Synthesis of simplified tedanolide analogues--connecting tedanolide to myriaporone and gephyronic acid

Southern belles! Simplified analogues of tedanolide, a natural product with picomolar activity against a range of tumor cell lines, were synthesized and evaluated for potency in mammalian cancer cells. The truncated analogues were found to retain significant activity in vitro (23 μmol mL(-1) for the example shown) compared with the parent compound tedanolide (0.33 nmol mL(-1)), and represent potential leads for the development of novel anticancer agents.     

How Simulations Reveal Dynamics,Disorder, and the Energy Landscapes of Biomolecular Function

Over the last 40 years, the area of computational molecular biophysics has grown and developed to the point where simulations can now provide detailed mechanistic insights, suggest theoretical principles that underpin function, and provide frameworks for understanding and interpreting experimental measurements. The success of molecular simulations has been the result of the unrelenting development of novel theoretical models, exponential growth in computational resources, and advances in structural biology techniques. Through the continued refinement and application of diverse classes of models, general themes in biomolecular dynamics are beginning to surface. In particular, molecular simulations are highlighting the pervasive role that order disorder events play in molecular biology. These dynamic modes of function are transforming our perspective on the role that entropy has in many large-scale biological processes.     

Helix 69: A Multitasking RNA Motif as a Novel Drug Target

High-resolution structures have shown that helix 69 (H69) of the large ribosomal subunit can assume variable conformational states during translation. Solution studies on small model RNAs, isolated subunits, and complete ribosomes also revealed a variety of H69 conformations. Specific nucleotides of H69 that undergo changes in their relative orientations within the ribosome structure play important roles in both translation and ribosome rescue. Furthermore, the presence of multiple pseudouridines influences the global conformational states of H69, and these highly conserved modifications play a role in regulating the positioning of key functional residues. Helix 69 has recently been identified as a novel antibiotic target site. Small molecules such as aminoglycosides bind to specific conformational states of H69 in bacterial ribosomes and affect the translation process. A variety of techniques have been employed to study H69, ranging from chemical synthesis to X-ray crystallography, highlighting the importance of a detailed evaluation of the underlying principles of RNA conformational dynamics and drug targeting.     

Towards Catalytic Antibiotics: Redesign of Aminoglycosides To Catalytically Disable Bacterial Ribosomes

The emergence of multidrug-resistant pathogens that are resistant to the majority of currently available antibiotics is a significant clinical problem. The development of new antibacterial agents and novel approaches is therefore extremely important. We set out to explore the potential of catalytic antibiotics as a new paradigm in antibiotics research. Herein, we describe our pilot study on the design, synthesis, and biological testing of a series of new derivatives of the natural aminoglycoside antibiotic neomycin B for their potential action as catalytic antibiotics. The new derivatives showed significant antibacterial activity against wild-type bacteria and were especially potent against resistant and pathogenic strains including Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus. Selected compounds displayed RNase activity even though the activity was not as high and specific as we would have expected. On the basis of the observed chemical and biochemical data, along with the comparative molecular dynamics simulations of the prokaryotic rRNA decoding site, we postulate that the rational design of catalytic antibiotics should involve not only their structure but also a comprehensive analysis of the rRNA A-site dynamics.     

Site-directed mutagenesis of Escherichia coli translation initiation factor IF1. Identification of the amino acids involved in its ribosomal binding and recycling

Starting from a synthetic modular gene (infA^*) encoding Escherichiacoli translation initiation factor IF1, we have constructedmutants in which amino acids are deleted from the carboxyl terminusor in which His29 or His34 are replaced by Tyr or Asp residues.The mutant proteins were overproduced, purified and tested invitro for their properties in several partial reactions of thetranslation initiation pathway and for their capacity to stimulateMS2 RNA-dependent protein synthesis. The results allow for theconclusion that: (i) Arg69 is part of the 30S ribosomal subunitbinding site of IF1 and its deletion results in the substantialloss of all IF1 functions; (ii) neither one of its two histidinesis essential for the binding of IF1 to the 30S ribosomal subunit,for the stimulation of fMet-tRNA binding to 30S or 70S ribosomalparticles or for MS2 RNA-dependent protein synthesis; but (iii)His29 is involved in the 50S subunit-induced ejection of IF1from the 30S ribosomal subunit.     

Binding of helix-threading peptides to E. coli 16S ribosomal RNA and inhibition of the S15-16S complex

Helix-threading peptides (HTPs) constitute a new class of small molecules that bind selectively to duplex RNA structures adjacent to helix defects and project peptide functionality into the dissimilar duplex grooves. To further explore and develop the capabilities of the HTP design for binding RNA selectively, we identified helix 22 of the prokaryotic ribosomal RNA 16S as a target. This helix is a component of the binding site for the ribosomal protein S15. In addition, the S15-16S RNA interaction is important for the ordered assembly of the bacterial ribosome. Here we present the synthesis and characterization of helix-threading peptides that bind selectively to helix 22 of E. coli 16S RNA. These compounds bind helix 22 by threading intercalation placing the N termini in the minor groove and the C termini in the major groove. Binding is dependent on the presence of a highly conserved purine-rich internal loop in the RNA, whereas removal of the loop minimally affects binding of the classical intercalators ethidium bromide and methidiumpropyl-EDTAFe (MPEFe). Moreover, binding selectivity translates into selective inhibition of formation of the S15-16S complex.     

Ribosomal frameshifting in yeast viruses

Proper maintenance of translational reading frame by ribosomes is essential for cell growth and viability. In the last 10 years it has been shown that a number of viruses induce ribosomes to shift reading frame in order to regulate the expression of gene products having enzymatic functions. Studies on ribosomal frameshifting in viruses of yeast have been particularly enlightening. The roles of viral mRNA sequences and secondary structures have been elucidated and a picture of how these interact with host chromosomal gene products is beginning to emerge. The efficiency of ribosomal frameshifting is important for viral particle assembly, and has identified ribosomal frameshifting as a potential target for antiviral agents. The availability of mutants of host chromosomal gene products involved in maintaining the efficiency of ribosomal frameshifting bodes well for the use of yeast in future studies of ribosomal frameshifting.