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.

Lehualides E-K, cytotoxic metabolites from the Tongan marine sponge Plakortis sp.

Spectroscopy-guided chemical analysis of a marine sponge from the genus Plakortis, collected in Tonga, yielded seven new metabolites of polyketide origin, lehualides E-K (5-11), four of which incorporate various sulfur functionalities. The structures of compounds 5-11 were elucidated by interpretation of spectroscopic data and spectral comparison with model compounds. The biological activities of compounds 6-9 were investigated against human promyeloid leukemic HL-60 cells and two yeast strains, wild-type and a drug-sensitive mutant.

Systematic chemical-genetic and chemical-chemical interaction datasets for prediction of compound synergism.

The network structure of biological systems suggests that effective therapeutic intervention may require combinations of agents that act synergistically. However, a dearth of systematic chemical combination datasets have limited the development of predictive algorithms for chemical synergism. Here, we report two large datasets of linked chemical-genetic and chemical-chemical interactions in the budding yeast Saccharomyces cerevisiae. We screened 5,518 unique compounds against 242 diverse yeast gene deletion strains to generate an extended chemical-genetic matrix (CGM) of 492,126 chemical-gene interaction measurements. This CGM dataset contained 1,434 genotype-specific inhibitors, termed cryptagens. We selected 128 structurally diverse cryptagens and tested all pairwise combinations to generate a benchmark dataset of 8,128 pairwise chemical-chemical interaction tests for synergy prediction, termed the cryptagen matrix (CM). An accompanying database resource called ChemGRID was developed to enable analysis, visualisation and downloads of all data. The CGM and CM datasets will facilitate the benchmarking of computational approaches for synergy prediction, as well as chemical structure-activity relationship models for anti-fungal drug discovery.

From large networks to small molecules.

Large-scale analysis of genetic and physical interaction networks has begun to reveal the global organization of the cell. Cellular phenotypes observed at the macroscopic level depend on the collective characteristics of protein and genetic interaction networks, which exhibit scale-free properties and are highly resistant to perturbation of a single node. The nascent field of chemical genetics promises a host of small-molecule probes to explore these emerging networks. Although the robust nature of cellular networks usually resists the action of single agents, they may be susceptible to rationally designed combinations of small molecules able to collectively shift network behavior.

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.

Prediction of Synergism from Chemical-Genetic Interactions by Machine Learning.

The structure of genetic interaction networks predicts that, analogous to synthetic lethal interactions between non-essential genes, combinations of compounds with latent activities may exhibit potent synergism. To test this hypothesis, we generated a chemical-genetic matrix of 195 diverse yeast deletion strains treated with 4,915 compounds. This approach uncovered 1,221 genotype-specific inhibitors, which we termed cryptagens. Synergism between 8,128 structurally disparate cryptagen pairs was assessed experimentally and used to benchmark predictive algorithms. A model based on the chemical-genetic matrix and the genetic interaction network failed to accurately predict synergism. However, a combined random forest and Naive Bayesian learner that associated chemical structural features with genotype-specific growth inhibition had strong predictive power. This approach identified previously unknown compound combinations that exhibited species-selective toxicity toward human fungal pathogens. This work demonstrates that machine learning methods trained on unbiased chemical-genetic interaction data may be widely applicable for the discovery of synergistic combinations in different species.

Chemical genetic and chemogenomic analysis in yeast.

Chemogenomics is the systematic genome-wide study of the cellular response to small molecule agents. Modern high-throughput genetic techniques allow massively parallel examination of the genetic effects of such biologically active small molecules (BASM). Here we present methodology for the identification and characterization of potentially bioactive compounds using the budding yeast Saccharomyces cerevisiae as a model organism. First, we present a method for screening libraries of compounds for growth inhibition in solid or liquid phase, followed by techniques for potency determination using a half-log dose response. Then the Deletion Mutant Array (DMA), a genome-wide library of single gene deletion strains, is used to probe the chemical genetic interactions of individual BASMs on genetic networks-a process that can be achieved with a solid phase pinning assay or a pooled liquid assay utilizing barcode microarray techniques. Finally, we offer some considerations for optimizing these protocols.

Networks of genes modulating the pleiotropic drug response in Saccharomyces cerevisiae.

The pleiotropic drug response (PDR) or multidrug resistance (MDR) are cellular defence mechanisms present in all species to deal with potential toxicity from environmental small molecule toxins or bioactives. The rapid induction of MDR by xenobiotics in mammalian cells and PDR in budding yeast (S. cerevisiae) has been well studied but how pathway specificity is achieved across different structural classes of xenobiotics is not well understood. As a novel approach to this problem we investigated the genome-wide network of genes modulating the yeast PDR. Fluorescently-tagged ABC pumps Pdr5p-GFP and Yor1p-GFP were used as real-time reporters for the Pdr1p/Pdr3p controlled response. Using the yeast non-essential gene deletion set fifty-four gene deletions that suppressed up-regulation of reporter fluorescence to the cell surface in the presence of atorvastatin were identified by high content confocal automated microscopy. Secondary validation using spot dilution assays to known PDR substrates and Western blot assays of Pdr5p expression confirmed 26 genes able to modulate the PDR phenotype. By analysis of network connectivity, an additional 10 genes that fell below the primary screen cut-off were predicted to be involved in PDR and confirmed as above. The PDR modulating genes taken together were enriched in signalling (Rho-GTPase, MAPK), Mediator complexes, and chromatin modification (subunits of ADA and SAGA complexes). Many of the gene deletions cause extra sensitivity in Δpdr1Δpdr3 strains strongly suggesting that there are alternative pathways to upregulate PDR, independently of Pdr1p/Pdr3p. We present here the first high-content microscopy screening for PDR modulators, and identify genes that are previously unsuspected regulators of PDR apparently contributing via network interactions.

Characterizing the laulimalide-peloruside binding site using site-directed mutagenesis of TUB2 in S. cerevisiae.

Baker's yeast, Saccharomyces cerevisiae, has significant sequence conservation with a core subset of mammalian proteins and can serve as a model for disease processes. The aim of this study was to determine whether yeast could be used as a model system to identify new agents that interact with the laulimalide-peloruside binding site on ß-tubulin. Agents that bind to this site cause stabilization of microtubules and interfere with cell division. Based on the location of the proposed laulimalide-peloruside binding site and of previously identified mutations shown to cause resistance in mammalian cells, we made the corresponding mutations in yeast and tested whether they conferred resistance to laulimalide and peloruside. Mutations A296T and R306H, which cause 6-fold and 40-fold increased resistance in human 1A9 ovarian carcinoma cells, respectively, also led to resistance in yeast to these compounds. Similarly, other mutations led to resistance or, in one case, increased sensitivity. Thus, we conclude that yeast is an appropriate model to screen for small molecule drugs that may be efficacious in cancer therapy in humans through the newly characterised laulimalide-peloruside binding site.

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.

Chemical genetic profiling of the microtubule-targeting agent peloruside A in budding yeast Saccharomyces cerevisiae.

Peloruside A, a microtubule-stabilising agent from a New Zealand marine sponge, inhibits mammalian cell division by a similar mechanism to that of the anticancer drug paclitaxel. Wild type budding yeast Saccharomyces cerevisiae (haploid strain BY4741) showed growth sensitivity to peloruside A with an IC(50) of 35µM. Sensitivity was increased in a mad2Δ (Mitotic Arrest Deficient 2) deletion mutant (IC(50)=19µM). Mad2 is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. Haploid mad2Δ cells were much less sensitive to paclitaxel than to peloruside A, possibly because the peloruside binding site on yeast tubulin is more similar to mammalian tubulin than the taxoid site where paclitaxel binds. In order to obtain information on the primary and secondary targets of peloruside A in yeast, a microarray analysis of yeast heterozygous and homozygous deletion mutant sets was carried out. Haploinsufficiency profiling (HIP) failed to provide hits that could be validated, but homozygous profiling (HOP) generated twelve validated genes that interact with peloruside A in cells. Five of these were particularly significant: RTS1, SAC1, MAD1, MAD2, and LSM1. In addition to its known target tubulin, based on these microarray 'hits', peloruside A was seen to interact genetically with other cell proteins involved in the cell cycle, mitosis, RNA splicing, and membrane trafficking.

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.

Evaluation of the Mycobacterium smegmatis and BCG models for the discovery of Mycobacterium tuberculosis inhibitors.

The objective of this study was to measure the efficacy of Mycobacterium smegmatis as a surrogate in vitro model for the detection of compounds which are inhibitory to the growth of Mycobacterium tuberculosis. A chemical screen of the LOPAC library for anti-mycobacterial compounds was performed using M. smegmatis. Parallel screens were conducted with another tuberculosis model, Mycobacterium bovis BCG, and with M. tuberculosis under identical growth conditions and the inhibitors detected across the three species were compared. 50% of compounds that were detected as active against M. tuberculosis were not detected using M. smegmatis compared to 21% of compounds using M. bovis BCG. To examine whether these findings were unique to LOPAC, screens were performed with the NIH Diversity Set and Spectrum Collection. An even higher proportion of M. tuberculosis inhibitors were not detected from the NIH Diversity Set and Spectrum Collection using M. smegmatis compared to M. bovis BCG. These data reveal that a significant proportion of M. tuberculosis inhibitors are missed in library screening with M. smegmatis. The basis of the variation in the inhibitory profiles of M. smegmatis and M. tuberculosis has yet to be fully determined, however, our genomic comparisons indicate that approximately 30% of M. tuberculosis proteins lack conserved orthologues in M. smegmatis compared to 3% being absent in M. bovis BCG. In conclusion, although M. smegmatis offers some technical benefits such as a shorter generation time and negligible risk to laboratory workers, it is significantly less effective in the detection of anti-M. tuberculosis compounds relative to M. bovis BCG. This limitation needs to be taken into consideration when selecting an in vitro screening model for tuberculosis drug discovery.

Chemical genetics reveals a complex functional ground state of neural stem cells.

The identification of self-renewing and multipotent neural stem cells (NSCs) in the mammalian brain holds promise for the treatment of neurological diseases and has yielded new insight into brain cancer. However, the complete repertoire of signaling pathways that governs the proliferation and self-renewal of NSCs, which we refer to as the 'ground state', remains largely uncharacterized. Although the candidate gene approach has uncovered vital pathways in NSC biology, so far only a few highly studied pathways have been investigated. Based on the intimate relationship between NSC self-renewal and neurosphere proliferation, we undertook a chemical genetic screen for inhibitors of neurosphere proliferation in order to probe the operational circuitry of the NSC. The screen recovered small molecules known to affect neurotransmission pathways previously thought to operate primarily in the mature central nervous system; these compounds also had potent inhibitory effects on cultures enriched for brain cancer stem cells. These results suggest that clinically approved neuromodulators may remodel the mature central nervous system and find application in the treatment of brain cancer.

Epstein-Barr virus BALF1 is a BCL-2-like antagonist of the herpesvirus antiapoptotic BCL-2 proteins.

Cellular BCL-2 family proteins can inhibit or induce programmed cell death in part by counteracting the activity of other BCL-2 family members. All sequenced gammaherpesviruses encode a BCL-2 homologue that potently inhibits apoptosis and apparently escapes some of the regulatory mechanisms that govern the functions of their cellular counterparts. Examples of these protective proteins include BHRF1 of Epstein-Barr virus (EBV) and KSBcl-2 of Kaposi's sarcoma-associated herpesvirus, also known as human herpesvirus 8. The gamma-1 subgroup of these viruses, such as EBV, encodes a second BCL-2 homologue. We have now found that this second BCL-2 homologue encoded by EBV, BALF1, inhibits the antiapoptotic activity of EBV BHRF1 and of KSBcl-2 in several transfected cell lines. However, BALF1 failed to inhibit the cellular BCL-2 family member, BCL-x(L). Thus, BALF1 acts as a negative regulator of the survival function of BHRF1, similar to the counterbalance observed between cellular BCL-2 family members. Unlike the cellular BCL-2 family antagonists, BALF1 lacked proapoptotic activity and could not be converted into a proapoptotic factor in a manner similar to cellular BCL-2 proteins by caspase cleavage or truncation of the N terminus. Coimmunoprecipitation experiments and immunofluorescence assays suggest that a minimal amount, if any, of the BHRF1 and BALF1 proteins colocalizes inside cells, suggesting that mechanisms other than direct interaction explain the suppressive function of BALF1.

BAD is a pro-survival factor prior to activation of its pro-apoptotic function.

The mammalian BAD protein belongs to the BH3-only subgroup of the BCL-2 family. In contrast to its known pro-apoptotic function, we found that endogenous and overexpressed BAD(L) can inhibit cell death in neurons and other cell types. Several mechanisms regulate the conversion of BAD from an anti-death to a pro-death factor, including alternative splicing that produces the N-terminally truncated BAD(S). In addition, caspases convert BAD(L) into a pro-death fragment that resembles the short splice variant. The caspase site that is selectively cleaved during cell death following growth factor (interleukin-3) withdrawal is conserved between human and murine BAD. A second cleavage site that is required for murine BAD to promote death following Sindbis virus infection, gamma-irradiation, and staurosporine treatment is not conserved in human BAD, consistent with the inability of human BAD to promote death with these stimuli. However, loss of the BAD N terminus by any mechanism is not always sufficient to activate its pro-death activity, suggesting that the N terminus is a regulatory domain rather than an anti-death domain. These findings suggest that BAD is more than an inert death factor in healthy cells; it is also a pro-survival factor, prior to its role in promoting cell death.