Orthoformimycin, a Selective Inhibitor of Bacterial Translation Elongation from Streptomyces Containing an Unusual Orthoformate Sonia I. Maffioli,* ,† Attilio Fabbretti, ‡ Letizia Brandi, ‡ Andreas Savelsbergh, § Paolo Monciardini, † Monica Abbondi, ∥,○ Rossana Rossi, ⊥ Stefano Donadio, † and Claudio O. Gualerzi* ,‡ † NAICONS, 20138 Milano, Italy ‡ Laboratory of Genetics, Department of Biosciences and Biotechnology, University of Camerino, 62032 Camerino, Italy § Institut fü r Medizinische Biochemie, Universitä t Witten/Herdecke, 58453 Witten, Germany ∥ Vicuron Pharmaceuticals, 21040 Gerenzano, Italy ⊥ ITB-CNR, 20090 Segrate, Italy * S Supporting Information ABSTRACT: Upon high throughput screening of 6700 microbial fermentation extracts, we discovered a compound, designated orthoformimycin, capable of inhibiting protein synthesis in vitro with high efficiency. The molecule, whose structure was elucidated by chemical, spectrometric, and spectroscopic methods, contains an unusual orthoformate moiety (hence the name) and belongs to a novel class of translation inhibitors. This antibiotic does not affect any function of the 30S ribosomal subunit but binds to the 50S subunit causing inhibition of translation elongation and yielding polypeptide products of reduced length. Analysis by fluorescence stopped flow kinetics revealed that EF-G- dependent mRNA translocation is inhibited by orthoformi- mycin, whereas, surprisingly, translocation of the aminoacyl-tRNA seems to be unaffected. N ature has designed a remarkable number of antimicrobial compounds that target specific processes in living cells. Among them, translation of mRNA into proteins is targeted by several classes of compounds of microbial origin, many of which have become important antibiotics in the fight against bacterial diseases. 1−3 Most of the known antibiotics bind to the ribosome, which thus constitutes a validated target for many synthetic and naturally occurring classes of drugs. 4 Although many antibiotics inhibit translation, several components and individual steps of translation remain untargeted by small molecules and thus offer an opportunity for identifying novel structural classes of ribosome inhibitors. The use of a target-based approach to discover translation inhibitors from microbial product extracts, making use of a bacterial cell-free extract programmed with a model mRNA, has previously been reported. 5,6 Additional tests in which trans- lation of the same mRNA in a eukaryotic cell-free extract led to the establishment of the selectivity of the inhibition, whereas the effect on poly(U)-directed poly-Phe synthesis allowed the discrimination of elongation versus nonelongation inhibitors thereby increasing the probability of chemical novelty. 6 This approach led to the identification of two new classes of ribosome inhibitors, represented by the tetrapeptide GE81112 7,8 and by the dityrosine-like cyclic peptide GE82832. 9,10 Here, we report the discovery, isolation, structure elucida- tion, and investigation of the mechanism of action of orthoformimycin, a structurally unique compound that inhibits bacterial protein synthesis by an unprecedented mechanism. ■ RESULTS AND DISCUSSION Identification of Orthoformimycin. Orthoformimycin was initially identified in a high throughput screen (HTS) of about 6700 microbial fermentation extracts, derived from ca. 4400 actinomycetes and from 450 filamentous fungi. The HTS test was aimed at identifying molecules capable of inhibiting in vitro translation of the universal 027IF2Cp(A) mRNA. 5 One of the extracts fulfilling the selection criteria (inhibition of the Escherichia coli but not of the Saccharomyces cerevisiae system) was further characterized. Upon HPLC fractionation, the active fraction showed an m/z of 540 [M + H] + , which did not apparently correspond to any known microbial product. The producer strain ID107558, characterized on the basis of its morphology and 16S rRNA gene sequence, was found to be closely related to the ubiquitous group of species related to Streptomyces griseus, 11 with >99.9% identity to S. griseus subsp. Received: June 8, 2013 Accepted: July 29, 2013 Articles pubs.acs.org/acschemicalbiology © XXXX American Chemical Society A dx.doi.org/10.1021/cb4004095 | ACS Chem. Biol. XXXX, XXX, XXX−XXX