The spliceosome is the macromolecular machine responsible for pre-mRNA splicing, an essential step in eukaryotic gene expression. During splicing, myriad subunits join and leave the spliceosome as it works on the pre-mRNA substrate. Strikingly, there are very few small molecules known to interact with the spliceosome. Splicing inhibitors are needed to capture transient spliceosome conformations and probe important functional components. Such compounds may also have chemotherapeutic applications, as links between splicing and cancer are increasingly uncovered. To identify new splicing inhibitors, we developed a high-throughput assay for in vitro splicing using a reverse transcription followed by quantitative PCR readout. In a pilot screen of 3080 compounds, we identified three small molecules that inhibit splicing in HeLa extract by interfering with different stages of human spliceosome assembly. Two of the compounds similarly affect spliceosomes in yeast extracts, suggesting selective targeting of conserved components. By examining related molecules, we identified chemical features required for the activity of two of the splicing inhibitors. In addition to verifying our assay procedure and paving the way to larger screens, these studies establish new compounds as chemical probes for investigating the splicing machinery.

Pladienolide B (PB) is a potent cancer cell growth inhibitor that targets the SF3B1 subunit of the spliceosome. There is considerable interest in the compound as a potential chemotherapeutic, as well as a tool to study SF3B1 function in splicing and cancer development. The molecular structure of PB, a bacterial natural product, contains a 12-member macrolide ring with an extended epoxide-containing side chain. Using a novel concise enantioselective synthesis, we created a series of PB structural analogs and the structurally related compound herboxidiene. We show that two methyl groups in the PB side chain, as well as a feature of the macrolide ring shared with herboxidiene, are required for splicing inhibition in vitro. Unexpectedly, we find that the epoxy group contributes only modestly to PB potency and is not absolutely necessary for activity. The orientations of at least two chiral centers off the macrolide ring have no effect on PB activity. Importantly, the ability of analogs to inhibit splicing in vitro directly correlated with their effects in a series of cellular assays. Those effects likely arise from inhibition of some, but not all, endogenous splicing events in cells, as previously reported for the structurally distinct SF3B1 inhibitor spliceostatin A. Together, our data support the idea that the impact of PB on cells is derived from its ability to impair the function of SF3B1 in splicing and also demonstrate that simplification of the PB scaffold is feasible.

Wolbachia are gram-negative, obligate, intracellular bacteria carried by a majority of insect species worldwide. Here we use a Wolbachia-infected Drosophila cell line and genome-wide RNA interference (RNAi) screening to identify host factors that influence Wolbachia titer. By screening an RNAi library targeting 15,699 transcribed host genes, we identified 36 candidate genes that dramatically reduced Wolbachia titer and 41 that increased Wolbachia titer. Host gene knockdowns that reduced Wolbachia titer spanned a broad array of biological pathways including genes that influenced mitochondrial function and lipid metabolism. In addition, knockdown of seven genes in the host ubiquitin and proteolysis pathways significantly reduced Wolbachia titer. To test the in vivo relevance of these results, we found that drug and mutant inhibition of proteolysis reduced levels of Wolbachia in the Drosophila oocyte. The presence of Wolbachia in either cell lines or oocytes dramatically alters the distribution and abundance of ubiquitinated proteins. Functional studies revealed that maintenance of Wolbachia titer relies on an intact host Endoplasmic Reticulum (ER)-associated protein degradation pathway (ERAD). Accordingly, electron microscopy studies demonstrated that Wolbachia is intimately associated with the host ER and dramatically alters the morphology of this organelle. Given Wolbachia lack essential amino acid biosynthetic pathways, the reliance of Wolbachia on high rates of host proteolysis via ubiquitination and the ERAD pathways may be a key mechanism for provisioning Wolbachia with amino acids. In addition, the reliance of Wolbachia on the ERAD pathway and disruption of ER morphology suggests a previously unsuspected mechanism for Wolbachia's potent ability to prevent RNA virus replication.

The retinoblastoma (Rb) tumor suppressor protein negatively regulates cell proliferation by binding and inhibiting E2F transcription factors. Rb inactivation occurs in cancer cells upon cyclin-dependent kinase (Cdk) phosphorylation, which induces E2F release and activation of cell cycle genes. We present a strategy for activating phosphorylated Rb with molecules that bind Rb directly and enhance affinity for E2F. We developed a fluorescence polarization assay that can detect the effect of exogenous compounds on modulating affinity of Rb for the E2F transactivation domain. We found that a peptide capable of disrupting the compact inactive Rb conformation increases affinity of the repressive Rb-E2F complex. Our results demonstrate the feasibility of discovering novel molecules that target the cell cycle and proliferation through directly targeting Rb rather than upstream kinase activity.

Pladienolide B (PB) is a potent cancer cell growth inhibitor that targets the SF3B1 subunit of the spliceosome. There is considerable interest in the compound as a potential chemotherapeutic, as well as a tool to study SF3B1 function in splicing and cancer development. The molecular structure of PB, a bacterial natural product, contains a 12-member macrolide ring with an extended epoxide-containing side chain. Using a novel concise enantioselective synthesis, we created a series of PB structural analogs and the structurally related compound herboxidiene. We show that two methyl groups in the PB side chain, as well as a feature of the macrolide ring shared with herboxidiene, are required for splicing inhibition in vitro. Unexpectedly, we find that the epoxy group contributes only modestly to PB potency and is not absolutely necessary for activity. The orientations of at least two chiral centers off the macrolide ring have no effect on PB activity. Importantly, the ability of analogs to inhibit splicing in vitro directly correlated with their effects in a series of cellular assays. Those effects likely arise from inhibition of some, but not all, endogenous splicing events in cells, as previously reported for the structurally distinct SF3B1 inhibitor spliceostatin A. Together, our data support the idea that the impact of PB on cells is derived from its ability to impair the function of SF3B1 in splicing and also demonstrate that simplification of the PB scaffold is feasible.

Investigating the mechanisms of action (MOAs) of bioactive compounds and the deconvolution of their cellular targets is an important and challenging undertaking. Drug resistance in model organisms such as S. cerevisiae has long been a means for discovering drug targets and MOAs. Strains are selected for resistance to a drug of interest, and the resistance mutations can often be mapped to the drug's molecular target using classical genetic techniques. Here we demonstrate the use of next generation sequencing (NGS) to identify mutations that confer resistance to two well-characterized drugs, benomyl and rapamycin. Applying NGS to pools of drug-resistant mutants, we develop a simple system for ranking single nucleotide polymorphisms (SNPs) based on their prevalence in the pool, and for ranking genes based on the number of SNPs that they contain. We clearly identified the known targets of benomyl (TUB2) and rapamycin (FPR1) as the highest-ranking genes under this system. The highest-ranking SNPs corresponded to specific amino acid changes that are known to confer resistance to these drugs. We also found that by screening in a pdr1Δ null background strain that lacks a transcription factor regulating the expression of drug efflux pumps, and by pre-screening mutants in a panel of unrelated anti-fungal agents, we were able to mitigate against the selection of multi-drug resistance (MDR) mutants. We call our approach "Mutagenesis to Uncover Targets by deep Sequencing", or "MUTseq", and show through this proof-of-concept study its potential utility in characterizing MOAs and targets of novel compounds.

Biofilms are a ubiquitous feature of microbial community structure in both natural and host environments; they enhance transmission and infectivity of pathogens and provide protection from human defense mechanisms and antibiotics. However, few natural products are known that impact biofilm formation or persistence for either environmental or pathogenic bacteria. Using the combination of a novel natural products library from the fish microbiome and an image-based screen for biofilm inhibition, we describe the identification of taurine-conjugated bile acids as inhibitors of biofilm formation against both Vibrio cholerae and Pseudomonas aeruginosa. Taurocholic acid (1) was isolated from the fermentation broth of the fish microbiome-derived strain of Rhodococcus erythropolis and identified using standard NMR and MS methods. Screening of the twelve predominant human steroidal bile acid components revealed that a subset of these compounds can inhibit biofilm formation, induce detachment of preformed biofilms under static conditions, and that these compounds display distinct structure-activity relationships against V. cholerae and P. aeruginosa. Our findings highlight the significance of distinct bile acid components in the regulation of biofilm formation and dispersion in two different clinically relevant bacterial pathogens, and suggest that the bile acids, which are endogenous mammalian metabolites used to solubilize dietary fats, may also play a role in maintaining host health against bacterial infection.

Determining mechanism of action (MOA) is one of the biggest challenges in natural products discovery. Here, we report a comprehensive platform that uses Similarity Network Fusion (SNF) to improve MOA predictions by integrating data from the cytological profiling high-content imaging platform and the gene expression platform Functional Signature Ontology, and pairs these data with untargeted metabolomics analysis for de novo bioactive compound discovery. The predictive value of the integrative approach was assessed using a library of target-annotated small molecules as benchmarks. Using Kolmogorov-Smirnov (KS) tests to compare in-class to out-of-class similarity, we found that SNF retains the ability to identify significant in-class similarity across a diverse set of target classes, and could find target classes not detectable in either platform alone. This confirmed that integration of expression-based and image-based phenotypes can accurately report on MOA. Furthermore, we integrated untargeted metabolomics of complex natural product fractions with the SNF network to map biological signatures to specific metabolites. Three examples are presented where SNF coupled with metabolomics was used to directly functionally characterize natural products and accelerate identification of bioactive metabolites, including the discovery of the azoxy-containing biaryl compounds parkamycins A and B. Our results support SNF integration of multiple phenotypic screening approaches along with untargeted metabolomics as a powerful approach for advancing natural products drug discovery.