Splicing is a process that occurs in the nuclei of all eukaryotic cells, removing non-coding sections from pre-mRNA in the final step of transcription. The process consists of two transesterification reactions carried out via interaction with the spliceosome - a large, highly dynamic RNA/protein complex essential in catalysing splicing. Fusidic acid has been shown previously to inhibit splicing, potentially by interaction with snRNA U5 of the spliceosome. This project attempts to elucidate the mechanism of binding, with a view to improving the inhibitory function of the compound. This was achieved by developing photo-crosslinking compounds which could be used to elucidate the protein structure of the binding site, and subsequently the interactions between U5 and fusidic acid. Chapter one discusses the nature of the splicing, as well as examples of photo-crosslinking ligands and their use in biological studies to date. The early sections of chapter three outline the synthesis of the photo-crosslinking compounds and subsequent incorporation into the skeleton of fusidic acid. The latter sections describe the investigations into the structural modifications to fusidic acid, and in particular how these affect the inhibitory function of the molecule. A number of pathways outlined here have been eliminated as unsuccessful in the functionalisation of fusidic acid. Attachment of both diazirinyl and benzophenone compounds was achieved in reasonable yield. While these compounds were insufficiently active to be taken further in the cross-linking study, active intermediates synthesised during the pathway to these compounds have been discovered. These were investigated further, and one compound in particular has shown promise as a potential therapy. Also explored was iodolactonization of fusidic acid, introducing restriction of movement to the side chain via cyclisation. The ambiguous structures of the products were confirmed using X-ray crystallography, and elimination of iodine was investigated to allow for further functionalisation at this position.

No description available.

Splicing is the process where pre-mRNA is converted to mRNA via two transesterification reactions. With this process unwanted sequences of nucleic acids, known as introns, are removed allowing only the coding nucleic acid sequences, exons, to remain. This process is catalysed by a dynamically assembled, highly complex macromolecular machine called the spliceosome, which is made up of five small nuclear ribonucleoproteins (snRNPs). To date, the spliceosome has defied conventional methods for conclusive characterisation, resulting in it being relatively poorly understood, although advances have been made.1, 2 Apart from being of interest due to the fact that splicing is an essential life process, it is also of interest medically. Disruption to the splicing process can produce incorrectly formed mRNA, which plays a part in many diseases.3 Small molecule inhibitors which bind to, and inhibit, the functions of individual proteins would “stall” the spliceosome,4 circumventing its dynamic nature. These inhibitors could also form the basis of new drugs, treating diseases which incorrectly formed mRNA can cause. Previously reported small molecule inhibitors have inhibited splicing at the early stages of spliceosome assembly.5-7 However, our target protein snu1148 belongs to the U5 snRNP, which is involved later on in the splicing cycle. Inhibition of Snu114 should, therefore, lead to accumulation of spliceosome complexes produced at later stages of the cycle. Homology studies of Snu114 indicated a strong correlation of amino acid sequences with ribosomal growth factors EF-2 and EF-G. This study allowed us to target Snu114 using known EF-2 and EF-G inhibitors, sordarin and fusidic acid, which were tested and found to have significant splicing inhibition activity. A series of derivatives of these parent compounds were then attempted in an effort to improve splicing inhibition activity and to analyse the structure-activity relationship of fusidic acid and sordarin as splicing inhibitors. The biosynthesis of sordarin proved to be difficult and only a few derivatives were synthesised, however an improvement was made to splicing inhibition activity by forming sordaricin 32. Various fusidic acid derivatives were successfully synthesised, leading to an analysis of the structure-activity relationship of fusidic acid as a splicing inhibitor. Most fusidic acid derivatives produced a lower splicing inhibition activity than fusidic acid. However, fusidic acid derivative 229 had an equivalent inhibition activity to that found for fusidic acid. This result leads us to believe that the C-3 hydroxyl moiety of fusidic acid would be an ideal area for modification in future studies.

Antibiotics that target the bacterial ribosome make up about half of all clinically used antibiotics. We have studied two ribosome targeting drugs: Fusidic acid and Viomycin. Fusidic acid inhibits bacterial protein synthesis by binding to elongation factor G (EF-G) on the ribosome, thereby inhibiting translocation of the bacterial ribosome. Viomycin binds directly to the ribosome and inhibits both the fidelity of mRNA decoding and translocation. We found that the mechanisms of inhibition of these two antibiotics were unexpectedly complex. Fusidic acid can bind to EF-G on the ribosome during three separate stages of translocation. Binding of the drug to the first and most sensitive state does not lead to stalling of the ribosome. Rather the ribosome continues unhindered to a downstream state where it stalls for around 8 seconds. Dissociation of fusidic acid from this state allows the ribosome to continue translocating but it soon reaches yet another fusidic acid sensitive state where it can be stalled again, this time for 6 seconds. Viomycin inhibits translocation by binding to the pre-translocation ribosome in competition with EF-G. If viomycin binds before EF-G it stalls the ribosome for 44 seconds, much longer than a normal elongation cycle. Both viomycin and fusidic acid probably cause long queues of ribosomes to build up on the mRNA when they bind. Viomycin inhibits translational fidelity by binding to the ribosome during initial selection. We found that the concentration of viomycin required to bind to the ribosome with a given probability during decoding is proportional to the accuracy of the codon∙anticodon pair being decoded. This demonstrated that long standing models about ribosomal accuracy cannot be correct. Finally, we demonstrated that a common viomycin resistance mutation increases the drug binding rate and decreases its dissociation rate. Our results demonstrate that ribosome targeting drugs have unexpectedly complex mechanisms of action. Both fusidic acid and viomycin preferentially bind to conformations of the ribosome other than those that they stabilize. This suggests that determining the structures of stable drug-bound states may not give sufficient information for drug design.

<p>Controlling bacterial infections with antibiotics is central to modern health care. However, increasing bacterial resistance to antibiotics threatens effective therapy. This thesis concerns the use of the antibiotic fusidic acid, and novel analogues of fusidic acid, to treat topical infections caused by the bacterial pathogen <i>Staphylococcu aureus</i>. It also addresses genetic mechanisms by which <i>S. aureus</i> develops resistance to fusidic acid.</p><p>Pre-clinical microbiological tests were made on two structurally different groups of fusidic acid analogues developed by Leo Pharma. These drugs were tested against <i>S. aureus</i> and <i>Streptococcus pyogenes</i> strains, measuring MIC, <i>in vitro</i> concentration-dependent bacteriocidal or bacteriostatic effects, and <i>in vivo</i> efficacy in clearing topical infections. We developed a new superficial skin infection animal model (the ‘tape-stripping model’) designed for testing topical antibiotics, including the novel fusidic acid analogues, against <i>S. aureus</i> and <i>S. pyogenes</i>. Some new compounds giving promising results will be further tested and developed by Leo Pharma. </p><p>Fusidic acid inhibits protein synthesis by binding to elongation factor EF-G on the ribosome. Previously described resistance mechanisms are mutations in the gene coding for EF-G (<i>fusA</i>), or, in some strains, the presence of a gene (<i>fusB, fusC</i> or <i>fusD</i>) coding for a protein that protects EF-G from fusidic acid.</p><p>We discovered two novel classes of spontaneous FusR mutants in <i>S. aureus</i> with the small colony variant (SCV) phenotype which is associated with persistent infections. The FusR SCV’s are very frequent, slow growing, cross-resistant to aminoglycosides, and auxotrophic for hemin or menadione. Some of the FusR SCV mutations are in structural domain V of EF-G (classic <i>fusA</i> mutations map overwhelmingly in domain III). The remaining FusR SCV’s are unmapped but their additive effect on MIC together with the <i>fusB</i> plasmid suggests the possibility that their mechanism of resistance is also associated with the translation machinery. </p>

Controlling bacterial infections with antibiotics is central to modern health care. However, increasing bacterial resistance to antibiotics threatens effective therapy. This thesis concerns the use of the antibiotic fusidic acid, and novel analogues of fusidic acid, to treat topical infections caused by the bacterial pathogen Staphylococcu aureus. It also addresses genetic mechanisms by which S. aureus develops resistance to fusidic acid. Pre-clinical microbiological tests were made on two structurally different groups of fusidic acid analogues developed by Leo Pharma. These drugs were tested against S. aureus and Streptococcus pyogenes strains, measuring MIC, in vitro concentration-dependent bacteriocidal or bacteriostatic effects, and in vivo efficacy in clearing topical infections. We developed a new superficial skin infection animal model (the ‘tape-stripping model’) designed for testing topical antibiotics, including the novel fusidic acid analogues, against S. aureus and S. pyogenes. Some new compounds giving promising results will be further tested and developed by Leo Pharma. Fusidic acid inhibits protein synthesis by binding to elongation factor EF-G on the ribosome. Previously described resistance mechanisms are mutations in the gene coding for EF-G (fusA), or, in some strains, the presence of a gene (fusB, fusC or fusD) coding for a protein that protects EF-G from fusidic acid. We discovered two novel classes of spontaneous FusR mutants in S. aureus with the small colony variant (SCV) phenotype which is associated with persistent infections. The FusR SCV’s are very frequent, slow growing, cross-resistant to aminoglycosides, and auxotrophic for hemin or menadione. Some of the FusR SCV mutations are in structural domain V of EF-G (classic fusA mutations map overwhelmingly in domain III). The remaining FusR SCV’s are unmapped but their additive effect on MIC together with the fusB plasmid suggests the possibility that their mechanism of resistance is also associated with the translation machinery.

The GTPase elongation factor G (EF-G) is an important player in the complex process of protein synthesis by bacterial ribosomes. Although extensively studied much remains to be learned about this fascinating protein. In the elongation phase, after incorporation of each amino acid into the growing peptide chain, EF-G translocates the ribosome along the mRNA template. In the recycling phase, when the synthesis of a protein has been completed, EF-G, together with ribosome recycling factor (RRF), splits the ribosome into its subunits. We developed the first in vitro assay for measuring the average time of a complete translocation step at any position along the mRNA. Inside the open reading frame, at saturating EF-G concentration and low magnesium ion concentration, translocation rates were fast and compatible with elongation rates observed in vivo. We also determined the complete kinetic mechanism for EF-G- and RRF-dependent splitting of the post-termination ribosome. We showed that splitting occurs only when RRF binds before EF-G and that the rate and GTP consumption of the reaction varies greatly with the factor concentrations. The antibiotic fusidic acid (FA) inhibits bacterial protein synthesis by binding to EF-G when the factor is ribosome bound, during translocation and ribosome recycling. We developed experimental methods and a theoretical framework for analyzing the effect of tight-binding inhibitors like FA on protein synthesis. We found that FA targets three different states during each elongation cycle and that it binds to EF-G on the post-termination ribosome both in the presence and absence of RRF. The stalling time of an FA-inhibited ribosome is about hundred-fold longer than the time of an uninhibited elongation cycle and therefore each binding event has a large impact on the protein synthesis rate and may induce queuing of ribosomes on the mRNA. Although ribosomes in the elongation and the recycling phases are targeted with similar efficiency, we showed that the main effect of FA in vivo is on elongation. Our results may serve as a basis for modelling of EF-G function and FA inhibition inside the living cell and for structure determination of mechanistically important intermediate states in translocation and ribosome recycling.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 4 uppsatser.

Protein synthesis, translation, performed by the ribosome, is a fundamental process of life and one of the main targets of antibacterial drugs. This thesis provides structural and biochemical understanding of three aspects of bacterial translation. Elongation factor G (EF-G) is the target for the antibiotic fusidic acid (FA). FA binds to EF-G only on the ribosome after GTP hydrolysis and prevents EF-G dissociation from the ribosome. Point mutations in EF-G can lead to FA resistance but are often accompanied by a fitness cost in terms of slower growth of the bacteria. Secondary mutations can compensate for this fitness cost while resistance is maintained. Here we present the crystal structure of the clinical FA drug target, Staphylococcus aureus EF-G, together with the mapping and analysis of all known FA-resistance mutations in EF-G. We also present crystal structures of the FA-resistant mutant F88L, the FA-hypersensitive mutant M16I and the FA-resistant but fitness-compensated double mutant F88L/M16I. Analysis of mutant structures together with biochemical data allowed us to propose that fitness loss and compensation are caused by effects on the conformational dynamics of EF-G on the ribosome. Aminoglycosides are another group of antibiotics that target the decoding region of the 30S ribosomal subunit. Resistance to aminoglycosides can be acquired by inactivation of the drugs via enzymatic modification. Here, we present the first crystal structure an aminoglycoside 3’’ adenyltransferase, AadA from Salmonella enterica. AadA displays two domains and unlike related structures most likely functions as a monomer. Frameshifts are deviations the standard three-base reading frame of translation. -1 frameshifting can be caused by normal tRNASer3 at GCA alanine codons and tRNAThr3 at CCA/CCG proline codons. This process has been proposed to involve doublet decoding using non-standard codon-anticodon interactions. In our study, we showed by equilibrium binding that these tRNAs bind with low micromolar Kd to the frameshift codons. Our results support the doublet-decoding model and show that non-standard anticodon loop structures need to be adopted for the frameshifts to happen. These findings provide new insights in antibiotic resistance and reading-frame maintenance and will contribute to a better understanding of the translation elongation process.

Protein synthesis is a highly complex process executed by the ribosome in coordination with mRNA, tRNAs and translational protein factors. Several antibiotics are known to inhibit bacterial protein synthesis by either targeting the ribosome or the proteins factors involved in translation. Fusidic acid (FA) is a bacteriostatic antibiotic that blocks polypeptide chain elongation by locking elongation factor-G (EF-G) on the ribosome. Mutations in fusA, the gene encoding bacterial EF-G, confer high-level of resistance towards FA.  Antibiotic resistance in bacteria is often associated with fitness loss, which is compensated by acquiring secondary mutations. In order to understand the mechanism of fitness loss and compensation in relation to FA resistance, we have characterized three S. aureus EF-G mutants with fast kinetics and crystal structures. Our results show that, the causes for fitness loss in the FA-resistant mutant F88L are resulting from significantly slower tRNA translocation and ribosome recycling. Analysis of the crystal structures, together with the results from our biochemical studies enabled us to propose that FA-resistant EF-G mutations causing fitness loss and compensation operate by affecting the conformational dynamics of EF-G on the ribosome. EF-G is a G-protein belonging to the GTPase super-family. In all the translational GTPases, a conserved histidine (H92 in E. coli EF-G) residue, located at the apex of switch II in the G-domain is believed to play a crucial role in ribosome-stimulated GTP hydrolysis and inorganic phosphate (Pi) release. Mutagenesis of H92 to alanine (A) and glutamic acid (E) showed different degree of defect in different steps of translation. Compared to wild type (WT) EF-G, mutant H92A showed a 10 fold defect in ribosome mediated GTP hydrolysis whereas the other mutant H92E showed a 100 fold defect. However, both the mutants are equally defective in single round Pi release (100 times slower than WT). When checked for their activity in mRNA translocation, H92A and H92E were 10 times and 100 times slower than WT respectively. Results from our tripeptide formation experiments revealed a 1000 fold defect for both mutants. Altogether, our results indicate that GTP hydrolysis occurs before tRNA translocation, whereas Pi release occurs probably after or independent of the translocation step. Further, our results confirm that, His92 has a vital role residue in ribosome-stimulated GTP hydrolysis and Pi release.