A Meroisoprenoid, Heptenolides, and C-Benzylated Flavonoids from Sphaerocoryne gracilis ssp. gracilis.

A new meroisoprenoid (1), two heptenolides (2 and 3), two C-benzylated flavonoids (4 and 5), and 11 known compounds (6-16) were isolated from leaf, stem bark, and root bark extracts of Sphaerocoryne gracilis ssp. gracilis by chromatographic separation. The structures of the new metabolites 1-5 were established by NMR, IR, and UV spectroscopic and mass spectrometric data analysis. (Z)-Sphaerodiol (7), (Z)-acetylmelodorinol (8), 7-hydroxy-6-hydromelodienone (10), and dichamanetin (15) inhibited the proliferation of Plasmodium falciparum (3D7, Dd2) with IC50 values of 1.4-10.5 µM, although these compounds also showed cytotoxicity against human embryonic kidney HEK-293 cells. None of the compounds exhibited significant disruption in protein translation when assayed in vitro.

A New Benzopyranyl Cadenane Sesquiterpene and Other Antiplasmodial and Cytotoxic Metabolites from Cleistochlamys kirkii.

Phytochemical investigations of ethanol root bark and stem bark extracts of Cleistochlamys kirkii (Benth.) Oliv. (Annonaceae) yielded a new benzopyranyl cadinane-type sesquiterpene (cleistonol, 1) alongside 12 known compounds (2-13). The structures of the isolated compounds were established from NMR spectroscopic and mass spectrometric analyses. Structures of compounds 5 and 10 were further confirmed by single crystal X-ray crystallographic analyses, which also established their absolute stereochemical configuration. The ethanolic crude extract of C. kirkii root bark gave 72% inhibition against the chloroquine-sensitive 3D7-strain malaria parasite Plasmodium falciparum at 0.01 µg/mL. The isolated metabolites dichamanetin, (E)-acetylmelodorinol, and cleistenolide showed IC50 = 9.3, 7.6 and 15.2 µM, respectively, against P. falciparum 3D7. Both the crude extract and the isolated compounds exhibited cytotoxicity against the triple-negative, aggressive breast cancer cell line, MDA-MB-231, with IC50 = 42.0 µg/mL (crude extract) and 9.6-30.7 µM (isolated compounds). Our findings demonstrate the potential applicability of C. kirkii as a source of antimalarial and anticancer agents.

Rocaglates as dual-targeting agents for experimental cerebral malaria.

Cerebral malaria (CM) is a severe and rapidly progressing complication of infection by Plasmodium parasites that is associated with high rates of mortality and morbidity. Treatment options are currently few, and intervention with artemisinin (Art) has limited efficacy, a problem that is compounded by the emergence of resistance to Art in Plasmodium parasites. Rocaglates are a class of natural products derived from plants of the Aglaia genus that have been shown to interfere with eukaryotic initiation factor 4A (eIF4A), ultimately blocking initiation of protein synthesis. Here, we show that the rocaglate CR-1-31B perturbs association of Plasmodium falciparum eIF4A (PfeIF4A) with RNA. CR-1-31B shows potent prophylactic and therapeutic antiplasmodial activity in vivo in mouse models of infection with Plasmodium berghei (CM) and Plasmodium chabaudi (blood-stage malaria), and can also block replication of different clinical isolates of P. falciparum in human erythrocytes infected ex vivo, including drug-resistant P. falciparum isolates. In vivo, a single dosing of CR-1-31B in P. berghei-infected animals is sufficient to provide protection against lethality. CR-1-31B is shown to dampen expression of the early proinflammatory response in myeloid cells in vitro and dampens the inflammatory response in vivo in P. berghei-infected mice. The dual activity of CR-1-31B as an antiplasmodial and as an inhibitor of the inflammatory response in myeloid cells should prove extremely valuable for therapeutic intervention in human cases of CM.

CRISPR/Cas9 Editing to Facilitate and Expand Drug Discovery.

INTRODUCTION: The ability of most laboratories to easily access CRISPR/Cas9 engineering tools has caused a revolution in biology. One of the areas that will continue to be impacted by genome editing is the drug discovery process. OBJECTIVE: CRISPR/Cas9 will not only serve to accelerate the drug discovery pipeline, but also streamline line it by identifying high-value targets, facilitating the validation of drug: target interactions and mechanisms of action, and stimulating the development of phenotype-based high throughput screens as alternatives to target-based assays. CONCLUSION: We review the literature and hurdles that have been overcome to develop the current generation of tools being used to enrich the drug discovery paradigm.

5,10b-Ethanophenanthridine amaryllidaceae alkaloids inspire the discovery of novel bicyclic ring systems with activity against drug resistant cancer cells.

Plants of the Amaryllidaceae family produce a large variety of alkaloids and non-basic secondary metabolites, many of which are investigated for their promising anticancer activities. Of these, crinine-type alkaloids based on the 5,10b-ethanophenanthridine ring system were recently shown to be effective at inhibiting proliferation of cancer cells resistant to various pro-apoptotic stimuli and representing tumors with dismal prognoses refractory to current chemotherapy, such as glioma, melanoma, non-small-cell lung, esophageal, head and neck cancers, among others. Using this discovery as a starting point and taking advantage of a concise biomimetic route to the crinine skeleton, a collection of crinine analogues were synthetically prepared and evaluated against cancer cells. The compounds exhibited single-digit micromolar activities and retained this activity in a variety of drug-resistant cancer cell cultures. This investigation resulted in the discovery of new bicyclic ring systems with significant potential in the development of effective clinical cancer drugs capable of overcoming cancer chemotherapy resistance.

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.

Inhibition of translation by cytotrienin A--a member of the ansamycin family.

The ansamycins are a diverse and often physiologically active group of compounds that include geldanamycin and rifamycin, inhibitors of heat shock protein 90 and prokaryotic DNA-dependent RNA synthesis, respectively. Cytotrienin A is an ansamycin-type small molecule with potent antiproliferative and proapoptotic properties. Here, we report that this compound inhibits eukaryotic protein synthesis by targeting translation elongation and interfering with eukaryotic elongation factor 1A function. We also find that cytotrienin A prevents HUVEC tube formation and diminishes microvessel formation in the chorioallantoic membrane assay. These results provide a molecular understanding into cytotrienin A's previously reported properties as an anticancer apoptosis-inducing drug.

Experimental design and statistical methods for improved hit detection in high-throughput screening.

Identification of active compounds in high-throughput screening (HTS) contexts can be substantially improved by applying classical experimental design and statistical inference principles to all phases of HTS studies. The authors present both experimental and simulated data to illustrate how true-positive rates can be maximized without increasing false-positive rates by the following analytical process. First, the use of robust data preprocessing methods reduces unwanted variation by removing row, column, and plate biases. Second, replicate measurements allow estimation of the magnitude of the remaining random error and the use of formal statistical models to benchmark putative hits relative to what is expected by chance. Receiver Operating Characteristic (ROC) analyses revealed superior power for data preprocessed by a trimmed-mean polish method combined with the RVM t-test, particularly for small- to moderate-sized biological hits.

Synergistic effect of inhibiting translation initiation in combination with cytotoxic agents in acute myelogenous leukemia cells.

We have previously shown that inhibition of translation initiation, using the small molecule inhibitor silvestrol, induces apoptosis in a pre-clinical murine lymphoma model when combined with daunorubicin. Silvestrol blocks ribosome recruitment by targeting the RNA helicase, eIF4A, which is required for this process. Here we investigate the sensitivity of acute myelogenous leukemia (AML) cell lines to protein synthesis inhibition in combination with the standard cytotoxic agents daunorubicin, etoposide, and cytarabine. Silvestrol shows synergy with standard-of-care agents in AML cell lines and synergizes with ABT-737, a small molecule inhibitor of Bcl-X(L) and Bcl-2. The in vitro synergy between silvestrol and the cytotoxic drugs used in AML therapy provides a basis for in vivo evaluation of these combinations.

Silibinin inhibits translation initiation: implications for anticancer therapy.

Silibinin is a nontoxic flavonoid reported to have anticancer properties. In this study, we show that silibinin exhibits antiproliferative activity on MCF-7 breast cancer cells. Exposure to silibinin leads to a concentration-dependent decrease in global protein synthesis associated with reduced levels of eukaryotic initiation factor 4F complex. Moreover, polysome profile analysis of silibinin-treated cells shows a decrease in polysome content and translation of cyclin D1 mRNA. Silibinin exerts its effects on translation initiation by inhibiting the mammalian target of rapamycin signaling pathway by acting upstream of TSC2. Our results show that silibinin blocks mammalian target of rapamycin signaling with a concomitant reduction in translation initiation, thus providing a possible molecular mechanism of how silibinin can inhibit growth of transformed cells.

Quassinoid inhibition of AP-1 function does not correlate with cytotoxicity or protein synthesis inhibition.

Several quassinoids were identified in a high-throughput screening assay as inhibitors of the transcription factor AP-1. Further biological characterization revealed that while their effect was not specific to AP-1, protein synthesis inhibition and cell growth assays were inconsistent with a mechanism of simple protein synthesis inhibition. Numerous plant extracts from the plant family Simaroubaceae were also identified in the same screen; bioassay-guided fractionation of one extract (Ailanthus triphylla) yielded two known quassinoids, ailanthinone (3) and glaucarubinone (4), which were also identified in the pure compound screening procedure.

Identifying small molecule inhibitors of eukaryotic translation initiation.

In eukaryotes, translation initiation is rate-limiting with much regulation exerted at the ribosome recruitment and ternary complex (eIF2.GTP.Met-tRNA(i)(Met)) formation steps. Although small molecule inhibitors have been extremely useful for chemically dissecting translation, there is a dearth of compounds available to study the initiation phase in vitro and in vivo. In this chapter, we describe reverse and forward chemical genetic screens developed to identify new inhibitors of translation. The ability to manipulate cell extracts biochemically, and to compare the activity of small molecules on translation of mRNA templates that differ in their factor requirements for ribosome recruitment, facilitates identification of the relevant target.

Homogenous time resolved fluorescence assay to identify modulators of cap-dependent translation initiation.

Eukaryotic initiation factor (eIF) 4F plays a key role in recruiting 40S ribosomes and associated factors to mRNA templates during translation initiation. The function of this heterotrimeric complex is to deliver an RNA helicase to the 5' cap proximal region of mRNAs in preparation for ribosome binding. To study the interaction between subunits of this complex, as well as identify small molecules that could interfere with their association, we developed a time resolved fluorescence assay that allows monitoring of interactions between two subunits of eIF4F. We have performed a small molecule chemical screen of >73,000 compounds using this assay.

Statistical practice in high-throughput screening data analysis.

High-throughput screening is an early critical step in drug discovery. Its aim is to screen a large number of diverse chemical compounds to identify candidate 'hits' rapidly and accurately. Few statistical tools are currently available, however, to detect quality hits with a high degree of confidence. We examine statistical aspects of data preprocessing and hit identification for primary screens. We focus on concerns related to positional effects of wells within plates, choice of hit threshold and the importance of minimizing false-positive and false-negative rates. We argue that replicate measurements are needed to verify assumptions of current methods and to suggest data analysis strategies when assumptions are not met. The integration of replicates with robust statistical methods in primary screens will facilitate the discovery of reliable hits, ultimately improving the sensitivity and specificity of the screening process.

High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer 1-based beta-arrestin2 recruitment assay.

In this study, the authors developed HEK293 cell lines that stably coexpressed optimal amounts of beta-arrestin2-Rluc and VENUS fusions of G protein-coupled receptors (GPCRs) belonging to both class A and class B receptors, which include receptors that interact transiently or stably with beta-arrestins. This allowed the use of a bioluminescence resonance energy transfer (BRET) 1- beta-arrestin2 translocation assay to quantify receptor activation or inhibition. One of the developed cell lines coexpressing CCR5-VENUS and beta-arrestin2- Renilla luciferase was then used for high-throughput screening (HTS) for antagonists of the chemokine receptor CCR5, the primary co-receptor for HIV. A total of 26,000 compounds were screened for inhibition of the agonist-promoted beta-arrestin2 recruitment to CCR5, and 12 compounds were found to specifically inhibit the agonist-induced beta-arrestin2 recruitment to CCR5. Three of the potential hits were further tested using other functional assays, and their abilities to inhibit CCR5 agonist-promoted signaling were confirmed. This is the 1st study describing a BRET1-beta-arrestin recruitment assay in stable mammalian cells and its successful application in HTS for GPCRs antagonists.

A new antimalarial quassinoid from Simaba orinocensis.

A new antimalarial quassinoid, namely, orinocinolide (1), was isolated from the root bark of Simaba orinocensis, together with the previously reported simalikalactone D (2). The structure of 1 was determined primarily from 1D and 2D NMR analysis, as well as by chemical derivatization. Compound 1 was found to be as equally potent as 2 against Plasmodium falciparum clones D6 and W2 (IC(50) 3.27 and 8.53 ng/mL vs 3.0 and 3.67 ng/mL, respectively), but was 4- and 28-fold less toxic than 2 against VERO cells (IC(50) 10 vs 2.3 microg/mL) and HL-60 (IC(50) 0.7 vs 0.025 microg/mL), respectively. In addition, 2 was >46- and >31-fold more potent than pentamidine and amphotericin B (IC(50) 0.035 vs 1.6 and 1.1 microg/mL) against Leishmania donovani, while 1 was inactive. Orinocinolide (1) inhibited growth of human cancer cells SK-MEL, KB, BT-549, and SK-OV-3, but was less potent than 2 (IC(50) 0.8-1.9 vs 0.3-1.0 microg/mL) against these cells.

Eukaryotic protein synthesis inhibitors identified by comparison of cytotoxicity profiles.

The National Cancer Institute (NCI) Human Tumor Cell Line Anti-Cancer Drug Screen has evaluated the cytotoxicity profiles of a large number of synthetic compounds, natural products, and plant extracts on 60 different cell lines. The data for each compound/extract can be assessed for similarity of cytotoxicity pattern, relative to a given test compound, using an algorithm called COMPARE. In applying a chemical biology approach to better understand the mechanism of eukaryotic protein synthesis, we used these resources to search for novel inhibitors of translation. The cytotoxicity profiles of 31 known protein synthesis inhibitors were used to identify compounds from the NCI database with similar activity profiles. Using this approach, two natural products, phyllanthoside and nagilactone C, were identified and characterized as novel protein synthesis inhibitors. Both compounds are specific for the eukaryotic translation apparatus, function in vivo and in vitro, and interfere with translation elongation. Our results demonstrate the feasibility of utilizing cytotoxicity profiles to identify new inhibitors of translation.