Natural Product Extract Libraries Research Articles

Depsides isolated from the Sri Lankan lichen Parmotrema sp. exhibit selective Plk1 inhibitory activity

Context: Mitotic kinase enzymes regulate critical stages of mitosis and are amenable to pharmacological inhibition. Since natural products have been a rich source of antimitotic inhibitors, we postulated that natural products would also provide effective inhibitors of mitotic kinases.Objective: To explore unique marine and terrestrial natural product sources for new anticancer drug leads, we screened our natural product extract library for polo-like kinase-1 (Plk1) kinase inhibitors.Materials and methods: Extracts of the lichen Parmotrema sp. (Parmeliaceae) exhibited in vitro inhibitory activity. Bioassay-guided fractionation of the Parmotrema sp. extract led to the isolation of depside inhibitors.Results: A new depside 1 has been isolated from the Sri Lankan lichen Parmotrema sp. along with the known metabolites 2 (β-collatolic acid) and 3 (β-alectoronic acid). The structure of depside 1 was elucidated by spectroscopic analysis. The three depsides 1–3 exhibited moderate inhibition of purified recombinant Plk1 kinase with IC50 of 2.8, 0.7, and 1.7 µM, respectively, at 1 µM ATP. Inhibitory activity was also observed at high concentrations of ATP, suggesting the potential for activity in a cellular environment. The depsides were also tested against a panel of 23 other recombinant kinases and were found to possess up to 30-fold selectivity toward Plk1.Discussion and conclusion: These data suggest that the depsides 1–3 may serve as core structures that can be further explored as potential inhibitors of Plk1 and other kinases.

Anti-obesity effects ofLysimachia foenum-graecumcharacterized by decreased adipogenesis and regulated lipid metabolism

Lysimachia foenum-graecum has been used as an oriental medicine with anti-inflammatory effect. The anti-obesity effect of L. foenum-graecum extract (LFE) was first discovered in our screening of natural product extract library against adipogenesis. To characterize its anti-obesity effects and to evaluate its potential as an anti-obesity drug, we performed various obesity-related experiments in vitro and in vivo. In adipogenesis assay, LFE blocked the differentiation of 3T3-L1 preadipocyte in a dose-dependent manner with an IC50 of 2.5 μg/ml. In addition, LFE suppressed the expression of lipogenic genes, while increasing the expression of lipolytic genes in vitro at 10 μg/ml and in vivo at 100 mg/kg/day. The anti-adipogenic and anti-lipogenic effect of LFE seems to be mediated by the inhibition of PPARγ and C/EBPα expression as shown in in vitro and in vivo, and the suppression of PPARγ activity in vitro. Moreover, LFE stimulated fatty acid oxidation in an AMPK-dependent manner. In high-fat diet (HFD)-induced obese mice (n = 8/group), oral administration of LFE at 30, 100, and 300 mg/kg/day decreased total body weight gain significantly in all doses tested. No difference in food intake was observed between vehicle- and LFE-treated HFD mice. The weight of white adipose tissues including abdominal subcutaneous, epididymal, and perirenal adipose tissue was reduced markedly in LFE-treated HFD mice in a dose-dependent manner. Treatment of LFE also greatly improved serum levels of obesity-related biomarkers such as glucose, triglycerides, and adipocytokines leptin, adiponectin, and resistin. All together, these results showed anti-obesity effects of LFE on adipogenesis and lipid metabolism in vitro and in vivo and raised a possibility of developing LFE as anti-obesity therapeutics.

Botryllamides: Natural Product Inhibitors of ABCG2

ABCG2 is a membrane-localized, human transporter protein that has been demonstrated to reduce the intracellular accumulation of substrates through ATP-dependent efflux. Highly expressed in placental syncytiotrophoblasts, brain microvasculature, and the gastrointestinal tract, ABCG2 has been shown to mediate normal tissue protection as well as limit oral bioavailability of substrate compounds. Development of ABCG2 inhibitors for clinical use may allow increased penetration of therapeutic agents into sanctuary sites and increased gastrointestinal absorption. Previously identified inhibitors have lacked potency or specificity or were toxic at concentrations needed to inhibit ABCG2; none are in clinical development. A previously developed high-throughput assay measuring inhibition of ABCG2-mediated pheophorbide a transport was applied to natural product extract libraries. Among the active samples were extracts from the marine ascidian Botryllus tyreus. Bioassay-guided fractionation resulted in purification of a series of botryllamides. Ten botryllamides were obtained, two of which (designated I and J) were novel. Activity against ABCG2 was confirmed by assessing the ability of the compounds to inhibit ABCG2-mediated BODIPY-prazosin transport in ABCG2-transfected HEK293 cells, compete with [(125)I]-iodoarylazidoprazosin (IAAP) labeling of ABCG2, stimulate ABCG2-associated ATPase activity, and reverse ABCG2-mediated resistance.

A Sensitive Coupled HPLC/Electrospray Mass Spectrometry Assay for SPM-1 Metallo-β-Lactamase Inhibitors

Antibiotic-resistant bacteria continue to threaten human health through multiple mechanisms, including hydrolytic inactivation of beta-lactam antibiotics by metallo-beta-lactamases (MBLs). The SPM-1 enzyme, originally identified from a Pseudomonas aeruginosa clinical isolate, is a Class B beta-lactamase responsible for resistance in bacteria against antibiotics such as penicillins, cephalosporins, and carbapenems. Unlike Class A, C, and D beta-lactamases, which employ a serine residue in their active site, Class B enzymes possess one or two Zn atoms in the active site that play both a structural and catalytic role. A beta-lactamase inhibitor with co-administration of a beta-lactam antibiotic has proven to be an effective treatment against antibiotic-resistant bacteria whose resistance is due to serine-based beta-lactamases (e.g., amoxicillin/clavulanic acid). A similar clinical approach has not yet been developed for resistant bacteria possessing MBLs. The identification and development of specific and effective MBL inhibitors to combat this resistance could extend the utility of currently prescribed antibiotics such as cephalosporins and carbapenems. To discover MBL inhibitors, compound libraries are screened typically by enzymatic hydrolysis of a chromogenic substrate such as nitrocefin monitored by absorbance. Spectrophotometric assays, while valuable, lack the sensitivity and selectivity to screen natural product extract libraries because of the strongly absorbing nature of some extracts and the dilute concentrations of active components. An assay is described herein that monitors the SPM-1-catalyzed hydrolysis of penicillin G by high-performance (high-pressure) liquid chromatography-electrospray mass spectroscopy, which permits investigations with greater sensitivity and selectivity allowing the screening of natural product extracts for inhibitors of MBLs.

Functional Cell-Based Screening and Saturation Transfer Double-Difference NMR Have Identified Haplosamate A as a Cannabinoid Receptor Agonist

A marine natural product extract library has been screened with a functional cell-based G-protein coupled receptor assay to find compounds capable of binding the human cannabinoid receptors CB1 and CB2. The methanol extract of the marine sponge Dasychalina fragilis collected in Papua New Guinea was active in the assay. Bioassay-guided fractionation of the extract identified the phosphorylated sterol sulfate haplosamate A (1) as a cannabinoid receptor agonist. The high water solubility of haplosamate A (1) allowed exploration of its binding interactions with the human cannabinoid receptors in whole insect cells by means of saturation transfer double-difference NMR spectroscopy. This technique confirmed that haplosamate A (1) binds selectively to these receptors.

Antimalarial activity of natural product extracts from Papua New Guinean and Australian plants against Plasmodium falciparum

In the search for new antimalarial compounds, a subset of a natural product extract library prepared from plant samples collected from Papua New Guinea and Australia was screened for in vitro activity against the chloroquine-sensitive 3D7 and chloroquine-resistant Dd2 strains of Plasmodium falciparum. Using the incorporation of ((3)H)-hypoxanthine into parasite nucleic acid as a marker of growth, 93 of the 794 extracts screened displayed >40% inhibition against 3D7 infected erythrocytes at 312 microge/mL. Antimalarial activity was confirmed in 48 of these extracts against both 3D7 and Dd2 infected erythrocytes at concentrations between 78 and 390 microge/mL, 14 of which caused >90% growth inhibition of 3D7 at the lowest concentration screened. Extracts were also tested for mammalian cell cytotoxicity to evaluate selectivity of action.

Discovery of metallo-β-lactamase inhibitors in extracts via an enzyme assay with HPLC/ES detection

Metallo-β-lactamases (MBL), such as the SPM-1 MBL, are enzymes responsible for the emerging antibiotic resistance in bacteria by hydrolyzing β-lactam based antibiotics such as penicillins and carbapenams. This resistance can be overcome by therapy combining an MBL inhibitor with a β-lactam antibiotic; the drug AugmentinTM combines amoxicillin with an inhibitor efficacious against serine based β-lactamases. Natural product extracts are relatively unexplored sources for MBL inhibitors but they can be identified through enzymatic screening followed by isolation. The discovery of a clinically useful MBL inhibitor would be highly significant, as one has not yet available. We are screening pure compounds from the NCI Diversity Set and natural product extracts against the SPM-1 enzyme. While enzyme inhibition assays are routinely performed on compound libraries, screening a natural product extract library is considerably more difficult due to the spectroscopic complexity of extract mixtures and the low concentrations of active components. Our method combines an SPM-1 enzyme inhibition assay with HPLC separation [1] and single ion ES-MS monitoring the concentration of penicillin G over time. This modification allowed the use of more therapeutically relevant substrates such as meropenem or kinetically superior substrates such as penicillin G [2] at biologically significant concentrations (achievable through the increased selectivity and sensitivity of the method). We confirmed the validity of this method against previously identified SPM-1 β-lactamase inhibitors and inactive extracts that have been spiked with these compounds. We are using this assay to screen extracts and have discovered one extract (Strophostyles helvola) with significant MBL inhibition.

Identification of Griseofulvin as an Inhibitor of Centrosomal Clustering in a Phenotype-Based Screen

A major drawback of cancer chemotherapy is the lack of tumor-specific targets which would allow for the selective eradication of malignant cells without affecting healthy tissues. In contrast with normal cells, most tumor cells contain multiple centrosomes, associated with the formation of multipolar mitotic spindles and chromosome segregation defects. Many tumor cells regain mitotic stability after clonal selection by the coalescence of multiple centrosomes into two functional spindle poles. To overcome the limitations of current cancer treatments, we have developed a cell-based screening strategy to identify small molecules that inhibit centrosomal clustering and thus force tumor cells with supernumerary centrosomes to undergo multipolar mitoses, and subsequently, apoptosis. Using a chemotaxonomic selection of fungi from a large culture collection, a relatively small but diverse natural product extract library was generated. Screening of this compound library led to the identification of griseofulvin, which induced multipolar spindles by inhibition of centrosome coalescence, mitotic arrest, and subsequent cell death in tumor cell lines but not in diploid fibroblasts and keratinocytes with a normal centrosome content. The inhibition of centrosome clustering by griseofulvin was not restricted to mitotic cells but did occur during interphase as well. Whereas the formation of multipolar spindles was dynein-independent, depolymerization of interphase microtubules seemed to be mechanistically involved in centrosomal declustering. In summary, by taking advantage of the tumor-specific phenotype of centrosomal clustering, we have developed a screening strategy that might lead to the identification of drugs which selectively target tumor cells and spare healthy tissues.

Highly Substituted Terphenyls as Inhibitors of Parasite cGMP-Dependent Protein Kinase Activity

Parasite cGMP-dependent protein kinase (PKG) is one of the validated biochemical targets for the treatment of coccidiosis. We screened our library of natural product extracts for inhibitors of parasite PKG for the discovery of anticoccidial leads. Terferol (1) and three new terphenyls (2, 3, and 4) were isolated using bioassay-guided fractionation of the microbial extract of a Phoma sp. by a high-throughput two-step isolation method employing LH-20 and reversed-phase HPLC. These compounds inhibited parasite PKG with IC(50) values in the range 0.9-5.8 microM.

Tenellones A and B from a Diaporthe sp.: Two Highly Substituted Benzophenone Inhibitors of Parasite cGMP-Dependent Protein Kinase Activity

Parasite cGMP-dependent protein kinase (PKG) has been recently validated as a biochemical target for the treatment of coccidiosis. To discover new anticoccidial leads, we have screened our library of natural product extracts for inhibitors of parasite PKG. Bioassay-guided fractionation of the microbial extracts has led to the discovery of tenellones A (2) and B (3), two new highly substituted benzophenones. The isolation, structure, and activity of these compounds are described.

Determination of Selectivity and Efficacy of Fatty Acid Synthesis Inhibitors

Type II fatty acid synthesis (FASII) is essential to bacterial cell viability and is a promising target for the development of novel antibiotics. In the past decade, a few inhibitors have been identified for this pathway, but none of them lend themselves to drug development. To find better inhibitors that are potential drug candidates, we developed a high throughput assay that identifies inhibitors simultaneously against multiple targets within the FASII pathway of most bacterial pathogens. We demonstrated that the inverse t(1/2) value of the FASII enzyme-catalyzed reaction gives a measure of FASII activity. The Km values of octanoyl-CoA and lauroyl-CoA were determined to be 1.1 +/- 0.3 and 10 +/- 2.7 microM in Staphylococcus aureus and Bacillus subtilis, respectively. The effects of free metals and reducing agents on enzyme activity showed an inhibition hierarchy of Zn2+ > Ca2+ > Mn2+ > Mg2+; no inhibition was found with beta-mercaptoethanol or dithiothreitol. We used this assay to screen the natural product libraries and isolated an inhibitor, bischloroanthrabenzoxocinone (BABX) with a new structure. BABX showed IC50 values of 11.4 and 35.3 microg/ml in the S. aureus and Escherichia coli FASII assays, respectively, and good antibacterial activities against S. aureus and permeable E. coli strains with minimum inhibitory concentrations ranging from 0.2 to 0.4 microg/ml. Furthermore, the effectiveness, selectivity, and the in vitro and in vivo correlations of BABX as well as other fatty acid inhibitors were elucidated, which will aid in future drug discovery.

Diversifying microbial natural products for drug discovery.

Historically, nature has provided the source for the majority of the drugs in use today. More than 20,000 microbial secondary metabolites have been described, but only a small percentage of these have been carried forward as natural product drugs. Natural products are in tough competition with large chemical libraries and with combinatorial chemistries. Hence, each step of a natural product program has to be more efficient than ever, starting from the collection of environmental samples and the selection of strains, to metabolic expression, genetic exploitation, sample preparation and chemical dereplication. This review will focus on approaches for diversifying microbial natural product strains and extract libraries, while decreasing genetic and chemical redundancy.

Natural product extracts that reduce accumulation of the Alzheimer's amyloid beta peptide: selective reduction in A beta42.

Alzheimer's disease (AD) is the most common form of dementia in the elderly. Without a treatment that significantly delays the progression of the disease over 14 million Americans are likely to be affected with AD by the middle of the 21st Century, presenting an enormous economic and social burden. Evidence gathered over the last two decades has implicated the abnormal accumulation of A beta, in particular the longer more amyloidogenic form A beta42, as a potential causative agent in the disease. To screen for compounds that reduce A beta accumulation we have established several high throughput, cell based screens capable of the sensitive and selective detection of A beta40 and A beta42. Using these screens we have analyzed a proprietary library of natural product extracts for their ability to influence A beta accumulation. Using this approach, we have identified several agents capable of influencing total A beta concentration. In addition, we have identified one extract that selectively reduces A beta42. Intracerebroventricular administration of this agent to mice results in a selective reduction in A beta42 in the brain.

High Resolution Screening of Plant Natural Product Extracts for Estrogen Receptor α and β Binding Activity Using an Online HPLC-MS Biochemical Detection System

A new screening technology that combines biochemical analysis with the resolution power of high-performance liquid chromatography (HPLC), referred to here as high-resolution screening (HRS) technique, is described. The capability of the HRS technology to analyze biologically active compounds in complex mixtures is demonstrated by screening a plant natural product extract library for estrogen receptor (ER) alpha and beta binding activity. The simultaneous structure elucidation of biologically active components in crude extracts was achieved by operating the HRS system in combination with mass spectrometry (MS). In contrast to conventional microtiter-type bioassays, the interactions of the extracts with the ER and the employed label, coumestrol, proceeded at high speed in a closed, continuous-flow reaction detection system, which was coupled directly to the outlet of a HPLC separation column. The reaction products of this homogeneous fluorescence enhancement-type assay were detected online using a flowthrough fluorescence detector. Primary screening of the extract library was performed in the fast-flow injection analysis mode (FlowScreening) wherein the chromatographic separation system was bypassed. The library was screened at high speed, using two assay lines in parallel. A total of 98% of the identified hits were confirmed in a traditional 96-well microplate-based fluorescence polarization assay, indicating the reliability of the FlowScreening process. Active extracts were reassayed in a transcriptional activation assay in order to assess the functional activity of the bioactive extracts. Only functional active extracts were processed in the more time-consuming HRS mode, which was operated in combination with MS. Information on the number of active compounds, their retention times, the molecular masses, and the MS/MS-fingerprints as a function of their biological activity was obtained from 50% of the functional active extracts in real time. This dramatically enhances the speed of biologically active compound characterization in natural product extracts compared to traditional fractionation approaches.

Development of a Cyanovirin-N–HIV-1 gpl20 Binding Assay for High Throughput Screening of Natural Product Extracts by Time-Resolved Fluorescence

The unique, high-affinity binding of cyanovirin-N (CV-N), a potent anti-human immunodeficiency virus (HIV) protein, to the HIV envelope glycoprotein gp120, was exploited to develop an HTS assay in an attempt to discover small-molecule mimetics of CV-N. A competition binding assay was developed using CV-N labeled with europium (Eu31). The labeling protocol did not significantly alter the gpl20 binding properties or the antiviral activity of CV-N. This report describes the assay development, validation, and results of screening a large library of aqueous and organic natural product extracts. The extracts were incubated with immobilized recombinant gpl20 in 96-well plates prior to the addition of Eu3+-labeled CV-N. Following a wash step, bound CV-N was measured by dissociation-enhanced time-resolved fluorometry of Eu3+. The assay proved to be robust, rapid, and reproducible, and was used to screen over 50,000 natural product extracts, and has resulted in the identification of several aqueous natural product extracts that inhibited CV-N–gp120 binding and also had anti-HIV activity.