Natural Product Library Research Articles

Inhibiting clathrin-mediated endocytosis of the leucine-rich G protein-coupled receptor-5 diminishes cell fitness

The leucine-rich G protein-coupled receptor-5 (LGR5) is expressed in adult tissue stem cells of many epithelia, and its overexpression is negatively correlated with cancer prognosis. LGR5 potentiates WNT/β-catenin signaling through its unique constitutive internalization property that clears negative regulators of the WNT-receptor complex from the membrane. However, both the mechanism and physiological relevance of LGR5 internalization are unclear. Therefore, a natural product library was screened to discover LGR5 internalization inhibitors and gain mechanistic insight into LGR5 internalization. The plant lignan justicidin B blocked the constitutive internalization of LGR5. Justicidin B is structurally similar to more potent vacuolar-type H+-ATPase inhibitors, which all inhibited LGR5 internalization by blocking clathrin-mediated endocytosis. We then tested the physiological relevance of LGR5 internalization blockade in vivo A LGR5-rainbow (LBOW) mouse line was engineered to express three different LGR5 isoforms along with unique fluorescent protein lineage reporters in the same mouse. In this manner, the effects of each isoform on cell fate can be simultaneously assessed through simple fluorescent imaging for each lineage reporter. LBOW mice express three different forms of LGR5, a wild-type form that constitutively internalizes and two mutant forms whose internalization properties have been compromised by genetic perturbations within the carboxyl-terminal tail. LBOW was activated in the intestinal epithelium, and a year-long lineage-tracing course revealed that genetic blockade of LGR5 internalization diminished cell fitness. Together these data provide proof-of-concept genetic evidence that blocking the clathrin-mediated endocytosis of LGR5 could be used to pharmacologically control cell behavior.

Mitochondrial uncoupler exerts a synthetic lethal effect against β-catenin mutant tumor cells.

The wingless/int‐1 (Wnt) signal transduction pathway plays a central role in cell proliferation, survival, differentiation and apoptosis. When β‐catenin: a component of the Wnt pathway, is mutated into an active form, cell growth signaling is hyperactive and drives oncogenesis. As β‐catenin is mutated in a wide variety of tumors, including up to 10% of all sporadic colon carcinomas and 20% of hepatocellular carcinomas, it has been considered a promising target for therapeutic interventions. Therefore, we screened an in‐house natural product library for compounds that exhibited synthetic lethality towards β‐catenin mutations and isolated nonactin, an antibiotic mitochondrial uncoupler, as a hit compound. Nonactin, as well as other mitochondrial uncouplers, induced apoptosis selectively in β‐catenin mutated tumor cells. Significant tumor regression was observed in the β‐catenin mutant HCT 116 xenograft model, but not in the β‐catenin wild type A375 xenograft model, in response to daily administration of nonactin in vivo. Furthermore, we found that expression of an active mutant form of β‐catenin induced a decrease in the glycolysis rate. Taken together, our results demonstrate that tumor cells with mutated β‐catenin depend on mitochondrial oxidative phosphorylation for survival. Therefore, they undergo apoptosis in response to mitochondrial dysfunction following the addition of mitochondrial uncouplers, such as nonactin. These results suggest that targeting mitochondria is a potential chemotherapeutic strategy for tumor cells that harbor β‐catenin mutations.

Identification of Bioaminergic Lead Compounds to Treat Parasitic Blood Flukes (Schistosoma Spp.) through High Throughput Screening Natural Product Libraries

Parasitic blood flukes (Schistosoma species of flatworms) infect over 200 million people worldwide, making them the second most costly parasite behind malaria. Despite this, the disease is currently treated with one broad spectrum drug, praziquantel, which has been in use for nearly 40 years. Concerns over reports of resistance in the field and the fact that the drug is ineffective against recently acquired infections highlight the need for research into new therapies to treat this disease. Towards this end, bioaminergic G‐protein coupled receptors (GPCRs) are attractive targets. In addition to the proven druggable track record of GPCRs, cAMP‐coupled bioaminergic signalling pathways control flatworm neuromuscular function and development. However, there are few reports of successful heterologous expression of flatworm GPCRs and no large scale drug screening studies. Therefore, we have recombinantly expressed a Schistosoma serotonin receptor required for parasite movement and developed an assay for high throughput screening of natural product libraries to identify anti‐schistosomal lead compounds.Homologs of a schistosome serotonin receptor were cloned for the three major species of Schistosoma parasites that account for human infections [S. mansoni (Sm.5HTR), S. haematobium (Sh.5HTR) and S. japonicum (Sj.5HTR)]. These receptors were stability expressed in mammalian HEK293 cells alongside a bioluminescent cAMP reporter to permit drug screening in 384 well format (Z′ score = 0.8, 6% coefficient of variation). Natural product libraries (>4000 compounds) were screened against this receptor at a fixed concentration (10μM). Agonist and antagonist primary hits were counter screened to exclude compounds with activity against endogenous mammalian GPCRs present in the HEK293 cell line, or those that compromised cell viability. Several classes of ligands were notable within the validated set of 29 hits ‐ benzylisoquinolines (10 compounds) and indoles (8 compounds). Further exploration of these chemical series identified potent schistosome 5HTR agonists (EC50 < 100nM) and antagonists (pKi ≤ 7). Structure activity relationships highlight key residues required for the efficacy of these pharmacophores at the schistosome receptor. For example, within the ergot‐alkaloid series of compounds, modification of the 2C position of the ergoline ring or saturation of the 9C–10C bond is crucial for agonist/antagonist activity. The most potent compounds were then screened against adult S. mansoni blood flukes; 5HTR agonists caused a dramatic hypermobilty in both male and female worms, while 5HTR antagonists paralyzed worms even in the presence of supra‐maximal concentrations of exogenous serotonin.These data represent the first reported high throughput screen of a flatworm GPCR, providing much needed leads for advancing in the pipeline for new anti‐schistosomal therapies. Additionally, this study evidences a workflow that may be adopted to rapidly screen other bioaminergic schistosome drug targets.Support or Funding InformationSupported by NIH (R21AI25821)

In silico approaches and chemical space of anti-P-type ATPase compounds for discovering new antituberculous drugs.

Tuberculosis (TB) is one of the most important public health problems around the world. The emergence of multi-drug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis strains has driven the finding of alternative anti-TB targets. In this context, P-type ATPases are interesting therapeutic targets due to their key role in ion homeostasis across the plasma membrane and the mycobacterial survival inside macrophages. In this review, in silico and experimental strategies used for the rational design of new anti-TB drugs are presented; in addition, the chemical space distribution based on the structure and molecular properties of compounds with anti-TB and anti-P-type ATPase activity is discussed. The chemical space distribution compared to public compound libraries demonstrates that natural product libraries are a source of novel chemical scaffolds with potential anti-P-type ATPase activity. Furthermore, compounds that experimentally display anti-P-type ATPase activity belong to a chemical space of molecular properties comparable to that occupied by those approved for oral use, suggesting that these kinds of molecules have a good pharmacokinetic profile (drug-like) for evaluation as potential anti-TB drugs.

Inhibition of IL-8 secretion on BxPC-3 and MIA PaCa-2 cells and induction of cytotoxicity in pancreatic cancer cells with marine natural products.

Pancreatic cancer presents one of the most negative prognosis of all cancers as it has usually metastasized by the time a patient is diagnosed. The American Cancer Society estimates that 93% of patients will die within 5 years of diagnosis, highlighting the need for new drugs to treat this disease. Interleukin 8 (IL-8) mediates the angiogenesis of tumors arising from Ras mutations, which are present in about 90% of pancreatic adenocarcinomas. Overexpression of IL-8 in pancreatic tumors is believed to promote tumor angiogenesis and to activate survival signaling pathways. A 96-well cell-based enzyme-linked immunosorbent assay was set up to screen the Harbor Branch Oceanographic Institute library of marine natural products to identify those with the ability to inhibit IL-8 production by BxPC-3 pancreatic cancer cells. Over 1000 fractions were screened, resulting in the identification of 10 known marine natural products with this ability. These compounds fall into four classes of compounds including the pyrroloiminoquinone alkaloids secobatzelline A and isobatzelline C; mycalamide A and B, onnamide A, discalamide A, and theopederin K from the mycalamide class of polyketides; the lipopeptide microcolin A; and the cyclic depsipeptides didemnin B and nordidemnin B. In addition, didemnin B, nordidemnin B, and theopederin K induce potent cytotoxicity against four pancreatic cancer cell lines tested. Many of these compounds have been previously reported to inhibit protein synthesis and the decrease in IL-8 production may be nonspecific. Nevertheless, this is a new activity for these compounds and inhibition of IL-8 secretion by pancreatic cancer cells can now be added to the previously reported antiangiogenic activities of the didemnins.

Live-Cell Imaging of Protease Activity: Assays to Screen Therapeutic Approaches.

Methodologies to image and quantify the activity of proteolytic enzymes have been developed in an effort to identify protease-related druggable pathways that are involved in malignant progression of cancer. Our laboratory has pioneered techniques for functional live-cell imaging of protease activity in pathomimetic avatars for breast cancer. We analyze proteolysis in the context of proliferation and formation of structures by tumor cells in 3-D cultures over time (4D). In order to recapitulate the cellular composition and architecture of tumors in the pathomimetic avatars, we include other tumor-associated cells (e.g., fibroblasts, myoepithelial cells, microvascular endothelial cells). We also model noncellular aspects of the tumor microenvironment such as acidic pericellular pH. Use of pathomimetic avatars in concert with various types of imaging probes has allowed us to image, quantify, and follow the dynamics of proteolysis in the tumor microenvironment and to test interventions that impact directly or indirectly on proteolytic pathways. To facilitate use of the pathomimetic avatars for screening of therapeutic modalities, we have designed and fabricated custom 3D culture chambers with multiple wells that are either individual or connected by a channel to allow cells to migrate between wells. Optical glass microscope slides underneath an acrylic plate allow the cultures to be imaged with an inverted microscope. Fluid ports in the acrylic plate are at a level above the 3D cultures to allow introduction of culture media and test agents such as drugs into the wells and the harvesting of media conditioned by the cultures for immunochemical and biochemical analyses. We are using the pathomimetic avatars to identify druggable pathways, screen drug and natural product libraries and accelerate entry of validated drugs or natural products into clinical trials.

Discovery of selective cystathionine β-synthase inhibitors by high-throughput screening with a fluorescent thiol probe.

A high-throughput assay was developed to identify inhibitors of cystathionine β-synthase (CBS), which is one of three key enzymes involved in H2S biosynthesis. Screening of 6491 natural compounds identified several selective CBS inhibitors, which suppressed the proliferation of HT29 cancer cells, with IC50 values of less than 10 μM.

Nanomolar Antimalarial Agents against Chloroquine-Resistant Plasmodium falciparum from Medicinal Plants and Their Structure-Activity Relationships.

Inspired by the discovery of the antimalarial drug artemisinin from a traditional Chinese medicine (TCM), a natural product library of 44 lindenane-type sesquiterpenoids was assessed for activities against the Dd2 chloroquine-resistant strain of the malaria parasite Plasmodium falciparum. These compounds were mainly isolated from plants of the Chloranthus genus, many species of which are named "Sikuaiwa" in TCM and have long been used to treat malaria. The compounds consisted of 41 sesquiterpenoid dimers and three monomers, including the 12 new dimers 1-12 isolated from Chloranthus fortunei. The results showed that 16 dimers exhibited potent antiplasmodial activities (<100 nM); in particular, compounds 1, 14, and 19 exhibited low nanomolar activities with IC50 values ranging from 1 to 7 nM, which is comparable to the potency of artemisinin, and selectivity index values toward mammalian cells greater than 500. A comprehensive structure-activity relationship study indicated that three functional groups are essential and two motifs can be modified.

New natural product carbonic anhydrase inhibitors incorporating phenol moieties

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the fundamental reaction of CO2 hydration in all living organisms, being actively involved in the regulation of a plethora of patho/physiological conditions. They represent a typical example of enzyme convergent evolution, as six genetically unrelated families of such enzymes were described so far. The need to find selective CA inhibitors (CAIs) triggered the investigation of natural product libraries, which proved to be a valid source of agents with such an activity, as demonstrated for the phenols, polyamines and coumarins. Herein we report an in vitro inhibition study of human (h) CA isoforms hCAs I, II, IV, VII and XII with a panel of natural polyphenols including flavones, flavonols, flavanones, flavanols, isoflavones and depsides, some of which extracted from Quercus ilex and Salvia miltiorrhiza. Several of the investigated derivatives showed interesting inhibition activity and selectivities for inhibiting some important isoforms over the off-target ones hCA I and II.

Identifying Natural Products (NPs) as potential UPR inhibitors for crop protection

As far as the future of agriculture is concerned, one major challenge will be to face an expected increase in health risks due to pesticides together with a lower efficiency of crop treatments. Therefore it is today necessary to develop new strategies to enhance the effectiveness and sustainability of current control methods. The so-called “Alternaria Leaf Spot“ is a common disease of crucifers caused by the fungal pathogen Alternaria brassicicola which affects different crops including cabbage, kale, Brussels sprout, cauliflower and broccoli. Indole phytoalexins camalexin and brassinin play in planta a key role in crop protection against this necrotrophic agent. However it has been shown that mutants become phytoalexin-resistant by activating at least three signaling pathways named as Cell Wall Integrity (CWI), High Osmolarity Glycerol (HOG) and Unfolded Protein Response (UPR) [1,2]. The latter is particularly involved in the fungus protection against phytoalexins since UPR deficient avirulent mutants of A. brassicicola appear as hypersensitive to camalexin and brassinin [3]. Since very few UPR inhibitors such as the synthetic STF-083010 [4] are known we decided to develop an original screening assay, detecting the production of a HAC1 fluorescence-induced protein, i.e. a transcriptional activator involved in the UPR pathway, in Saccharomyces cerevisiae cultures (Figure 1). The preliminary screening of an in-house NPs library [c.a. 70 compounds (polyphenols, terpenoids and alkaloids)] clearly revealed aescin (Aesculus hippocastanum)] as a potential UPR inhibitor.

Natural Compounds as Occult Ototoxins? Ginkgo biloba Flavonoids Moderately Damage Lateral Line Hair Cells.

Several drugs, including aminoglycosides and platinum-based chemotherapy agents, are well known for their ototoxic properties. However, FDA-approved drugs are not routinely tested for ototoxicity, so their potential to affect hearing often goes unrecognized. This issue is further compounded for natural products, where there is a lack of FDA oversight and the manufacturer is solely responsible for ensuring the safety of their products. Natural products such as herbal supplements are easily accessible and commonly used in the practice of traditional eastern and alternative medicine. Using the zebrafish lateral line, we screened a natural products library to identify potential ototoxins. We found that the flavonoids quercetin and kaempferol, both from the Gingko biloba plant, demonstrated significant ototoxicity, killing up to 30% of lateral line hair cells. We then examined a third Ginkgo flavonoid, isorhamnetin, and found similar levels of ototoxicity. After flavonoid treatment, surviving hair cells demonstrated reduced uptake of the vital dye FM 1-43FX, suggesting that the health of the remaining hair cells was compromised. We then asked if these flavonoids enter hair cells through the mechanotransduction channel, which is the site of entry for many known ototoxins. High extracellular calcium or the quinoline derivative E6 berbamine significantly protected hair cells from flavonoid damage, implicating the transduction channel as a site of flavonoid uptake. Since known ototoxins activate cellular stress responses, we asked if reactive oxygen species were necessary for flavonoid ototoxicity. Co-treatment with the antioxidant D-methionine significantly protected hair cells from each flavonoid, suggesting that antioxidant therapy could prevent hair cell loss. How these products affect mammalian hair cells is still an open question and will be the target of future experiments. However, this research demonstrates the potential for ototoxic damage caused by unregulated herbal supplements and suggests that further supplement characterization is warranted.

Activity of novel inhibitors of Staphylococcus aureus biofilms.

Staphylococcus aureus is one of the most important pathogens causing chronic biofilm infections. These are becoming more difficult to treat owing to drug resistance, particularly because S. aureus biofilms limit the efficacy of antimicrobial agents, leading to high morbidity and mortality. In the present study, we screened for inhibitors of S. aureus biofilm formation using a natural product library from the Korea Chemical Bank (KCB). Screening by crystal violet-based biomass staining assay identified hit compounds. Further examination of antibiofilm properties of these compounds was conducted and led to the identification of celastrol and telithromycin. In vitro, both celastrol and telithromycin were toxic to planktonic S. aureus and also active against a clinical methicillin-resistant S. aureus (MRSA) isolate. The effect of the compounds on preformed biofilms of clinical MRSA isolates was evaluated by confocal laser scanning microscopy (CLSM), which revealed the absence of typical biofilm architecture. In addition, celastrol and telithromycin inhibited the production of extracellular protein at selected sub-MIC concentrations, which revealed the reduced extracellular polymeric substance (EPS) secretion. Celastrol exhibited greater cytotoxicity than telithromycin. These data suggest that the hit compounds, especially telithromycin, could be considered novel inhibitors of S. aureus biofilm. Although the mechanisms of the effects on S. aureus biofilms are not fully understood, our data suggest that telithromycin could be a useful adjuvant therapeutic agent for S. aureus biofilm-related infections.

Standardized LC×LC-ELSD Fractionation Procedure for the Identification of Minor Bioactives via the Enzymatic Screening of Natural Extracts.

To identify natural bioactive compounds from complex mixtures such as plant extracts, efficient fractionation for biological screening is mandatory. In this context, a fully automated workflow based on two-dimensional liquid chromatography (2D-LC × LC) was developed, allowing for the production of hundreds of semipure fractions per extract. Moreover, the ELSD response was used for online sample weight estimation and automated concentration normalization for subsequent bioassays. To evaluate the efficiency of this protocol, an enzymatic assay was developed using AMP-activated protein kinase (AMPK). The activation of AMPK by nonactive extracts spiked with biochanin A, a known AMPK activator, was enhanced greatly when the fractionation workflow was applied compared to screening crude spiked extracts. The performance of the workflow was further evaluated on a red clover (Trifolium pratense) extract, which is a natural source of biochanin A. In this case, while the crude extract or 1D chromatography fractions failed to activate AMPK, semipure fractions containing biochanin A were readily localized when produced by the 2D-LC×LC-ELSD workflow. The automated fractionation methodology presented demonstrated high efficiency for the detection of bioactive compounds at low abundance in plant extracts for high-throughput screening. This procedure can be used routinely to populate natural product libraries for biological screening.

Chemical profiling of anti-hepatocellular carcinoma constituents from Caragana tangutica Maxim. by off-line semi-preparative HPLC-NMR

An EtOAc fraction from the roots of Caragana tangutica Maxim. (CTEA) displayed promising anti-hepatocellular carcinoma (HCC) activity during screening of a traditional Chinese ethnic herb library against HepG2 and Hep3B cell lines. HPLC-based activity profiling of CTEA by combination of MS-guided large-scale semi-preparative HPLC and NMR methods led to the identification of a new pterocarpan glycoside, (-)-maackiain 3-O-6′-O-methyl malonyl-β-d-glucopyranoside (1), together with three known pterocarpan glycosides, (-)-maackiain 3-O-β-d-glucopyranoside (2), 3-O-6′-O-acrylyl-β-d-galactopyranoside (3), and (-)-maackiain 3-O-6′-O-acetyl-β-d-glucopyranoside (4). Compound 1 was isolated during a drug discovery programme aimed at identifying new anti-HCC leads from a natural product library. Anti-HCC study showed that all four compounds exhibited cytotoxic activity with IC50 values range of 29.1–53.5 μg/mL against HepG2 and Hep3B cell lines.

Discovery of a new antifungal agent ASP2397 using a silkworm model of Aspergillus fumigatus infection.

Natural products are the major source of currently available drugs. However, screening natural product presents several challenges, including the time-consuming and labor-intensive steps required for the isolation of a drug from crude extracts as well as the differences between the activities of compounds in vitro and in vivo. To address these challenges, we used silkworm larvae infected with Aspergillus fumigatus to screen a natural products library for potent drugs to treat invasive aspergillosis. A rationally designed library was constructed using numerous, geographically diverse fungal species and then screened to collect extracts of microorganisms that had detectable anti-Aspergillus activity. We evaluated this library using cultures of A. fumigatus and a silkworm model system of A. fumigatus infection. With this model, we identified the novel antifungal compound ASP2397 that not only cured infected silkworm larvae but also increased the rates of survival of mice infected with A. fumigatus. These findings strongly support the utility of the silkworm screening system for the simple and rapid isolation of antibiotics from natural products libraries.

Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs.

Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC50 values against T. brucei below 10 μM. Four X-ray crystal structures and docking studies explained the observed structure-activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 μM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability.

A high throughput screen of a natural product library for the discovery of novel antibiotics that target Enterococcus faecalis.

Antibiotic resistance is a natural phenomenon that serves as a threat to public health. The investigation and discovery of novel antibiotic compounds is essential for battling the trend of decreasing antibiotic efficacy. The National Center for Natural Products Research (NCNPR) has assembled a library of organic compounds derived from plants, fungi, marine, and algae species with potential antibacterial properties. In partnership with NCNPR, we have developed a rapid and effective method for screening large quantities of organic compounds for antibacterial efficacy against Enterococcus faecalis. E. faecalis is a gram positive opportunistic pathogen responsible for various hospital acquired infections. To identify extracts that were inhibitory against E. faecalis, bacteria were inoculated into 96-well plates containing liquid growth medium. A small volume of each compound was added to a corresponding well in the 96-well plate. After 24 hour incubation at 37o C, bacteria growth was measured using a plate reader.Wells lacking treatment or containing antibiotics recognized as inhibitors of E. faecalis were used to identify normal or inhibited growth levels correspondingly. Compounds are currently being evaluated for their ability to inhibit bacterial growth. Further investigations will involve the characterization and a detailed analysis of the antimicrobial efficacy of these compounds.

High throughput screening in an in vitro infection model of tularemia to identify novel antibiotics and immunotheraputics.

Francisella tularensis is a pathogenic coccobacillus endemic to North America and is classified by the United States government as a Tier 1 bioterrorism agent. The intentional release of antibiotic-resistant strains of F. tularensis could be disastrous. Since a vaccine against F. tularensis licensed for human use does not exist, we are substantially vulnerable to to this pathogen. However, the development of new therapeutics that target tularemia will make our nation safer against a potential terror attack and naturally resistant F. tularensis. Here, we investigated a library of natural extracts to identify novel therapies that are effective against F. tularensis infections. We have previously engineered F. tularensis to express a red fluorescent protein (LVS/ pTC3D) during intracellular infection. LVS/pTC3D was used to infect THP-1 cells (a monocyte line) seeded into a 96-well plate. To identify novel antibiotics and immunomodulatory therapies, each well was treated with an extract from a cataloged natural product library. Several extracts that led to a reduction in fluorescence over time were selected for further investigation. Current investigations seek to determine whether library extracts directly inhibit bacterial growth in rich culture media (no host cells) or decrease replication in THP-1 cells through immunomodulation. Any extract that inhibited growth in both scenarios would likely be a traditional small molecule antibiotic. Extracts that ONLY inhibit intracellular bacterial growth within the THP-1 cells would likely be doing so by augmenting the host immune responses. Because these act on the host cells and not the bacterium, the potential for the development of antimicrobial resistance is eliminated.

A high throughput screen of a natural product library for the discovery of novel antibiotics with efficiacy against Klebsiella pneumoniae.

Klebsiella pneumoniae is a gram negative bacterium in the enterobacteriaceae family. This organism is an opportunistic pathogen that has been associated with antibiotic resistance in recent years. The discovery of new antibiotics is critical to combat this rising trend in resistance. The National Center for Natural Products Research (NCNPR) has assembled a library of over 2500 extracts derived from plants, fungi, marine, and algae species with potential antibacterial properties. In partnership with NCNPR, we have developed a rapid and effective method for screening large quantities of organic compounds for antibacterial sensitivity against K. pneumoniae. Bacteria were inoculated into a microtiter plate containing liquid growth medium. A small volume of each compound was added to a corresponding well in the microtiter dish.After incubation at 37°C, bacteria growth was measured using a plate reader. Wells lacking treatment or containing antibiotics known to be effective at inhibiting K. pneumoniae were used to identify normal or inhibited growth levels respectively. At least four extracts exhibited potential for inhibiting K. pneumoniae: blanket flower, smallflower globemallow, Arizona walnut, and tree fern. Future investigations will involve the characterization and a detailed analysis of the antimicrobial efficacy of these compounds.

A Grand Challenge. 2. Phenotypic Profiling of a Natural Product Library on Parkinson's Patient-Derived Cells.

Harnessing the inherent biological relevance of natural products requires a method for the recognition of biological effects that may subsequently lead to the discovery of particular targets. An unbiased multidimensional profiling method was used to examine the activities of natural products on primary cells derived from a Parkinson's disease patient. The biological signature of 482 natural products was examined using multiparametric analysis to investigate known cellular pathways and organelles implicated in Parkinson's disease such as mitochondria, lysosomes, endosomes, apoptosis, and autophagy. By targeting several cell components simultaneously the chance of finding a phenotype was increased. The phenotypes were then clustered using an uncentered correlation. The multidimensional phenotypic screening showed that all natural products, in our screening set, were biologically relevant compounds as determined by an observed phenotypic effect. Multidimensional phenotypic screening can predict the cellular function and subcellular site of activity of new compounds, while the cluster analysis provides correlation with compounds with known mechanisms of action. This study reinforces the value of natural products as biologically relevant compounds.