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International audience; Natural products serve as chemical blueprints for most antibiotics in clinical use. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class of antibiotics1. Virginiamycin acetyltransferase (Vat) enzymes are resistance proteins that provide protection against streptogramins2, potent antibiotics against Gram-positive bacteria that inhibit the bacterial ribosome3. Owing to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogues that overcome the resistance conferred by Vat enzymes have not been previously developed2. Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with extensive structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogues to the bacterial ribosome at high resolution, revealing binding interactions that extend into the peptidyl tRNA-binding site and towards synergistic binders that occupy the nascent peptide exit tunnel. One of these analogues has excellent activity against several streptogramin-resistant strains of Staphylococcus aureus, exhibits decreased rates of acetylation in vitro, and is effective at lowering bacterial load in a mouse model of infection. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.

BackgroundIbezapolstat (ACX-362E) is the first DNA polymerase IIIC inhibitor undergoing clinical development for the oral treatment of Clostridioides difficile infection (CDI).MethodsIn this study, the in vitro activity of ibezapolstat was evaluated against a panel of 104 isolates of C. difficile, including those with characterized ribotypes (e.g. 027 and 078) and those producing toxin A or B and was shown to have similar activity to those of comparators against these strains.ResultsThe overall MIC50/90 (mg/L) for ibezapolstat against evaluated C. difficile was 2/4, compared with 0.5/4 for metronidazole, 1/4 for vancomycin and 0.5/2 for fidaxomicin. In addition, the bactericidal activity of ibezapolstat was evaluated against actively growing C. difficile by determining the MBC against three C. difficile isolates. Time–kill kinetic assays were additionally performed against the three C. difficile isolates, with metronidazole and vancomycin as comparators.ConclusionsThe killing of C. difficile by ibezapolstat was observed to occur at concentrations similar to its MIC, as demonstrated by MBC:MIC ratios and reflected in time–kill kinetic assays. This activity highlights the therapeutic potential of ibezapolstat for the treatment of CDI.

Background Pulmonary infections caused by Nontuberculous Mycobacteria (NTM) are increasing in prevalence and are associated with high mortality and morbidity. Members of the Mycobacterium avium complex (MAC; primarily M. avium and M. intracellulare) and M. abscessus are most commonly associated with NTM pulmonary disease. Treatment options are limited and new agents with potent activity are needed. In this study, the activity of SPR719, a novel aminobenzimidazole, against NTM is reported. MIC range and MIC50/90 summary table Methods The susceptibility of 58 non-consecutive, non-duplicate clinical NTM isolates was determined in accordance with the Clinical and Laboratory Standards Institute (CLSI) standard M24. Isolates included 20 rapidly-growing mycobacteria (10 M. abscessus/chelonae Group, 6 M. fortuitum Group, and 4 M. mucogenicum Group) and 38 slow-growing mycobacteria (28 MAC and 10 M. kansasii). SPR719 and comparators clarithromycin (CLA), amikacin (AMK), moxifloxacin (MXF), rifabutin (RFB), minocycline (MIN), and imipenem (IPM) were evaluated. Minimum bactericidal concentrations (MBC) for SPR719, CLA, and AMK were determined in accordance with CLSI M26. Results The activity of SPR719 and comparators by MIC range and MIC50/90 (µg/mL) is summarized in the accompanying table. SPR719 activity was not affected by resistance to CLA, AMK, or MXF. MBC:MIC ratios for SPR719 and CLA were typically >8 which indicates a bacteriostatic mode of action; AMK MBC:MIC ratios were typically ≤ 4 indicative of bactericidal activity. Conclusion SPR719 had potent activity by both MIC50/90 and MIC range across the evaluated NTM species. The SPR719 activity against clinically relevant MAC and M. abcessus/chelonae Group isolates was comparable or superior to the evaluated comparators, and SPR719 was active against isolates resistant to currently utilized agents. These results highlight the potential of SPR719 in the treatment of NTM pulmonary disease. Disclosures Nicole S. Cotroneo, BS, Spero Therapeutics (Employee, Shareholder) Ian Critchley, PhD, Spero Therapeutics (Employee, Shareholder) Michael Pucci, PhD, Spero Therapeutics (Employee, Shareholder) Suzanne Stokes, PhD, Spero Therapeutics (Employee, Shareholder)

Natural products serve as chemical blueprints for the majority of classes of antibiotics in our clinical arsenal. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class. Virginiamycin acetyltransferases (Vats) are resistance proteins that provide protection against streptogramins, potent Gram-positive antibiotics that inhibit the bacterial ribosome. Due to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogs that overcome Vat resistance have not been previously accessible. Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with unprecedented structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogs to the bacterial ribosome at high resolution (2.4-2.8 Å), revealing new binding interactions that extend into the peptidyl tRNA binding site and towards synergistic binders that occupy the nascent peptide exit tunnel. Two of these analogs have excellent activity against a streptogramin-resistant strain of S. aureus expressing VatA and exhibit decreased acetylation rates in vitro. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.

Summaryα-Neurexins are essential synaptic adhesion molecules implicated in autism spectrum disorder and schizophrenia. The α-neurexin extracellular domain consists of six LNS domains interspersed by three EGF-like repeats and interacts with many different proteins in the synaptic cleft. To understand how α-neurexins might function as synaptic organizers, we solved the structure of the neurexin 1α extracellular domain (n1α) to 2.65 Å. The L-shaped molecule can be divided into a flexible repeat I (LNS1-EGF-A-LNS2), a rigid horseshoe-shaped repeat II (LNS3-EGF-B-LNS4) with structural similarity to so-called reelin repeats, and an extended repeat III (LNS5-EGF-B-LNS6) with controlled flexibility. A 2.95 Å structure of n1α carrying splice insert SS#3 in LNS4 reveals that SS#3 protrudes as a loop and does not alter the rigid arrangement of repeat II. The global architecture imposed by conserved structural features enables α-neurexins to recruit and organize proteins in distinct and variable ways, influenced by splicing, thereby promoting synaptic function.

Neurexins and neuroligins play an essential role in synapse function, and their alterations are linked to autistic spectrum disorder. Interactions between neurexins and neuroligins regulate inhibitory and excitatory synaptogenesis in vitro through a "splice-insert signaling code." In particular, neurexin 1beta carrying an alternative splice insert at site SS#4 interacts with neuroligin 2 (found predominantly at inhibitory synapses) but much less so with other neuroligins (those carrying an insert at site B and prevalent at excitatory synapses). The structure of neurexin 1beta+SS#4 reveals dramatic rearrangements to the "hypervariable surface," the binding site for neuroligins. The splice insert protrudes as a long helix into space, triggers conversion of loop beta10-beta11 into a helix rearranging the binding site for neuroligins, and rearranges the Ca(2+)-binding site required for ligand binding, increasing its affinity. Our structures reveal the mechanism by which neurexin 1beta isoforms acquire neuroligin splice isoform selectivity.