Genomic and Targeted Approaches Unveil the Cell Membrane as a Major Target of the Antifungal Cytotoxin Amantelide A.

Amantelide A, a polyhydroxylated macrolide isolated from a marine cyanobacterium, displays broad-spectrum activity against mammalian cells, bacterial pathogens, and marine fungi. We conducted comprehensive mechanistic studies to identify the molecular targets and pathways affected by amantelide A. Our investigations relied on chemical structure similarities with compounds of known mechanisms, yeast knockout mutants, yeast chemogenomic profiling, and direct biochemical and biophysical methods. We established that amantelide A exerts its antifungal action by binding to ergosterol-containing membranes followed by pore formation and cell death, a mechanism partially shared with polyene antifungals. Binding assays demonstrated that amantelide A also binds to membranes containing epicholesterol or mammalian cholesterol, thus suggesting that the cytotoxicity to mammalian cells might be due to its affinity to cholesterol-containing membranes. However, membrane interactions were not completely dependent on sterols. Yeast chemogenomic profiling suggested additional direct or indirect effects on actin. Accordingly, we performed actin polymerization assays, which suggested that amantelide A also promotes actin polymerization in cell-free systems. However, the C-33 acetoxy derivative amantelide B showed a similar effect on actin dynamics in vitro but no significant activity against yeast. Overall, these studies suggest that the membrane effects are the most functionally relevant for amantelide A mechanism of action.

The nucleus is irreversibly shaped by motion of cell boundaries in cancer and non-cancer cells.

Actomyosin stress fibers impinge on the nucleus and can exert compressive forces on it. These compressive forces have been proposed to elongate nuclei in fibroblasts, and lead to abnormally shaped nuclei in cancer cells. In these models, the elongated or flattened nuclear shape is proposed to store elastic energy. However, we found that deformed shapes of nuclei are unchanged even after removal of the cell with micro-dissection, both for smooth, elongated nuclei in fibroblasts and abnormally shaped nuclei in breast cancer cells. The lack of shape relaxation implies that the nuclear shape in spread cells does not store any elastic energy, and the cellular stresses that deform the nucleus are dissipative, not static. During cell spreading, the deviation of the nucleus from a convex shape increased in MDA-MB-231 cancer cells, but decreased in MCF-10A cells. Tracking changes of nuclear and cellular shape on micropatterned substrata revealed that fibroblast nuclei deform only during deformations in cell shape and only in the direction of nearby moving cell boundaries. We propose that motion of cell boundaries exert a stress on the nucleus, which allows the nucleus to mimic cell shape. The lack of elastic energy in the nuclear shape suggests that nuclear shape changes in cells occur at constant surface area and volume.

A Tryptoline Ring-Distortion Strategy Leads to Complex and Diverse Biologically Active Molecules from the Indole Alkaloid Yohimbine.

High-throughput screening (HTS) is the primary driver to current drug-discovery efforts. New therapeutic agents that enter the market are a direct reflection of the structurally simple compounds that make up screening libraries. Unlike medically relevant natural products (e.g., morphine), small molecules currently being screened have a low fraction of sp3 character and few, if any, stereogenic centers. Although simple compounds have been useful in drugging certain biological targets (e.g., protein kinases), more sophisticated targets (e.g., transcription factors) have largely evaded the discovery of new clinical agents from screening collections. Herein, a tryptoline ring-distortion strategy is described that enables the rapid synthesis of 70 complex and diverse compounds from yohimbine (1); an indole alkaloid. The compounds that were synthesized had architecturally complex and unique scaffolds, unlike 1 and other scaffolds. These compounds were subjected to phenotypic screens and reporter gene assays, leading to the identification of new compounds that possessed various biological activities, including antiproliferative activities against cancer cells with functional hypoxia-inducible factors, nitric oxide inhibition, and inhibition and activation of the antioxidant response element. This tryptoline ring-distortion strategy can begin to address diversity problems in screening libraries, while occupying biologically relevant chemical space in areas critical to human health.

Pitiamides A and B, Multifunctional Fatty Acid Amides from Marine Cyanobacteria.

Two geometric isomers related to pitiamide A, termed 1E-pitiamide B (1) and 1Z-pitiamide B (2), were isolated from a marine cyanobacterium collected from the shallow reef flat at Piti Bomb Holes, Guam, Mariana Islands. The structures of these analogues were elucidated using 1D and 2D NMR analysis. Pitiamide A, which has been previously described, but has not been investigated in bioassays, was co-isolated. Pitiamides A and B were subjected to a biological evaluation and they both showed antiproliferative effects on HCT116 cells with IC50 values of 1-5 µM. Pitiamide A was investigated individually and caused plasma membrane hyperpolarization and an increase of intracellular calcium in HCT116 cells.

The cellular target specificity of pateamine A.

The natural product pateamine A (pateamine) from the sponge Mycale hentscheli is active against a wide range of dividing cells and has been shown to inhibit the functions of the eukaryotic initiation factor 4A (eIF4A). We have identified that pateamine is additionally able to modulate the formation of actin filaments and microtubules in vitro but at higher concentrations than required for inhibition of eIF4A. Cell cycle analysis confirmed that actin and tubulin are not major mediators of the cellular activity of pateamine. The range of targets identified demonstrates the value of multiple approaches to determining the mode of action of biologically active compounds.

The protein synthesis inhibitors mycalamides A and E have limited susceptibility toward the drug efflux network.

The mycalamides belong to a family of protein synthesis inhibitors noted for antifungal, antitumour, antiviral, immunosuppressive, and nematocidal activities. Here we report a systematic analysis of the role of drug efflux pumps in mycalamide resistance and the first isolation of mycalamide E. In human cell lines, neither P-glycoprotein overexpression nor the use of efflux pump inhibitors significantly modulated mycalamide A toxicity in the systems tested. In Saccharomyces cerevisiae, it appears that mycalamide A is subject to efflux by the principle mediator of xenobiotic efflux, Pdr5p along with the major facilitator superfamily pump Tpo1p. Mycalamide E showed a similar efflux profile. These results suggest that future drugs based on the mycalamides are likely to be valuable in situations where efflux pump-based resistance leads to failure of other chemotherapeutic approaches, although efflux may be a mediator of resistance in antifungal applications.

A Complementary Chemical and Genomic Screening Approach for Druggable Targets in the Nrf2 Pathway and Small Molecule Inhibitors to Overcome Cancer Cell Drug Resistance.

Resistance to chemotherapy is a major obstacle in the treatment of a wide array of different types of cancer. Chemotherapeutic drug resistance is achieved by cancer cells by a variety of different mechanisms, which can be either compound specific or general. An emerging mechanism for nonspecific chemotherapeutic drug resistance relies on hyperactivity of the transcription factor Nrf2. Normally Nrf2 levels are tightly regulated by the ubiquitin-proteasome system; however, mutations in genes responsible for this regulation are common in many cancer types, resulting in increased expression of Nrf2, activation of its downstream target genes, and resistance to a variety of chemotherapeutic agents. For this reason, there has been considerable interest in the discovery of small molecule inhibitors of Nrf2 capable of attenuating this resistance mechanism. To this end, we have screened two commercially available libraries of known biologically active small molecules to identify potential Nrf2 inhibitors. To increase the breadth of this screen we have also screened an RNAi library that targets the majority of the druggable genome to also identify Nrf2-inhibitor targets that are not currently targeted by small molecules. To complement the commercial chemical and genomic library screening, we screened a small collection of proprietary natural products isolated from marine cyanobacteria, which included actin targeting and uncharacterized but biologically active compounds. Through these efforts, we have identified three classes of compounds: cardiac glycosides, Stat3 inhibitors, and actin disrupting agents as Nrf2 inhibitors that are able to attenuate Nrf2 activity and synergize with chemotherapeutic agents in the non-small-cell lung cancer cell line A549. In addition, we found that grassypeptolide A exerts Nrf2 modulatory activity via a thus far uncharacterized mechanism. Moreover, we have identified a set of putative Nrf2 targets comprising the transcription factors TWIST1 and ELF4, the protein kinase NEK8, the TAK1 kinase regulator TAB1, and the dual specific phosphatase DUSP4. This study broadens the range of mechanisms through which inhibition of Nrf2 activity can be achieved, which will facilitate the characterization of novel Nrf2 inhibitors and allow the design of target specific screening procedures with which to identify more.

Poly(N-(2-Hydroxypropyl) Methacrylamide)-Valproic Acid Conjugates as Block Copolymer Nanocarriers.

We report nanoassemblies based on block copolymers of N-(2-hydroxypropyl) methacrylamide (HPMA) in which drug cleavage enhances the biological compatibility of the original polymer carrier by regeneration of HPMA units. Drug release via ester hydrolysis suggests this approach offers potential for stimuli-responsive drug delivery under acidic conditions.

Pleiotropic drug-resistance attenuated genomic library improves elucidation of drug mechanisms.

Identifying Saccharomyces cerevisiae genome-wide gene deletion mutants that confer hypersensitivity to a xenobiotic aids the elucidation of its mechanism of action (MoA). However, the biological activities of many xenobiotics are masked by the pleiotropic drug resistance (PDR) network which effluxes xenobiotics that are PDR substrates. The PDR network in S. cerevisiae is almost entirely under the control of two functionally homologous transcription factors Pdr1p and Pdr3p. Herein we report the construction of a PDR-attenuated haploid non-essential DMA (PA-DMA), lacking PDR1 and PDR3, which permits the MoA elucidation of xenobiotics that are PDR substrates at low concentrations. The functionality of four key cellular processes commonly activated in response to xenobiotic stress: oxidative stress response, general stress response, unfolded stress response and calcium signalling pathways were assessed in the absence of PDR1 and PDR3 genes and were found to unaltered, therefore, these key chemogenomic signatures are not lost when using the PA-DMA. Efficacy of the PA-DMA was demonstrated using cycloheximide and latrunculin A at low nanomolar concentrations to attain chemical genetic profiles that were more specific to their known main mechanisms. We also found a two-fold increase in the number of compounds that are bioactive in the pdr1Δpdr3Δ compared to the wild type strain in screening the commercially available LOPAC(1280) library. The PA-DMA should be particularly applicable to mechanism determination of xenobiotics that have limited availability, such as natural products.

The novel equisetin-like compound, TA-289, causes aberrant mitochondrial morphology which is independent of the production of reactive oxygen species in Saccharomyces cerevisiae.

Tetramic acids constitute a large class of natural products isolated from a variety of different fungal and bacterial species. While the presence of the distinctive 2,4-pyrrolidinedione ring system defines this class of compounds, these compounds are widely diverse both structurally and in the biological activities that they display. Equisetin-like compounds are tetramic acids that have been shown to be growth inhibitory towards bacteria, fungi, yeasts and mammalian cell lines; however, the mechanisms inhibiting prokaryotic and eukaryotic cell growth have not been fully explained. Here we report the isolation and biological characterisation of a novel equisetin-like tetramic acid named tetramic acid-289 (TA-289) produced by a Fusarium sp. fungus. This compound displayed pH- and carbon source-dependent cytotoxic effects in Saccharomyces cerevisiae and caused an irreversible cell cycle block via a microtubule independent mechanism. To fully elucidate a mechanism, we used an unbiased approach employing chemogenomic profiling of the yeast deletion library and demonstrated that TA-289 hypersensitive deletion strains are also sensitive to oxidants, respiratory inhibitors and have abnormal mitochondrial morphology. In support of the hypothesis that TA-289 perturbs mitochondrial function, we demonstrated the ability of this compound to generate reactive oxygen species only during fermentative growth, an effect reliant on an intact electron transport chain. In addition, mitochondrial morphological defects were detected upon exposure to TA-289 independent of the increase in oxidative stress. The generation of reactive oxygen species was not the sole cause of cell death by TA-289, as only partial amelioration of cell death was achieved by the deletion of genes encoding components of the electron transport chain, despite these deletions causing attenuation of the magnitude of oxidative stress. We propose that TA-289 induces cell death via the direct inhibition of a mitochondrially localised target or targets, and that the mitochondrial morphology defect and ROS production observed in this study is a direct consequence of the induction of cell death. This study highlights the complex interplay between mitochondrial function, cell death and the generation of reactive oxygen species when elucidating the mode-of-action of compounds that cause oxidative stress and cell death, and further deepens the mystery surrounding the molecular basis of the activity of equisetin-like compounds.

Laulimalide and peloruside A inhibit mitosis of Saccharomyces cerevisiae by preventing microtubule depolymerisation-dependent steps in chromosome separation and nuclear positioning.

The activity and mechanism of action of two microtubule-stabilising agents, laulimalide and peloruside A, were investigated in Saccharomyces cerevisiae. In contrast to paclitaxel, both compounds displayed growth inhibitory activity in yeast with wild type TUB2 and were susceptible to the yeast pleiotropic drug efflux pumps, as evidenced by the increased sensitivity of a pump transcription factor knockout strain, pdr1Δpdr3Δ. Laulimalide (IC50=3.7 µM) was 5-fold more potent than peloruside A (IC50=19 µM) in this knockout strain. Bud index assays and flow cytometry revealed a G2/M block as seen in mammalian cells subsequent to treatment with these compounds. Furthermore, peloruside A treatment caused an increase in the number of cells with polymerised spindle microtubules. These results indicate an anti-mitotic action of both compounds with tubulin the likely target. This conclusion was supported by laulimalide and peloruside chemogenomic profiling using a yeast deletion library in the pdr1Δpdr3Δ background. The chemogenomic profiles of these compounds indicate that, in contrast to microtubule destabilising agents like nocodazole and benomyl, laulimalide and peloruside A inhibit mitotic processes that are reliant on microtubule depolymerisation, consistent with their ability to stabilise microtubules. Gene deletion strains hypersensitive to laulimalide and peloruside A represent possible targets for drugs that can synergize with microtubule stabilising agent and be of potential use in combination therapy for the treatment of cancer or other diseases.

Molecular basis for fungicidal action of neothyonidioside, a triterpene glycoside from the sea cucumber, Australostichopus mollis.

Neothyonidioside is a triterpene glycoside (TG) isolated from the sea cucumber, Australostichopus mollis, that is potently cytotoxic to S. cerevisiae, but does not permeabilize cellular membranes. We mutagenized S. cerevisiae and isolated a neothionidioside-resistant (neo(R)) strain. Using synthetic genetic array mapping and sequencing, we identified NCP1 as the resistance locus. Quantitative HPLC revealed that neo(R)/ncp1 mutants have reduced ergosterol content. Ergosterol added to growth media reversed toxicity, demonstrating that neothionidioside binds directly to ergosterol, similar to the polyene natamycin. Ergosterol synthesis inhibitors ketoconazole and atorvastatin conferred resistance to neothionidioside in a dose-dependent manner showing that a threshold ergosterol concentration is required for toxicity. A genome-wide screen of deletion mutants against neothionidioside revealed hypersensitivity of many of the component genes in the ESCRT complexes relating to multivesicular body formation. Confocal microscopy of cells stained with a vital dye showed blockage at this step. Thus, we propose neothionidioside may affect membrane curvature and fusion capability in the endosome-vacuole pathway.