Identification of inhibitors for MDM2 ubiquitin ligase activity from natural product extracts by a novel high-throughput electrochemiluminescent screen

High-throughput screening technologies have revolutionized the manner in which potential therapeutics are identified. Although they are the source of lead compounds for ~65% of anticancer and antimicrobial drugs approved by the Food and Drug Administration between 1981 and 2002, natural products have largely been excluded from modern screening programs. This is due, at least in part, to the inherent difficulties in testing complex extract mixtures, which often contain nuisance compounds, in modern bioassay systems. In this article, the authors present a novel electrochemiluminescent assay system for inhibition of MDM2 activity that is suitable for testing natural product extracts in high-throughput screening systems. The assay was used to screen more than 144,000 natural product extracts. The authors identified 1 natural product, sempervirine, that inhibited MDM2 auto-ubiquitination, MDM2-mediated p53 degradation, and led to accumulation of p53 in cells. Sempervirine preferentially induced apoptosis in transformed cells expressing wild-type p53, suggesting that it could be a potential lead for anticancer therapeutics.     

Antimutagenic compounds and their possible mechanisms of action

Mutagenicity refers to the induction of permanent changes in the DNA sequence of an organism, which may result in a heritable change in the characteristics of living systems. Antimutagenic agents are able to counteract the effects of mutagens. This group of agents includes both natural and synthetic compounds. Based on their mechanism of action among antimutagens, several classes of compounds may be distinguished. These are compounds with antioxidant activity; compounds that inhibit the activation of mutagens; blocking agents; as well as compounds characterized with several modes of action. It was reported previously that several antitumor compounds act through the antimutagenic mechanism. Hence, searching for antimutagenic compounds represents a rapidly expanding field of cancer research. It may be observed that, in recent years, many publications were focused on the screening of both natural and synthetic compounds for their beneficial muta/antimutagenicity profile. Thus, the present review attempts to give a brief outline on substances presenting antimutagenic potency and their possible mechanism of action. Additionally, in the present paper, a screening strategy for mutagenicity testing was presented and the characteristics of the most widely used antimutagenicity assays were described.     

Induction of apoptosis against cancer cell lines by four ascomycetes (endophytes) from Malaysian rainforest

Endophytic fungi have been shown to be a promising source of biologically active natural products. In the present study, extracts of four endophytic fungi isolated from plants of the National Park, Pahang were evaluated for their cytotoxic activity and the nature of their active compounds determined. Those extracts exhibiting activity with IC(50) values less than 17 μg/ml against HCT116, MCF-7 and K562 cell lines were shown to induce apoptosis in these cell lines. Molecular analysis, based on sequences of the rDNA internal transcribed spacers ITS1 and ITS4, revealed all four endophytic fungi to be ascomycetes: three sordariomycetes and a dothideomycete. Six known compounds, cytochalasin J, dechlorogriseofulvin, demethylharzianic-acid, griseofulvin, harzianic acid and 2-hexylidene-3-methyl-succinic acid were identified from a rapid dereplication technique for fungal metabolites using an in-house UV library. The results from the present study suggest the potential of endophytic fungi as cytotoxic agents, and there is an indication that the isolates contain bioactive compounds that mainly kill cancer cells by apoptosis.     

Discovery and development of heat shock protein 90 inhibitors

Heat shock protein 90 (Hsp90) is an important target in cancer because of its role in maintaining transformation and has recently become the focus of several drug discovery and development efforts. While compounds with different modes of action are known, the focus of this review is on those classes of compounds which inhibit Hsp90 by binding to the N-terminal ATP pocket. These include natural product inhibitors such as geldanamycin and radicicol and synthetic inhibitors comprised of purines, pyrazoles, isoxazoles and other scaffolds. The synthetic inhibitors have been discovered either by structure-based design, high throughput screening and more recently using fragment-based design and virtual screening techniques. This review will discuss the discovery of these different classes, as well as their development as potential clinical agents.     

Cytotoxic,immunosuppressive, trypanocidal and antileishmanial activities of Basidiomycota fungi present in Atlantic Rainforest in Brazil

One hundred three Basidiomycota fungi representing 84 species and 17 families were collected from different Atlantic Rainforest in Brazil. Their basidiomes and fermentation broth extracts were screened in a bioassay panel that included three human cancer cells lines, human peripheral blood mononuclear cells (PBMCs), the enzyme trypanothione reductase (TryR) from Trypanosoma cruzi, and amastigote forms of Leishmania amazonensis. Forty-two extracts representing 21 genera and 35 species presented activities higher than 60% in one or more assays employed in this study. Eighteen extracts were toxic to one or more human cancer cell lines. Extracts from Lentinus strigosus CCB 178 and Lentinus sp. UFMGCB 38 showed selectivity towards cancer cells as they showed only a minor impact on PBMCs. Six extracts suppressed PBMCs proliferation and showed low toxic effect to cancer cells. Thirty-four extracts inhibited the activity of the TryR. Of these, five showed low toxicity towards PBMCs. Extracts from Gymnopilus areolatus, Irpex lacteus, L. strigosus, Nothopanus hygrophanus, Pleurotus flabellatus, and unidentified Basidiomycetes were toxic to L. amazonensis. The results of this screening reinforce the potential of Basidiomycota fungi as sources of bioactive natural products that may be developed into new therapeutic agents for cancer and neglected diseases such as trypanosomiasis and leishmaniasis.     

Rapid identification of elaiophylin and geldanamycin inStreptomyces fermentation broths using CPC coupled with a photodiode array detector and LC-MS methodologies

During the course of screening microbial broth extracts in various high through-put bioassays (eg receptor binding or enzyme inhibition), several actinomycete cultures were discovered to produce active metabolites. The natural products elaiophylin and/or geldanamycin are produced by severalStreptomyces violaceusniger strains, and the bioactivity of the extracts from these cultures was frequently associated with the fractions containing these metabolites. CPC coupled to a photodiode array detector and LC-MS techniques were applied to these broth extracts to ascertain rapidly when these natural products were present. These methodologies allowed us to identify the metabolites quickly in the crude extract, and the application demonstrated further the utility of CPC-photodiode array detection and LC-MS as powerful, initial analytical tools in analyses of the complex metabolite profiles produced by microorganisms.     

Novel bioactive natural products from bacteria via bioprospecting,genome mining and metabolic engineering

For over seven decades, bacteria served as a valuable source of bioactive natural products some of which were eventually developed into drugs to treat infections, cancer and immune system-related diseases. Traditionally, novel compounds produced by bacteria were discovered via conventional bioprospecting based on isolation of potential producers and screening their extracts in a variety of bioassays. Over time, most of the natural products identifiable by this approach were discovered, and the pipeline for new drugs based on bacterially produced metabolites started to run dry. This mini-review highlights recent developments in bacterial bioprospecting for novel compounds that are based on several out-of-the-box approaches, including the following: (i) targeting bacterial species previously unknown to produce any bioactive natural products, (ii) exploring non-traditional environmental niches and methods for isolation of bacteria and (iii) various types of ‘genome mining’ aimed at unravelling genetic potential of bacteria to produce secondary metabolites. All these approaches have already yielded a number of novel bioactive compounds and, if used wisely, will soon revitalize drug discovery pipeline based on bacterial natural products.     

Identification of Traditional Medicinal Plant Extracts with Novel Anti-Influenza Activity

The emergence of drug resistant variants of the influenza virus has led to a need to identify novel and effective antiviral agents. As an alternative to synthetic drugs, the consolidation of empirical knowledge with ethnopharmacological evidence of medicinal plants offers a novel platform for the development of antiviral drugs. The aim of this study was to identify plant extracts with proven activity against the influenza virus. Extracts of fifty medicinal plants, originating from the tropical rainforests of Borneo used as herbal medicines by traditional healers to treat flu-like symptoms, were tested against the H1N1 and H3N1 subtypes of the virus. In the initial phase, in vitro micro-inhibition assays along with cytotoxicity screening were performed on MDCK cells. Most plant extracts were found to be minimally cytotoxic, indicating that the compounds linked to an ethnomedical framework were relatively innocuous, and eleven crude extracts exhibited viral inhibition against both the strains. All extracts inhibited the enzymatic activity of viral neuraminidase and four extracts were also shown to act through the hemagglutination inhibition (HI) pathway. Moreover, the samples that acted through both HI and neuraminidase inhibition (NI) evidenced more than 90% reduction in virus adsorption and penetration, thereby indicating potent action in the early stages of viral replication. Concurrent studies involving Receptor Destroying Enzyme treatments of HI extracts indicated the presence of sialic acid-like component(s) that could be responsible for hemagglutination inhibition. The manifestation of both modes of viral inhibition in a single extract suggests that there may be a synergistic effect implicating more than one active component. Overall, our results provide substantive support for the use of Borneo traditional plants as promising sources of novel anti-influenza drug candidates. Furthermore, the pathways involving inhibition of hemagglutination could be a solution to the global occurrence of viral strains resistant to neuraminidase drugs.     

Exploring anti-TB leads from natural products library originated from marine microbes and medicinal plants

Multidrug-resistant tuberculosis (MDR-TB) and TB–HIV co-infection have become a great threat to global health. However, the last truly novel drug that was approved for the treatment of TB was discovered 40?years ago. The search for new effective drugs against TB has never been more intensive. Natural products derived from microbes and medicinal plants have been an important source of TB therapeutics. Recent advances have been made to accelerate the discovery rate of novel TB drugs including diversifying strategies for environmental strains, high-throughput screening (HTS) assays, and chemical diversity. This review will discuss the challenges of finding novel natural products with anti-TB activity from marine microbes and plant medicines, including biodiversity- and taxonomy-guided microbial natural products library construction, target- and cell-based HTS, and bioassay-directed isolation of anti-TB substances from traditional medicines.     

Investigating the antiangiogenic potential of Rumex vesicarius (humeidh), anticancer activity in cancer cell lines and assessment of developmental toxicity in zebrafish embryos

Recent trends in anticancer therapy is to use therapeutic agents which not only kill the cancer cell, but are less toxic to surrounding normal cells/tissue. One approach is to cut the nutrient supply to growing tumor cells, by blocking the formation of new blood vessels around the tumor. As the phytochemicals and botanical crude extracts have proven their efficacy as natural antiangiogenic agents with minimum toxicities, there is need to explore varieties of medicinal plants for novel antiangiogenic compounds.Rumex vesicarius L. (Humeidh), is an annual herbal plant with proven medicinal values. The antiangiogenic potential, and developmental toxicity of humeidh in experimental animal models has never been studied before. The crude extracts were prepared from the roots, stems, leaves and flowers of Rumex vesicarius L. in methanol, chloroform, ethyl acetate and n-hexane. The developmental toxicity screening in zebrafish embryos, has revealed that Rumex vesicarius was not toxic to zebrafish embryos. The chloroform stem extract showed significant level of antiangiogenic activity in zebrafish angiogenic assay on a dose dependent manner. Thirty five (35) bioactive compounds were identified by gas chromatography mass spectrophotometry (GC–MS) analysis in the stem extract of Rumex vesicarius. Propanoic acid, 2-[(trimethylsilyl)oxy]-, trimethylsilyl ester, Butane, 1,2,3-tris(trimethylsiloxy), and Butanedioic acid, bis(trimethylsilyl) ester were identified as major compound present in the stem of R. vasicarius.The anticancer activity of roots, stem, leaves and flowers crude extract was evaluated in human breast cancer (MCF7), human colon carcinoma (Lovo, and Caco-2), human hepatocellular carcinoma (HepG2) cell lines. Most of the crude extracts did not show significant level of cytotoxicity in tested cancer cells line, except, chloroform extract of stem which exhibited strong anticancer activity in all tested cancer cells with IC₅₀ values in micro molar range.Based on these results, it is recommended that formulation prepared from R. vesicarius can further be tested in clinical trials in order to explore its therapeutic potential as an effective and safe natural anticancer product.     

Methyl jasmonate: A plant stress hormone as an anti-cancer drug

Jasmonates act as signal transduction intermediates when plants are subjected to environmental stresses such as UV radiation, osmotic shock and heat. In the past few years several groups have reported that jasmonates exhibit anti-cancer activity in vitro and in vivo and induce growth inhibition in cancer cells, while leaving the non-transformed cells intact. Recently, jasmonates were also discovered to have cytotoxic effects towards metastatic melanoma both in vitro and in vivo.Three mechanisms of action have been proposed to explain this anti-cancer activity. The bio-energetic mechanism – jasmonates induce severe ATP depletion in cancer cells via mitochondrial perturbation. Furthermore, methyl jasmonate (MJ) has the ability to detach hexokinase from the mitochondria. Second, jasmonates induce re-differentiation in human myeloid leukemia cells via mitogen-activated protein kinase (MAPK) activity and were found to act similar to the cytokinin isopentenyladenine (IPA). Third, jasmonates induce apoptosis in lung carcinoma cells via the generation of hydrogen peroxide, and pro-apoptotic proteins of the Bcl-2 family.Combination of MJ with the glycolysis inhibitor 2-deoxy-d-glucose (2DG) and with four conventional chemotherapeutic drugs resulted in super-additive cytotoxic effects on several types of cancer cells. Finally, jasmonates have the ability to induce death in spite of drug-resistance conferred by either p53 mutation or P-glycoprotein (P-gp) over-expression.In summary, the jasmonates are anti-cancer agents that exhibit selective cytotoxicity towards cancer cells, and thus present hope for the development of cancer therapeutics.     

Single-Stranded siRNAs Activate RNAi in Animals

The therapeutic utility of siRNAs is limited by the requirement for complex formulations to deliver them to tissues. If potent single-stranded RNAs could be identified, they would provide a simpler path to pharmacological agents. Here, we describe single-stranded siRNAs (ss-siRNAs) that silence gene expression in animals absent lipid formulation. Effective ss-siRNAs were identified by iterative design by determining structure-activity relationships correlating chemically modified single strands and Argonaute 2 (AGO2) activities, potency in cells, nuclease stability, and pharmacokinetics. We find that the passenger strand is not necessary for potent gene silencing. The guide-strand activity requires AGO2, demonstrating action through the RNAi pathway. ss-siRNA action requires a 5' phosphate to achieve activity in?vivo, and we developed a metabolically stable 5'-(E)-vinylphosphonate (5'-VP) with conformation and sterioelectronic properties similar to the natural phosphate. Identification of potent ss-siRNAs offers an additional option for RNAi therapeutics and an alternate perspective on RNAi mechanism.     

Plant-based anticancer molecules: a chemical and biological profile of some important leads

A number of natural products, with diverse chemical structures, have been isolated as anticancer agents. Several potential lead molecules such as camptothecin, vincristine, vinblastine, taxol, podophyllotoxin, combretastatins, etc. have been isolated from plants and many of them have been modified to yield better analogues for activity, toxicity or solubility. Several successful molecules like topotecan, irinotecan, taxotere, etoposide, teniposide, etc. also have emerged as drugs upon modification of these natural leads and many more are yet to come. In this review, the authors have focused on four important anticancer leads, that is, camptothecin, taxol, combretastatin A-4 and podophyllotoxin. Their chemistry, structure and activity relationships, biological activities, modes of action, analogue synthesis and future prospects have been discussed.     

Synthetic analogs of indole-containing natural products as inhibitors of sortase A and isocitrate lyase

Guided by the inhibitory activities of indole-containing natural products against isocitrate lyase (ICL) from Candida albicans and sortase A (SrtA) from Staphylococcus aureus, a series of compounds structurally analogous to natural products were synthesized. Eight SrtA inhibitors and an ICL inhibitor having higher activities than the natural products were discovered by screening the enzyme inhibitory activities of synthesized compounds. Among the SrtA inhibitors discovered, six exhibited higher activities than p-hydroxymercuribenzoic acid, which suggests that these compounds have great potential as alternative antibacterial agents.     

Plant extracts as modulators of genotoxic effects

Higher plants used extensively in traditional medicines are increasingly being screened for their role in modulating the activity of environmental genotoxicants. The property of preventing carcinogenesis has been reported in many plant extracts. The observation of a close association between carcinogenesis and mutagenesis has extended the survey to include plant extracts and plant products able to modify the process of mutagenesis, which involves alteration in the genetic material. Natural plant products may, apart from inducing mutations, modify the action of other known mutagens on the living organisms by 1) activating the existing mutagens within the cell, 2) inhibiting the production of mutagens in the cell, 3) synergising the activity of existing mutagens, or 4) activating the promutagens within the cell into mutagens. This review deals with data obtained in the course of research on the modulatory effects of plant extracts on mutagenesis and clastogenesis, two genotoxic phenomena associated with carcinogenesis. In screening for antimutagenic effects, extracts of different plant parts have been used, ranging from leafy vegetables, fruits, and underground storage organs to whole plants. The extracts were prepared mainly in water or organic solvents. Several of these assays have indicated the involvement of certain factors that are intrinsic components of the extracts, ranging from specific compounds like ascorbic acid to vegetable fibres which could act as nonspecific redox agents, free radical scavengers, or ligands for binding metals or toxic principles. The possible ways in which inhibitors of mutagenesis can act include the inhibition of interaction between genes and biochemically reactive mutagens and the inhibition of metabolic activation of indirectly acting mutagens. The effects of toxicants can be observed at the level of chromosomes (clastogenesis) through alterations in chromosome structure (chromosomal aberrations) and number (aneuploidy, polyploidy). A wide range of short-term and long-term screening procedures is available. The most common ones use higher plants or rodents in vivo as test systems for monitoring chromosomal aberrations. Experiments with a number of crude vegetable and fruit extracts have demonstrated their anticlastogenic activities against known cytotoxic agents. The individual components of the extracts—e.g., sulfhydryl and flavonoid compounds, gallic acid, ellagic acid, mucic acid, citric acid, reducing sugars, tannin—are observed to have an additive interaction with the major constituents chlorophyll and ascorbic acid, when modulating the effects of the clastogens. Under certain conditions, plant products may induce mutagenic effects, due to the presence of multiple biological properties. Some inhibitors can stimulate simultaneously both enhancing and detoxifying mechanisms, e.g., inducers of coordinated enzyme activities. Many oxidants can, depending on the redox potential, either accept or donate electrons, rendering them protective or harmful. Plants also play an active role in the accumulation, metabolism, and environmental distribution of xenobiotics. The property of plants to activate promutagens that may enter the food chain is of great significance in view of the large number and types of chemicals to which the plants are exposed. A promutagen is a chemical that is not mutagenic itself but that can be biologically transformed by a plant system into a mutagen. Several methods for studying promutagens from plants were developed both in vivo and in vitro, including plant cell-free systems. Both mutagens and antimutagens can be extracted from the same plant extract depending on the nature of solvents used for extraction. Interaction between inhibitors may lead to synergistic effects. Such combined action may take place through the different inhibitors acting at different levels or being localised at different cellular areas. The greater protection afforded by crude plant extract as compared with an equivalent amount of the purified or synthetic ingredients, as observed withPhyllanthus emblica L. andBeta vulgaris L. var.benghalensis Hort., may be related to this phenomenon. Specific biological action of a drug is due to its specific binding to a functional molecular receptor. In complex plant extracts, the variable observed effects can be attributed to the many chemically reactive species that are formed during the processing and ingestion of the extract, which could act as non-specific redox agents, scavengers of free radicals, and ligands for binding to toxicants. The final effects are obviously the outcome of interactions between the components and their individual and collective interaction with the toxicant. The specificity and efficacy of such responses will be influenced also by the physiological factors influencing the plants and the process of administration of the extract. In utilizing pharmacologically active herbs, both beneficial and potential adverse effects must be taken into account. The actual dose and form of the plant also need to be worked out.     

Stimulators of translation identified during a small molecule screening campaign

In screening a library of natural and synthetic products for eukaryotic translation modulators, we identified two natural products, isohymenialdisine and hymenialdisine, that exhibit stimulatory effects on translation. The characterization of these compounds led to the insight that mRNA used to program the translation extracts during high-throughput assay setup was leading to phosphorylation of eIF2α, a potent negative regulatory event that is mediated by one of four kinases. We identified double-stranded RNA-dependent protein kinase (PKR) as the eIF2α kinase that was being activated by exogenously added mRNA template. Characterization of the mode of action of isohymenialdisine revealed that it directly acts on PKR by inhibiting autophosphorylation, perturbs the PKR–eIF2α phosphorylation axis, and can be modeled into the PKR ATP binding site. Our results identify a source of “false positives” for high-throughput screen campaigns using translation extracts, raising a cautionary note for this type of screen.