Lantibiotics offer an untapped pipeline for the development of novel antibiotics to treat serious Gram-positive (+) infections including Clostridium difficile. Mutacin 1140 (MU1140) is a lantibiotic produced by Streptococcus mutans and acts via a novel mechanism of action, which may limit the development of resistance. This study sought to identify a lead compound for the treatment of C. difficile associated diarrhea (CDAD). Compounds were selected from a saturation mutagenesis library of 418 single amino acid variants of MU1140. Compounds were produced by small scale fermentation, purified, characterized and then subjected to a panel of assays aimed at identifying the best performers. The screening assays included: in vitro susceptibility testing [MIC against Micrococcus luteus, Clostridium difficile, vancomycin-resistant enterococci (VRE), Staphylococcus aureus, Streptococcus pneumonia, Mycobacterium phlei, and Pseudomonas aeruginosa; cytotoxicity screening on HepG2 hepatocytes; in vitro pharmacological profiling with the Safety Screen 44TM, metabolic and chemical stability in biologically relevant fluids (FaSSGF, FaSSIF and serum); and efficacy in vivo]. Several lantibiotic compounds had better MIC against C. difficile, compared to vancomycin, but not against other bacterial species tested. The Safety Screen 44TM in vitro pharmacological profiling assay suggested that this class of compounds has relatively low overall toxicity and that compound OG253 (MU1140, Phe1Ile) is not likely to present inadvertent off-target effects, as evidenced by a low promiscuity score. The in vitro cytotoxicity assay also indicated that this class of compounds was characterized by low toxicity; the EC50 of OG253 was 636 mg/mL on HepG2 cells. The half-life in simulated gastric fluid was >240 min. for all compound tested. The stability in simulated intestinal fluid ranged between a half-life of 5 min to >240 min, and paralleled the half-life in serum. OG253 ultimately emerged as the lead compound based on superior in vivo efficacy along with an apparent lack of relapse in a hamster model of infection. The lessons learned from this report are applicable to therapeutic lanthipeptides in general and may assist in the design of novel molecules with improved pharmacological, therapeutic and physicochemical profiles. The data presented also support the continued clinical development of OG253 as a novel antibiotic against CDAD that could prevent recurrence of the infection.

Lantibiotics continue to offer an untapped pipeline for the development of novel antibiotics. We report here the discovery of a novel lantibiotic for the treatment of C. difficile infection (CDI). The leads were selected from a library of over 300 multiple substitution variants of the lantibiotic Mutacin 1140 (MU1140). Top performers were selected based on testing for superior potency, solubility, manufacturability, and physicochemical and/or metabolic stability in biologically-relevant systems. The best performers in vitro were further evaluated orally in the Golden Syrian hamster model of CDAD. In vivo testing ultimately identified OG716 as the lead compound, which conferred 100% survival and no relapse at 3 weeks post infection. MU1140-derived variants are particularly attractive for further clinical development considering their novel mechanism of action.

Lantibiotics represent a large untapped pipeline of attractive scaffolds for the development of novel antibiotics. Saturation mutagenesis was employed to substitute every amino acid of a lantibiotic called mutacin 1140 (MU1140), creating an unbiased expression library of 418 variants that was used to study the permissiveness to mutagenesis and the "drugability" of several compounds. Contrasting previous reports, the results from this study supported that not all residues involved in lanthionine bridge formation were critical for maintaining optimal activity. While substitutions in lanthionine bridges in Ring A, C, and D invariably lead to inactive variants, permissive substitutions in Abu8 and Ala11 (Ring B) were observed, albeit infrequently. Further, the data generated suggested that the unsaturated bond from Dha5 (Ser5) may not be critically involved in Lipid-II binding but still important for conferring optimal activity. This study identified additional permissive mutations of Ser5, including Ser5His, Ser5Met, Ser5Gln, and Ser5Leu. In contrast, no permissive substitutions were identified for Dhb14, which suggested that this residue may be critical for optimal activity. Novel blueprints are proposed for directing further development of MU1140 variants and other lantibiotics, which may enable the rational design, development, manufacture, and formulation of an entirely new class of anti-infectives.

Lantibiotics present an attractive scaffold for the development of novel antibiotics. We report here a novel lantibiotic for the treatment of Clostridium difficile infection. The lead compounds were selected from a library of over 700 single-and multiple-substitution variants of the lantibiotic mutacin 1140 (MU1140). The best performers in vitro and in vivo were further used to challenge Golden Syrian hamsters orally in a Golden Syrian hamster model of Clostridium difficile-associated disease (CDAD) in a dose-response format, resulting in the selection of OG716 as the lead compound. This lantibiotic was characterized by a 50% effective dose of 23.85 mg/kg of body weight/day (10.97 mol/kg/day) in this model. Upon oral administration of the maximum feasible dose (Ն1,918 mg/kg/day), no observable toxicities or side effects were noted, and no effect on intestinal motility was observed. Compartmentalization to the gastrointestinal tract was confirmed. MU1140-derived variants offer a large pipeline for the development of novel antibiotics for the treatment of several indications and are particularly attractive considering their novel mechanism of action. Based on the currently available data, OG716 has an acceptable profile for further development for the treatment of CDAD.

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