Myotonic dystrophy (DM) is one of the most common forms of muscular dystrophy. DM is an autosomal dominant disease caused by a toxic gain of function RNA. The toxic RNA is produced from expanded non-coding CTG/CCTG repeats, and these CUG/CCUG repeats sequester the Muscleblind-like (MBNL) family of RNA binding proteins. The MBNL proteins are regulators of alternative splicing, and their sequestration has been linked with mis-splicing events in DM. A previously reported screen for small molecules found that pentamidine was able to improve splicing defects associated with DM. Biochemical experiments and cell and mouse model studies of the disease indicate that pentamidine and related compounds may work through binding the CTG*CAG repeat DNA to inhibit transcription. Analysis of a series of methylene linker analogs of pentamidine revealed that heptamidine reverses splicing defects and rescues myotonia in a DM1 mouse model.

The enzymatic activities of three proteins encoded by the thienamycin gene cluster of Streptomyces cattleya (ThnR, ThnH, and ThnT) have been shown to incrementally cleave CoA to afford the active side-chain component of the ␤-lactam antibiotic thienamycin. These results supersede proposals based on earlier radiochemical incorporation experiments. For 20 years it has been thought that cysteine was directly incorporated into the antibiotic. Specific, stepwise truncation of CoA to 4-phosphopantetheine, pantetheine, and finally cysteamine was observed with ThnR, ThnH, and ThnT, respectively, in a series of coupled enzymatic assays. Pantetheinylated carbapenams were synthesized to address possible thienamycin biosynthetic intermediates and were shown to be effective substrates for the pantetheine-cleaving enzyme ThnT. Finally, a fourth gene, thnF, was shown to encode a protein capable of N-acetylating a model compound containing cysteamine in the presence of acetyl-CoA, consistent with the production of the S. cattleya cometabolite, N-acetylthienamycin. Taken together, these four enzymes are proposed to siphon CoA from primary metabolism to create the side chains for the predominant S. cattleya carbapenems, thienamycin and N-acetylthienamycin, in a process likely to be general for the broader class of these antibiotics.␤-lactam antibiotics ͉ carbapenem ͉ pyrophosphatase ͉ acylase ͉ phosphatase

Carbapenems are a clinically important antibiotic family. More than 50 naturally occurring carbapenam/ems are known and are distinguished primarily by their C-2/C-6 side chains where many are only differentiated by the oxidation states of these substituents. With a limited palette of variations the carbapenem family comprises a natural combinatorial library, and C-2/C-6 oxidation is associated with increased efficacy. We demonstrate that ThnG and ThnQ encoded by the thienamycin gene cluster in Streptomyces cattleya oxidize the C-2 and C-6 moieties of carbapenems, respectively. ThnQ stereospecifically hydroxylates PS-5 (5) giving N-acetyl thienamycin (2). ThnG catalyzes sequential desaturation and sulfoxidation of PS-5 (5), giving PS-7 (7) and its sulfoxide (9). The enzymes are relatively substrate selective but are proposed to give rise to the oxidative diversity of carbapenems produced by S. cattleya, and orthologues likely function similarly in allied streptomyces. Elucidating the roles of ThnG and ThnQ will focus further investigations of carbapenem antibiotic biosynthesis.

Myotonic Dystrophy type 1 (DM1) is a disease characterized by errors in alternative splicing, or “mis-splicing”. The causative agent of mis-splicing in DM1 is an inherited CTG repeat expansion located in the 3′ untranslated region of the DM protein kinase gene. When transcribed, CUG repeat expansion RNA sequester MBNL proteins, which constitute an important family of alternative splicing regulators. Sequestration of MBNL proteins results in the mis-splicing of its regulated transcripts. Previous work has demonstrated that pentamidine, a diamidine which is currently FDA-approved as an anti-parasitic agent, was able to partially reverse mis-splicing in multiple DM1 models, albeit at toxic concentrations. In this study, we characterized a series of pentamidine analogues in order to determine their ability to reverse mis-splicing and their toxicity in vivo. Experiments in cell and mouse models demonstrated that compound 13, also known as furamidine, effectively reversed mis-splicing with equal efficacy and reduced toxicity compared to pentamidine.

Approximately 50 naturally occurring carbapenem β-lactam antibiotics are known. All but one of these have been isolated from Streptomyces species and are disubstituted structural variants of a simple core that is synthesized by Pectobacterium carotovorum (Erwinia carotovora), a phylogenetically distant plant pathogen. While the biosynthesis of the simple carbapenem, (5R)-carbapen-2-em-3-carboxylic acid, is impressively efficient requiring only three enzymes, CarA, CarB and CarC, the formation of thienamycin, one of the former group of metabolites from Streptomyces, is markedly more complex. Despite their phylogenetic separation, bioinformatic analysis of the encoding gene clusters suggests that the two pathways could be related. Here we demonstrate with gene swapping, stereochemical and kinetics experiments that CarB and CarA and their S. cattleya orthologues, ThnE and ThnM, respectively, are functionally and stereochemically equivalent, although their catalytic efficiencies differ. The biosynthetic pathways, therefore, to thienamycin, and likely to the other disubstituted carbapenems, and to the simplest carbapenem, (5R)-carbapen-2-em-3-carboxylic acid, are initiated in the same manner, but share only two common steps before diverging.