Nonsense mutations promote premature translational termination and cause anywhere from 5-70% of the individual cases of most inherited diseases. Studies on nonsense-mediated cystic fibrosis have indicated that boosting specific protein synthesis from <1% to as little as 5% of normal levels may greatly reduce the severity or eliminate the principal manifestations of disease. To address the need for a drug capable of suppressing premature termination, we identified PTC124-a new chemical entity that selectively induces ribosomal readthrough of premature but not normal termination codons. PTC124 activity, optimized using nonsense-containing reporters, promoted dystrophin production in primary muscle cells from humans and mdx mice expressing dystrophin nonsense alleles, and rescued striated muscle function in mdx mice within 2-8 weeks of drug exposure. PTC124 was well tolerated in animals at plasma exposures substantially in excess of those required for nonsense suppression. The selectivity of PTC124 for premature termination codons, its well characterized activity profile, oral bioavailability and pharmacological properties indicate that this drug may have broad clinical potential for the treatment of a large group of genetic disorders with limited or no therapeutic options.

Background Ataluren was developed to restore functional protein production in genetic disorders caused by nonsense mutations, which are the cause of cystic fibrosis (CF) in 10% of patients.. Methods This randomized, double-blind, placebo-controlled study enrolled 238 patients ≥6 years with nmCF to receive oral ataluren 10 mg/kg in the morning, 10 mg/kg mid-day, and 20 mg/kg in the evening or matching placebo for 48 weeks. The primary endpoint was relative change in % predicted forced expiratory volume in one second (FEV1) at Week 48; the secondary endpoint was the rate of pulmonary exacerbations. This study is registered with ClinicalTrials.gov, number NCT00803205. Findings There was no statistically significant difference in relative change from baseline in % predicted FEV1between ataluren and placebo at Week 48(-2•5% vs -5•5%, p=0.1235). The rate of pulmonary exacerbations was not statistically different between treatment arms (rate ratio 0.77 (95% CI 0.57, 1.05), p=0.0992). However, post hoc analysis of the subgroup of patients not using chronic inhaled tobramycin showed a 5.7% difference in relative change from baseline in % predicted FEV1 between ataluren and placebo at Week 48 (-0.7% vs -6.4%, nominal p=0•008, adjusted for multiplicity p = 0•024) and 40% fewer exacerbations in ataluren-treated patients (OR 0.60 (95% CI 0•42, 0•86), nominal p=0•006, adjusted for multiplicity p = 0•018). Interpretation While there was no statistically significant improvement in lung function or exacerbation rate in the ITT population of cystic fibrosis patients with nonsense mutations treated with ataluren, treatment might be beneficial for nmCF patients not receiving chronic inhaled tobramycin.

Direct inoculation of DNA, in the form of purified bacterial plasmids that are unable to replicate in mammalian cells but are able to direct cell synthesis of foreign proteins, is being explored as an approach to vaccine development. Here, a highly attenuated Shigella vector invaded mammalian cells and delivered such plasmids into the cytoplasm of cells, and subsequent production of functional foreign protein was measured. Because this Shigella vector was designed to deliver DNA to colonic mucosa, the method is a potential basis for oral and other mucosal DNA immunization and gene therapy strategies.

Small molecules that affect specific protein functions can be valuable tools for dissecting complex cellular processes. Peptidoglycan synthesis and degradation is a process in bacteria that involves multiple enzymes under strict temporal and spatial regulation. We used a set of small molecules that inhibit the transglycosylation step of peptidoglycan synthesis to discover genes that help to regulate this process. We identified a gene responsible for the susceptibility of Escherichia coli cells to killing by glycolipid derivatives of vancomycin, thus establishing a genetic basis for activity differences between these compounds and vancomycin.

Ramoplanin (Figure 1) is a cyclic glycolipodepsipeptide antibiotic that kills gram positive bacteria by inhibiting cell wall biosynthesis. Ramoplanin was shown to block the conversion of Lipid I to Lipid II, 1 a reaction that is catalyzed by the intracellular GlcNAc transferase, MurG (Scheme 1). It was proposed that ramoplanin inhibits MurG by complexing Lipid I, which prevents it from being utilized as a substrate. Below we show that ramoplanin also inhibits the polymerization of Lipid II; therefore, we propose that another mechanism by which ramoplanin can kill bacterial cells is through inhibition of the transglycosylation step of peptidoglycan synthesis. Using a synthetic analogue of Lipid II, we present evidence that enzyme inhibition by ramoplanin involves substrate binding. Ramoplanin undergoes a conformational change upon substrate binding, and the resulting complexes self-associate to form fibrils. The significance of fibril formation is discussed.The mechanism of action of ramoplanin has been investigated in permeabilized bacterial cells and membrane preparations by following the incorporation of radiolabel from a precursor into various intermediates along the pathway to peptidoglycan. 1-3 A limitation of these assays is that if one enzymatic step is blocked, then no information can be obtained about subsequent steps. Thus, because ramoplanin prevents the formation of Lipid II, it is not possible to determine whether it also inhibits the polymerization of Lipid II. We reinvestigated the ability of ramoplanin to block Lipid II polymerization using a modified membrane assay 4 in which the transglycosylases are selectively inhibited to permit the buildup of radiolabeled Lipid II. Following removal of the inhibitor, peptidoglycan synthesis commences. The effect of ramoplanin on Lipid II polymerization was evaluated by monitoring the amount of radioactive peptidoglycan formed in the presence of increasing concentrations of ramoplanin. Ramoplanin blocks the polymerization of Lipid II and thus is an inhibitor of the transglycosylation step of peptidoglycan synthesis (Figure 2).Ramoplanin was proposed to act by complexing substrates required for peptidoglycan synthesis. 1 Unfortunately, difficulties in isolating Lipid intermediates from bacterial cells have hindered studies of their interactions with ramoplanin. 5,6 Moreover, the natural Lipid intermediates contain a 55 carbon polyprenol chain that renders them insoluble in water, and thus difficult to use in biophysical studies of complex formation. We recently developed a synthetic route to a soluble Lipid I analogue (1) to use in studying MurG, 7 the GlcNAc transferase that converts Lipid I to Lipid II. Using purified MurG, we have now made the corresponding Lipid II analogue 2 from 1, as shown (Scheme 2). 8,9 Compound 2 is identical to natural Lipid II except that the 55 carbon chain has been replaced with a 10 carbon unit so that the compound is freely water soluble. 10 The ability of ramoplanin to interact with 2 was investigated by NMR (Figure 3). Ti...