauren Forgrave,1 Trevor N. Clark,1 John A. Johnson1 and Christopher A. Gray.1,2 1 Department of Biological Sciences, University of New Brunswick, Saint John 2 Department of Chemistry, University of New Brunswick, Fredericton Presenting author: Undergraduate student Medicinal plants that have been traditionally used by the First Nations people of Canada have proven to be a rich source of natural products. Reports of the isolation of antibiotic natural products from Canadian specimens of Chimaphila and Moneses led us to investigate the natural products chemistry of related Pyrola species that had displayed antimycobacterial activity in our bioassay screens. Limited quantities of plant material precluded the isolation of the antimycobacterial constituents of the extracts although it did lead to the isolation of homoarbutin and isohomoarbutin from Pyrola elliptica and homoarbutin, isohomoarbutin and hyperoside from Pyrola americana. This presentation will discuss the isolation of the glycosides and the challenges that were encountered in the elucidation and confirmation of their structures.
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Abstracts for Poster Presentations Screening of a fungal library for fungicide adjuvants against the necrotrophic fungus Botrytis cinerea
Mustafa Tahir1, Kate McQuillan2, Martin Lanteigne1, Hebelin Correa2 and Russell Kerr1 1Department of Biology, University of Prince Edward Island, Charlottetown, PE, 2Nautilus Biosciences Inc. Charlottetown, PE Botrytis cinerea is an infectious pathogen affecting many plant species and is known to be highly resistant to conventional antifungal drugs or fungicides. According to recent studies, natural products have been shown to be effective antifungal agents. Approximately 90 fungal strains from the Nautilus fungal library were examined for potential fungicide adjuvant activity that would play a role in increasing the biological efficiency of four commercial fungicides: tebuconazole, fludioxonil, iprodione and pyraclostrobin against spores of B. cinerea. An adjuvant is defined as a compound that is inactive in the antifungal screen but significantly improves the efficacy of a known antifungal agent. The 90 fungal strains were fermented in selective media, and the broth and mycelium produced by each was extracted using ethyl acetate as a solvent. Extracts were then fractionated using C18 solid phase extraction cartridges. These samples were then analyzed by LCHRMS and tested in the antifungal assay alone and in conjunction with the four commercial fungicides to discover potential adjuvants.
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Use of Metabolomics and Antimicrobial Screening to Identify Putatively New Natural Products
Vernon C. Ptycia-Lamky, Brad Haltli, Martin Lanteigne and Russell G. Kerr Atlantic Veterinary College, University of Prince Edward Island The marine ecosystems provide a vast reservoir of microbes that produce structurally unique and biologically active compounds. However, the re-discovery of known compounds has become a major issue with traditional methods. Therefore, this project utilizes both antimicrobial bioassay-guided fractionation and metabolomic analysis as an efficient method to discover novel compounds. Twenty Gram-positive marine microbes that were found to produce natural products with potent antimicrobial activity from a preliminary screening of 3660 fractions/crude extracts were selected for secondary evaluation. Each strain was refermented in their original conditions and the extracts were prepared for both liquid chromatography-high resolution mass spectrometry and antimicrobial assay analysis. The preliminary metabolomic findings indicated that four Streptomyces strains: RKBH-B7, RKBH-B122, RKJM-36 and RKND-689 produced putatively novel compounds. Additionally, the antimicrobial bioassays of RKND-689 produced a fraction with strong activity against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and Staphylococcus warnerii, while RKJM-36 was selectively active against Methicillin-resistant Staphylococcus aureus. For this reason, the scale-up fermentations, antimicrobial bioassay-guided fractionations and purifications are currently underway to obtain sufficient pure product for structural elucidation.
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The lipopeptide cystargamide stimulates the production of silent natural products in some Streptomyces species Zacharie Maw, Brad Haltli, and Russell G. Kerr Department of Biomedical Science, Atlantic Veterinary College, University of Prince Edward Island Genome mining of some bacteria has revealed that many natural products are not expressed under standard cultivation conditions and thus remain undiscovered;; these are referred to as silent natural products. One avenue to induce the production of silent natural products is the use of messenger small-molecules that can stimulate their expression. The cyclic-lipopeptide cystargamide, produced by the actinomycete Kitasatospora cystarginea, structurally bears similarities to other lipopeptides such as surfactin while also having a 2,3-epoxy fatty acid chain. Pure cystargamide fed to production media of Streptomyces spp. cultures caused changes in their metabolomic profile characterized by LC-HRMS and analyzed using peak detection methods followed by statistical analysis in R. In a preliminary study, with 20 strains of Streptomyces spp. where strains were treated with cystargamide, 1 strain showed de novo induction of a group of known antibiotics while many others upregulated production of other natural products compared to controls. To evaluate the potential of other surface active type molecules in inducing silent natural product expression, a panel of Streptomyces spp. with a group of synthetic and microbial derived surfactant lipopeptides will be tested for their ability to stimulate natural product production.
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Porous, Pyrogenic Material as a Vector for Targeted, Beneficial Microbe Delivery to the Rhizosphere Kaitlyn Blatt-Janmaat, Dr. Clarissa Sit* Saint Mary’s University
The soil microbiome is one of the most complex and least understood microbial communities on earth, with over 1000 identifiable strains of bacteria and fungi present in a one gram aliquot.1 The plant-microbe interactions that occur within the rhizosphere are drawing more attention as the role these microbes play in plant health is explored and understood. Microbes in the rhizosphere have been attributed to crucial nutrient cycling and disease resistance;; both of which are exceptionally beneficial to plant growth. As the demand for agricultural products increases with the growing population, the benefits of these interactions seem incredibly attractive. Addition of beneficial microorganisms for enhancing plant growth is not a new idea, but most applications are topical and solution-based, resulting in microbial loss with rainfall or lack of soil penetration. Biochar, a porous material generated through plant waste pyrolysis, is an attractive solution to this issue as the porous structure makes it an ideal vessel for microbial transport. The inoculation of forest waste biochar has been accomplished with multiple strains of beneficial bacteria and imaged via SEM. The treatment of the model agricultural crop, Vitis vinifera (wine grape), with the generated inoculate will be discussed. 1. Morgan et al. J. Exp. Bot. 2005, 56(417): 1729-1739
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Pairwise testing of Lentzea strains against Mycobacterium smegmatis to search for new anti-tuberculosis compound Julie Anne Dayrit, Clarissa S. Sit* Saint Mary’s University Tuberculosis remains one of the top ten causes of death worldwide. Therefore, discovery of new antibiotic compounds is essential for counteracting the evolving antibiotic resistance of strains of Mycobacterium tuberculosis and related species. Previous studies have shown that a soil bacterium, Lentzea kentuckyensis, can biosynthesize lassomycin, a peptide that has the ability to kill multi-drug resistant M. tuberculosis. To investigate whether other Lentzea strains produce anti-mycobacterial compounds, commercially available Lentzea albida and Lentzea albiodocapillata will be grown alongside Mycobacterium smegmatis to check for inhibitory activity. Because M. smegmatis is a non-pathogenic mycobacteria strain, we are using it in the lab as a proxy for M. tuberculosis. In the process of culturing the strains in the lab, I investigated the growth of L. albida and L. albiodocapillata on agar containing cycloheximide, a compound produced by Streptomyces griseus which is routinely used for selection against yeast and fungi. The objective of this approach is to determine how cycloheximide affects the growth and metabolomic profiles of the Lentzea strains. Any Lentzea strain that exhibits activity against Mycobacterium smegmatis, e.g. slows or inhibits the growth of Mycobacterium smegmatis, will be cultured on a larger scale and will be extracted to search for active compounds. The extract will then be fractionated using reversed-phase HPLC to separate the compounds present. Each fraction will be tested against M. smegmatis to isolate the active compound and will be analyzed using LC-MS and NMR. We will cross-reference our hits against the reported molecules in the literature. Any novel compounds we characterize will provide a better understanding of how Lentzea strains interact with and defend themselves against competing microbes, such as mycobacteria.
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Isolation and identification of 3,5-dihydroxyhexanoic acid oligomers from an unidentified sterile endophyte of the brown alga Scytosiphon lomentaria Nicholas J. Morehouse,1 Andrew J. Flewelling,1 John A. Johnson,1 Christopher A. Gray1,2
Presenting Author: Undergraduate student 1 Department of Biological Sciences, University of New Brunswick, Saint John 2 Department of Chemistry, University of New Brunswick, Fredericton Endophytic fungi of marine macroalgae represent an excellent source of antimicrobial natural products. An extract of an unidentified sterile fungus isolated from the brown alga Scytosiphon lomentaria was selected for further study based on its unique bioactivity profile. Bioassay guided fractionation of the extract led to the isolation of what was initially considered to be 3,5-dihydroxyhexanoic acid. However, difficulties in obtaining confirmatory mass spectrometric data in conjunction with unexpected outcomes of routine chemical transformations suggested that our proposed structure was incorrect. Further work has suggested that the bioactive fraction is in fact an inseparable mixture of 3,5-dihydroxyhexanoic acid oligomers (n = 2-27). This poster will discuss the isolation and structure elucidation of the oligomers and present a strategy that will be employed to confirm their identity and absolute stereochemistry.
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The NPRG fungal endophyte library
Julie Therrien,1 Allyson Bos,1 Matthew L. Clinton,2 Kathleen Complak,1 Samantha Cox,1 Trevor N. Clark,1 Katelyn Ellsworth,1 Andrew J. Flewelling,1 Lauren Forgrave,1 Nicholas Morehouse,1 Artabaz Nazari,1 Kelsey Pendleton,1 Marija Valjanovska,1 John A. Johnson1 and Christopher A. Gray.1,2 1 Department of Biological Sciences, University of New Brunswick, Saint John 2 Department of Chemistry, University of New Brunswick, Fredericton Presenting Author: Undergraduate student Natural products discovery programmes rely on comprehensive collections of biological material and corresponding libraries of extracts. Endophytic fungi of medicinal plants and marine macroalgae are being increasingly recognised as a source of novel and functional chemical diversity. The Natural Products Research Group (NPRG) at UNB has compiled a library of extracts derived from endophytes isolated from Canadian medicinal plants and subarctic and temperate marine macroalgae. This poster will provide an overview of the library, its curation and the metabolomics and bioactivity profiling methods that are being used to identify and prioritize extracts of interest.
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Anticancer natural products from traditionally used Canadian medicinal plants Allyson Bos1, Haoxin Li1, Stéphanie Jean2,3, Gilles A. Robichaud2,3, John A. Johnson1, Christopher A. Gray1,4 1 Department of Biological Sciences, University of New Brunswick, Saint John, NB 2 Department of Chemistry and Biochemistry, Université de Moncton, NB 3 Atlantic Cancer Research Institute, Moncton, NB 4 Department of Chemistry, University of New Brunswick, Fredericton, NB Presenting author: Research Associate Breast cancer is the most commonly diagnosed malignant neoplasm among the female population worldwide and, despite significant advances in screening technologies and therapies, it remains the second leading cause of cancer related deaths in Canadian women. It is therefore imperative that we continue to develop novel and more specific anticancer agents of greater efficacy and diminished toxicity. Combining natural products research with ethnopharmacology is an effective strategy for identifying potential anticancer drug candidates. The ethnobotanical knowledge of the Canadian First Nations is therefore an important resource for identifying plants that produce natural product drug leads. Bioassay screening of a library of thirty-five Canadian medicinal plant extracts identified eleven extracts that were potent inducers of apoptosis in an aggressive human breast carcinoma cell line (MDA-MB-231). Bioassay guided fractionation of the seven most active extracts (Aralia nudicaulis, Juniperus communis, Nuphar lutea, Populus tremuloides, Hypericum perforatum, Moneses uniflora, and Orthilia secunda) resulted in the isolation of sixteen natural products, most of which have not previously been reported to be pro-apoptotic. Although these results confirm the importance of our ethnopharmacological approach, our studies of medicinal plants has predominantly resulted in the isolation of known natural products. In addition to our medicinal plant extracts, we have conducted preliminary bioassay screening of a library of endophytic fungal extracts derived from medicinal plants. Our data indicate that these represent a promising source of pro-apoptotic natural products and, therefore, in the future we will be focussing our attention on these endophytes in an effort to discover novel chemical entities with anticancer activity.
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Synthetic Methodology Development Towards Pyrroles and Aza-BODIPYs Roberto-Manuel Diaz-Rodriguez, Sophie Margaret Gaube and Alison Thompson Dalhousie University Pyrroles and their derivatives are found widely in physiological systems and natural products, from enzymes to pigments to therapeutically useful metabolites. The synthesis and functionalization of pyrrole-containing compounds is the focus of this work.
Aza-BODIPY dyes have begun to garner attention due to their substantial red shift in absorbance (ca. 100nm) and corresponding near-IR fluorescence compared to meso-carbon BODIPYs;; these properties make them attractive for biological imaging applications. Previous work in the Thompson group is expanded upon here by facilitating substitutions at boron using Cl-BODIPY as an intermediate.
Previous work in the Thompson group investigated the reduction of 2-thionopyrrole esters to 2-formyl pyrroles in a single-step process using Raney nickel. The viability of using a Knorr-type synthesis for large-scale 2-thionopyrrole ester production was investigated and the reactivity of the product was explored.
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Maps of UPEI and Charlottetown
UPEI Campus map with SSDE and free parking area encircled.
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Map of Charlottetown with UPEI and event venues marked with pins.
