The Molecular Pharmacology of Pateamine A

Pateamine A is a cytotoxic terpenoid isolated from the marine sponge Mycale hentscheli that induces apoptosis in mammalian cell lines and is growth inhibitory to yeasts and fungi, yet shows no inhibitory action in prokaryotes. The targets of pateamine in mammalian cell lines were isolated and identified using a combination of affinity chromatography and mass spectrometry, putative targets included the DEAD-Box helicase eIF4A family of proteins, β-tubulin and actin. In vitro assessment of tubulin and actin polymerization showed pateamine was able to affect them only at high micromolar concentrations, whereas the effect on eIF4A in vitro was shown by others to occur at nanomolar concentrations. Additionally, pateamine was shown to inhibit cap-dependent protein synthesis in vivo, suggesting eIF4A as a primary target. The generation of a pateamine resistance-conferring mutation in the yeast eIF4A encoding gene TIF1, suggested further that eIF4A is a primary target in both mammalian and yeast cells, and allows the speculation of the position of the binding site for pateamine on the N-terminal lobe of eIF4A and the proposal of potential covalent interaction between this drug and its target. Given the size of the DEAD-Box helicase family, all of which share considerable homology with the eIF4As, FAL1 especially which is essential for rRNA maturation, a chemogenomic screen was performed in an attempt to establish the breadth of functional interactions of pateamine. The results of hierarchical clustering of these screen results suggest that pateamine has a mode-of-action distinct from other compounds screened previously, despite its effect on protein synthesis it failed to cluster with any other protein synthesis inhibitors regardless of their separate mechanisms, though, as a class, protein synthesis inhibitors were not found to form a discrete cluster in any of the variations of cluster analysis performed. Functional analysis, by GO term enrichment, of the genes whose deletions are hypersensitive to pateamine indicates that deletions of genes involved in numerous aspects of RNA metabolism affect pateamine sensitivity, however clear results regarding the involvement of FAL1 or any other non-eIF4A target in pateamine’s mode-of-action were not found

The Molecular Pharmacology of Pateamine A

Pateamine A is a cytotoxic terpenoid isolated from the marine sponge Mycale hentscheli that induces apoptosis in mammalian cell lines and is growth inhibitory to yeasts and fungi, yet shows no inhibitory action in prokaryotes. The targets of pateamine in mammalian cell lines were isolated and identified using a combination of affinity chromatography and mass spectrometry, putative targets included the DEAD-Box helicase eIF4A family of proteins, β-tubulin and actin. In vitro assessment of tubulin and actin polymerization showed pateamine was able to affect them only at high micromolar concentrations, whereas the effect on eIF4A in vitro was shown by others to occur at nanomolar concentrations. Additionally, pateamine was shown to inhibit cap-dependent protein synthesis in vivo, suggesting eIF4A as a primary target. The generation of a pateamine resistance-conferring mutation in the yeast eIF4A encoding gene TIF1, suggested further that eIF4A is a primary target in both mammalian and yeast cells, and allows the speculation of the position of the binding site for pateamine on the N-terminal lobe of eIF4A and the proposal of potential covalent interaction between this drug and its target. Given the size of the DEAD-Box helicase family, all of which share considerable homology with the eIF4As, FAL1 especially which is essential for rRNA maturation, a chemogenomic screen was performed in an attempt to establish the breadth of functional interactions of pateamine. The results of hierarchical clustering of these screen results suggest that pateamine has a mode-of-action distinct from other compounds screened previously, despite its effect on protein synthesis it failed to cluster with any other protein synthesis inhibitors regardless of their separate mechanisms, though, as a class, protein synthesis inhibitors were not found to form a discrete cluster in any of the variations of cluster analysis performed. Functional analysis, by GO term enrichment, of the genes whose deletions are hypersensitive to pateamine indicates that deletions of genes involved in numerous aspects of RNA metabolism affect pateamine sensitivity, however clear results regarding the involvement of FAL1 or any other non-eIF4A target in pateamine’s mode-of-action were not found

The uses of genome-wide yeast mutant collections

We assess five years of usage of the major genome-wide collections of mutants from Saccharomyces cerevisiae: single deletion mutants, double mutants conferring 'synthetic' lethality and the 'TRIPLES' collection of mutants obtained by random transposon insertion. Over 100 experimental conditions have been tested and more than 5,000 novel phenotypic traits have been assigned to yeast genes using these collections

MicroRNAs in cellular transformation and tumorigenesis

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2009.Vita.Includes bibliographical references.MicroRNAs (miRNAs) are a novel class of small (approximately 23 nucleotides long), highly conserved, non-coding RNAs that function by broadly regulating gene expression. In animals, this regulation is achieved via interaction with target messenger RNAs (mRNAs), largely through their imperfect base pairing with the 3' untranslated regions (3' UTRs) of these target transcripts. Through this imperfect base pairing, miRNAs induce a repression of mRNA translation that is frequently coupled to enhanced turnover of the targeted transcript. This miRNA-mediated repression is highly related to that of RNA interference (RNAi), in which small non-coding RNAs exhibit perfect base pairing with target mRNA transcripts, leading to the endonucleolytic cleavage and degradation of these targeted mRNAs. Computational algorithms have been designed to predict putative miRNA binding sites within mRNAs. Using these predictions, it has been suggested that more than half of all mRNAs within mammals are under the control of miRNAs. Some of the earliest discovered miRNAs (characterized by genetic studies in the nematode Caenorhabditis elegans) were found to control the proliferation and differentiation of the cells in which they were expressed. As altered control of proliferation and differentiation frequently manifest in cancer in mammals, it was suggested that miRNAs might contribute to the development of tumorigenesis.(cont.) In fact, several miRNAs are frequently deleted or amplified in human cancer and miRNA expression profiling studies have shown widespread reductions in steady-state miRNA levels in human cancers relative to normal tissue. These observations have implied a role for miRNAs in tumorigenesis. However, there is a paucity of functional studies demonstrating a role for miRNAs in oncogenic transformation. In the studies described below, we first provide strong evidence for the global loss of miRNAs in human cancers functionally enhancing cellular transformation and tumorigenesis. This enhanced transformation only occurred within tumor cells, suggesting that inhibiting miRNA biogenesis would not be sufficient to induce tumor formation. Moreover, we demonstrate that inhibition of miRNA processing in cancer must be incomplete, as Dicerl, a component of the miRNA processing pathway, suppresses tumorigenesis via haploinsufficiency. Finally, we examine the role of a specific miRNA family, the let-7 family, in the development of non-small cell lung cancer by showing that let-7 can suppress tumorigenesis via inhibition of its targets K-Ras and HMGA2. Taken together, these findings offer promise, not just for understanding the relationship of miRNAs and cancer, but for developing therapeutic agents against the disease.by Madhu S. Kumar.Ph.D

High Throughput Drug Discovery in S. Cerevisiae: the Characterisation of FC-592 and FC-888

The discovery and characterisation of novel small molecule drug candidates is a medical priority. Recent advances in synthetic organic chemistry allow the de novo production of diversity oriented synthetic compound libraries and synthetic modification of natural products to provide candidate compounds for screening as potential therapeutics, bioactive agents or genetic probes. Small drugs function through interaction with complex genetic networks and pathways. However, it is difficult to characterise these interactions on a genome wide level to achieve understanding of drug mechanism. Here, discovery based approaches are utilised to achieve system wide parsing of biological mechanism, in an attempt to characterise the action of novel synthetic compounds and natural product derivatives. Chemical genomic analysis allows for such understanding by examining growth profiles of a genomic deletion library of Saccharomyces cerevisiae mutants in the presence of sub-inhibitory concentrations of drug. The gene targets of small molecule compounds can be identified by noting deletion strains which display increased sensitivity, indicating chemical interaction with the associated gene network. In addition, the development and characterisation of resistant mutants can be used to identify putative drug targets. In this strategy, characterisation of the mechanism of resistance gives insight into drug mode-of-action. This study develops a high throughput yeast inhibition assay to identify bioactive compounds from a synthetic organic compound library, and attempts to characterise mechanism of action by establishing a profile of each compound’s interaction with these gene networks; and mapping a resistance mutation to provide evidence of inhibitory mechanism. Two candidate compounds are identified, FC-592 and FC-888. FC-592 displayed cytostatic inhibition. Further, yeast tag microarray homozygous profiling (HOP), chemical structure analysis, and cell-cycle analysis via flow cytometry for this compound provided evidence for a mechanism of poor specificity that targets glycoprotein biosynthesis and the secretory (Sec) pathway, as well as the cell-division cycle (CDC) pathway. Attempts to characterise a mutant resistant to this compound via synthetic genetic array mapping were unsuccessful when the resistance mutation proved to mediate a slow growth phenotype, abrogating the Synthetic Genetic Array Mapping approach utilised. Pending further analysis, it is suggested that this compound could have a role as a genetic probe in future exploration of the Sec and CDC pathways. Chemical structure analysis and a non-specific HOP screen chemigenomic profile suggested that FC-888 is an alkylating agent with a broad affinity for cellular nucleophiles. The compound demonstrates cytotoxic activity, and its efflux is not mediated by the pleiotropic drug resistance (PDR) network. It is suggested that the compound could find utility as a probe dissecting processes related to cellular defence against non-DNA specific alkylation

Ribosomal proteins in zebrafish haematopoiesis and human disease

PhDSeveral congenital disorders of human haematopoiesis including Diamond- Blackfan anaemia result from heterozygous loss of genes involved in ribosome biogenesis. Further, hemizygosity for ribosomal protein gene RPS14 has been implicated in the pathogenesis of myelodysplastic syndrome with loss of 5q, suggesting that genes involved in ribosome biogenesis may act as both haploinsufficient tumour suppressors and regulators of normal haematopoiesis. Ribosome biogenesis is highly conserved through evolution and readily studied in simple organisms such as yeasts. However the zebrafish provides a wellestablished genetic model system which is ideally suited to rapid assessment of vertebrate haematopoiesis. I have therefore used the zebrafish to study genes involved in ribosome biogenesis and their effects on developmental haematopoiesis relevant to human disease. Presented in this work is investigation of the effect of disruption of 4 genes known to be involved in ribosome biogenesis on zebrafish haematopoiesis. Firstly, I describe a gene, Dead-box 18 (ddx18), identified in a forward genetic screen, whose disruption results in defective haematopoiesis and embryonic lethality. Secondly, I have studied the effects of loss of zebrafish orthologues of the human nucleophosmin gene (NPM1), the most frequently mutated gene in human acute myeloid leukaemia. Loss of Npm1 resulted in aberrant numbers of myeloid cells. Heterologous overexpression of mutated NPM1(NPMc+) resulted in increased production of haematopoietic stem cells suggesting a role for NPMc+ in pathogenesis of AML. Finally, I have shown that loss of Rps14 and Rps19 result in anaemia in developing zebrafish and have investigated p53-independent mechanisms for this effect. The findings described herein demonstrate that disruption of normal ribosome biogenesis frequently results in abnormal developmental haematopoiesis. Further genetic assessment of these tissue-specific pathways deregulated by loss of normal ribosome function may represent an important common mechanism underlying the pathogenesis of congenital and acquired disorders of haematopoiesis, and may provide novel pathways for therapeutic targeting

Chemical and Biological Aspects of Secondary Metabolites from Tongan Marine Sponges

This thesis describes the isolation and structural elucidation of 17 new secondary metabolites from Tongan marine sponges including examples of alkaloids, polyketides and terpenoids. In the process of this work, 19 sponge specimens were subjected to preliminary NMR-guided investigation. Nine organisms were selected for further analysis on the basis of the structural novelty perceived within the HMBC spectrum of crude fractions generated by the first chromatographic purification of their crude extracts, and the apparent rarity of the specimen. Investigation of two different demosponge specimens afforded the γ-hydroxybutenolide sesterterpenes (23 and 24), and small quantities of the potently cytotoxic alkaloid 14-bromohomofascaplysin (29). The analysis of two samples of a dictyoceratid sponge yielded the new labdane diterpenes luakuliides A–C (33–36), characterised by a bridging hemi-acetal function on the B-ring of the labdane bicycle. Luakuliide A (33) and its methyl acetal derivative 34 were found to display interesting immunomodulatory activity. Seven new α-pyrone polyketides, lehualides E–K (69–75), were isolated from a Plakortis sp. Lehualides H–K (72–75) display a range of sulfur functionalities, the natures of which were determined by spectroscopic comparison with synthesised model compounds. Another plakinid sponge specimen contained four new polyketides (95–98), all of which possess different cyclic peroxide moieties. Cyclic peroxides 95, 97 and 98 displayed potent cytoxicity against human promyelocytic leukemia cells (HL- 60). Chemical genetic and phenoytypic profiling studies of 95 were undertaken in Saccharomyces cerevisiae yeast using the homozygous diploid and heterozygous diploid deletion libraries. These studies indicate that 95 acts to disrupt Ca2+ homeostasis, leading to elevation of intracellular Ca2 levels