Collection Of Natural Products Research Articles

The US National Cancer Institute’s natural products repository; origins and utility

The US National Cancer Institute's natural products collection program has been running since 1986 and over the years the materials collected and processed have been stored in a repository that as a result of initial planning, has permitted the establishment of a national resource that is now being utilized as a drug discovery tool for any disease of interest to the NIH by researchers world-wide. This paper describes the history of the program.

The Benzo[c]phenanthridine Alkaloid, Sanguinarine, Is a Selective, Cell-active Inhibitor of Mitogen-activated Protein Kinase Phosphatase-1

Mitogen-activated protein kinase phosphatase-1 (MKP-1) is a dual specificity phosphatase that is overexpressed in many human tumors and can protect cells from apoptosis caused by DNA-damaging agents or cellular stress. Small molecule inhibitors of MKP-1 have not been reported, in part because of the lack of structural guidance for inhibitor design and definitive assays for MKP-1 inhibition in intact cells. Herein we have exploited a high content chemical complementation assay to analyze a diverse collection of pure natural products for cellular MKP-1 inhibition. Using two-dimensional Kolmogorov-Smirnov statistics, we identified sanguinarine, a plant alkaloid with known antibiotic and antitumor activity but no primary cellular target, as a potent and selective inhibitor of MKP-1. Sanguinarine inhibited cellular MKP-1 with an IC50 of 10 microM and showed selectivity for MKP-1 over MKP-3. Sanguinarine also inhibited MKP-1 and the MKP-1 like phosphatase, MKP-L, in vitro with IC50 values of 17.3 and 12.5 microM, respectively, and showed 5-10-fold selectivity for MKP-3 and MKP-1 over VH-1-related phosphatase, Cdc25B2, or protein-tyrosine phosphatase 1B. In a human tumor cell line with high MKP-1 levels, sanguinarine caused enhanced ERK and JNK/SAPK phosphorylation. A close congener of sanguinarine, chelerythrine, also inhibited MKP-1 in vitro and in whole cells, and activated ERK and JNK/SAPK. In contrast, sanguinarine analogs lacking the benzophenanthridine scaffold did not inhibit MKP-1 in vitro or in cells nor did they cause ERK or JNK/SAPK phosphorylation. These data illustrate the utility of a chemical complementation assay linked with multiparameter high content cellular screening.

Functional expression of mammalian opioid receptors in insect cells and high-throughput screening platforms for receptor ligand mimetics

Lepidopteran cell lines have been engineered to constitutively express high levels of mouse delta opioid receptors either alone or in combination with human Galpha16 protein. Biochemical and pharmacological studies demonstrate that these lines contain all the mediator G proteins and downstream effectors required for opioid receptor function, including phospholipase C, and that expression of exogenous Galpha16 does not contribute significantly to increased receptor responses upon activation. The activation of the phospholipase C pathway in the transformed cells upon stimulation with known receptor ligands results in easily and quantitatively measurable increases in free intracellular calcium, which can be monitored by automated fluorescent methods, while the addition of specific antagonists blocks the agonist-induced responses. Therefore, the transformed lepidopteran cell lines can be used as sensitive high-throughput screening platforms for fast detection of delta opioid receptor ligand mimetics (agonists and antagonists) in collections of natural products and synthetic compounds.

Decrypting the Biochemical Function of an Essential Gene from Streptococcus pneumoniae Using ThermoFluor® Technology

The protein product of an essential gene of unknown function from Streptococcus pneumoniae was expressed and purified for screening in the ThermoFluor affinity screening assay. This assay can detect ligand binding to proteins of unknown function. The recombinant protein was found to be in a dimeric, native-like folded state and to unfold cooperatively. ThermoFluor was used to screen the protein against a library of 3000 compounds that were specifically selected to provide information about possible biological functions. The results of this screen identified pyridoxal phosphate and pyridoxamine phosphate as equilibrium binding ligands (K(d) approximately 50 pM, K(d) approximately 2.5 microM, respectively), consistent with an enzymatic cofactor function. Several nucleotides and nucleotide sugars were also identified as ligands of this protein. Sequence comparison with two enzymes of known structure but relatively low overall sequence homology established that several key residues directly involved in pyridoxal phosphate binding were strictly conserved. Screening a collection of generic drugs and natural products identified the antifungal compound canescin A as an irreversible covalent modifier of the enzyme. Our investigation of this protein indicates that its probable biological role is that of a nucleoside diphospho-keto-sugar aminotransferase, although the preferred keto-sugar substrate remains unknown. These experiments demonstrate the utility of a generic affinity-based ligand binding technology in decrypting possible biological functions of a protein, an approach that is both independent of and complementary to existing genomic and proteomic technologies.

Activity of quinones on colletotrichum species.

The antifungal activity of 1,4-naphthoquinones, 1,2-naphthoquinones, 1,4-benzoquinones, and anthraquinones from our natural products collection was tested by direct bioautography to identify natural products with potential use in agricultural fungal pathogen control. Quinones demonstrated good to moderate antifungal activity against Colletotrichum spp. Colletotrichum fragariae was the most sensitive species to quinone-based chemistry, Colletotrichum gloeosporioides had intermediate sensitivity, while Colletotrichum acutatum was the species least sensitive to these compounds.

Antitumor potential of natural products from marine ascidians of the Gibraltar Strait: A survey

The chemical study of a selection of marine ascidians from the Gibraltar Strait has led to the characterization of a collection of natural products of diverse biosynthetic origin. The structures were elucidated by spectroscopic methods and, in certain instances, the absolute stereochemistry was determined using chiral auxiliaries following the Mosher method. In particular, the new compounds isolated from the ascidians Phallusia fumigata (Grübe, 1864), Botryllus leachi (Savigny, 1816), Stolonica socialis (Hartmayer, 1903), and Pseudodistoma obscurum (Pères, 1959) are presented and key aspects of the structural elucidation are discussed. In order to anticipate a possible antitumor application, the cytotoxicity of the new compounds was evaluated against cell lines P-388 (suspension culture of a lymphoid neoplasm from a DBA/2 mouse), A-549 (monolayer culture of a human lung carcinoma), HT-29 (monolayer culture of a human colon carcinoma), and MEL-28 (monolayer culture of a human melanoma).

Binding affinities and geometries of various metal ligands in peptide deformylase inhibitors

Removal of the N-terminal formyl group from newly synthesized proteins by the enzyme peptide deformylase (PDF) is essential for normal growth of bacteria but not higher organisms. Recently, PDF has been explored as a target for novel antibiotics. Screening a collection of natural products for antimicrobial activity identified actinonin and two matlystatin analogs as potent PDF inhibitors. A number of synthetic analogs of these natural products were prepared and their inhibitory potency determined. Previous work has shown that PDF is an iron metalloproteinase also containing a catalytic glutamic acid residue. Ligation of the ferrous cation is an essential feature of potent inhibitors. The structures of actinonin, a matlystatin analog and a synthetic inhibitor complexed with PDF were determined by crystallography. A quantum mechanics/molecular mechanics (QM/MM) method was used to reproduce the geometry of known complexes, to predict the protonation state in the active site and to predict the geometry of additional complexes. The requirement for protonation of the active site glutamate anion is an important factor in understanding the potency of inhibitors with acidic iron-ligating groups such as hydroxamate and carboxylate. Even though potent inhibitors of PDF have been discovered, their bacteriostatic mechanism of action and the rapid development of resistance in vitro may limit their potential as antibacterial drugs.

Coscinosulfate, a CDC25 phosphatase inhibitor from the sponge Coscinoderma mathewsi

The dual specificity CDC25 phosphatases dephosphorylate two inhibitory phospho-amino acids of cyclin-dependent kinases, a major family of cell cycle regulators. CDC25 inhibitors constitute new anti-mitotic agents with potential anticancer activity. While screening through a collection of natural products derived from marine organisms for CDC25A inhibitors, we purified and identified coscinosulfate 1, a sesquiterpene sulfate from the New Caledonian sponge Coscinoderma matthewsi, along with 4. The purified compound 1 displayed significant inhibitory activity towards CDC25A (IC50: 3μM).

Natural product yields small molecule that mimics insulin

Screening 50,000 mixtures from Merck's collection of synthetic and natural products, researchers at the pharmaceutical company have uncovered a small organic molecule that lowers blood glucose leve...

A non-pungent triprenyl phenol of fungal origin, scutigeral, stimulates rat dorsal root ganglion neurons via interaction at vanilloid receptors.

1. A [3H]-resiniferatoxin (RTX) binding assay utilizing rat spinal cord membranes was employed to identify novel vanilloids in a collection of natural products of fungal origin. Of the five active compounds found (scutigeral, acetyl-scutigeral, ovinal, neogrifolin, and methyl-neogrifolin), scutigeral (Ki=19 microM), isolated from the edible mushroom Albatrellus ovinus, was selected for further characterization. 2. Scutigeral induced a dose-dependent 45Ca uptake by rat dorsal root ganglion neurons with an EC50 of 1.6 microM, which was fully inhibited by the competitive vanilloid receptor antagonist capsazepine (IC50=5.2 microM). 3. [3H]-RTX binding isotherms were shifted by scutigeral (10-80 microM) in a competitive manner. The Schild plot of the data had a slope of 0.8 and gave an apparent Kd estimate for scutigeral of 32 microM. 4. Although in the above assays scutigeral mimicked capsaicin, it was not pungent on the human tongue up to a dose of 100 nmol per tongue, nor did it provoke protective wiping movements in the rat (up to 100 microM) upon intraocular instillation. 5. In accord with being non-pungent, scutigeral (5 microM) did not elicit a measurable inward current in isolated rat dorsal root ganglion neurons under voltage-clamp conditions. It did, however, reduce the proportion of neurons (from 61 to 15%) that responded to a subsequent capsaicin (1 microM) challenge. In these neurons, scutigeral both delayed (from 27 to 72 s) and diminished (from 5.0 to 1.9 nA) the maximal current evoked by capsaicin. 6. In conclusion, scutigeral and its congeners form a new chemical class of vanilloids, the triprenyl phenols. Scutigeral promises to be a novel chemical lead for the development of orally active, non-pungent vanilloids.

Polyketide System Cloned, Characterized

Minnesota researchers have cloned and characterized a massive polyketide-antibiotic biosynthetic gene cluster with some unique properties that make it a promising system for use in combinatorial biosynthesis. The work has potential applicability to the development of new antibiotics effective against drug-resistant bacteria. Polyketides are macrocyclic (largering) natural products that have been a prolific source of therapeutic drugs, such as erythromycin (C&EN, March 9, page 29). Combinatorial biosynthesis is a set of techniques in which the genes of host microorganisms are manipulated to produce libraries (collections) of natural products that can be tested for biological activity (C&EN, Sept. 14, page 29). Several groups currently are trying to use combinatorial biosynthesis and polyketide gene systems as a route to the discovery of novel biologically active agents. The new study focused on a 60-kilobase polyketide-antibiotic gene cluster in the bacterium Streptomyces venezuelae . It was a collaborativ...

633 New drug development at the end of the second millenium

A limited number of cancer patients can be cured with currently available chemotherapy. Therefore, the search for and subsequent development of more effective and selective agents for cancer remains one of the major challenges in medicine today. Over the last decade our knowledge on the molecular basis of cellular processes like proliferation, differentiation, and apoptosis and their importance in cancer biology and treatment has increased dramatically. Also specific pathways of neoplastic transformation and processes involved in tumour vascularization have been elucidated. Key proteins involved in such processes are currently investigated as possible targets for therapeutic intervention. Among these the E2F transcription factor family, the cyclin dependent kinases, the tumour suppressor gene P53, the enzyme telomerase and others have attracted enormous interest recently. This is illustrated by the rapid growth of mechanism based screens. The availability of large chemicalor natural product collections recently extended by combinatorial libraries and the presence of advanced technology allow high throughput screening of several thousands of compounds per week. Nevertheless, in vitro and in vivo models exhibiting presence or absence of such mechanisms will remain necessary to demonstrate activity at cellular and tumour level. Moreover, by studying drug activity in relation to the level of expression of different targets in tumour cell line panels, the advantages of both random and mechanism based approaches can be combined. Despite several advantages, the human tumour xenograft model in immunodeficient mice or comparable models cannot be used for such first line testing, because of the high costs and relatively large quantities of drug required. By performing limited "rodent only" toxicological research on potential anticancer agents and careful planning of subsequent steps of the drug development process significant unnecessary time loss up to several years between the discovery of a new drug and its ultimate use in clinical medicine can be avoided. This will certainly lead to an even more rapid clinical evaluation of exciting new concepts in the coming years. Several examples of new drugs already under clinical trial will be presented.

III. On the resin of ficus rubiginosa, and a new homologue of benzylic alcohol

Among the interesting collection of natural products contributed to the Paris Universal Exhibition of 1855 by the British Colonies, was a gum presenting some remarkable qualities. This substance was stated by the exhibitor to be a new species of gutta percha, in consequence of its becoming plastic when masticated, and on this account it attracted considerable attention. It had been contributed by Dr. Stephenson, of Manning River, N. S. W., who had obtained it from a species of Ficus , known as F. rubiginosa . The zealous Commissioner for New South Wales, Mr. (now Sir William) M c Arthur, brought this gum under our notice with the view of obtaining some information respecting its chemical properties. A few preliminary experiments, which were made in Paris, at once showed us that this substance had very little in common with gutta percha, and, moreover, its chemical properties were found to be entirely different from those of that gum. Although our experiments did not allow us to hold out hopes to Mr. M c Arthur of the gum becoming a valuable article of exportation from the colony, they offered some inducement to us to make a more rigorous investigation of it on purely scientific grounds. On communicating this view to Mr. M c Arthur, he liberally placed at our disposal the remainder of the specimen exhibited.