OG716: Designing a fit-for-purpose lantibiotic for the treatment of <i>Clostridium difficile</i> infections

<div><p>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 <i>C</i>. <i>difficile</i> 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 <i>in vitro</i> were further evaluated orally in the Golden Syrian hamster model of CDAD. <i>In vivo</i> 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.</p></div

Hamster CDAD study design.

<p>Hamster CDAD study design.</p

Structural features of MU1140 and select lead compounds.

<p>Panel (A) depicts the primary amino acid sequence of MU1140. The second generation of MU1140 variants designed in the current study focused on the residues highlighted in gray. Panel (B) depicts the structure of unusual amino acids. Panel (C) tabulates the substitutions of lead compounds carried through <i>in vivo</i> efficacy studies. Legend: amino acids, AA.</p

MIC characteristics of selected compounds against <i>C</i>. <i>difficile</i>¹.

<p>MIC characteristics of selected compounds against <i>C</i>. <i>difficile</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197467#t003fn001" target="_blank">¹</a>.</p

Efficacy of lead compounds assessed <i>in vivo</i>.

<p>Golden Syrian hamsters (N = 6 per group) were infected on Day 1 and received a single subcutaneous injection of Clindamycin (10 mg/Kg) on Day 2. Test compounds at 20 mg/Kg in 5% mannitol were administered by oral gavage 3 times per day (TID), starting on Day 2 at 18 hours after Clindamycin treatment, for 5 consecutive days (Days 2 through 6). Vancomycin (positive control) was administered at 20 mg/kg QD in parallel, and the infection control group was dosed with vehicle alone. See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197467#sec002" target="_blank">Materials and Methods</a></b>for details.</p

Illustration of the relative zones of inhibition of screened compounds versus MU1140 and OG253.

<p>Controls are boxed in the upper-left corner and include blanks (Column 1, negative control), MU1140 (Column 2, positive control) and OG253 (Column 3, comparator).</p

Triage strategy used in this study.

<p>High-throughput activity screening used robotic spotting and an optical scanner for the determination of the zones of inhibition. MIC testing provided specific activity values for the determination of the potency. Stability testing performed using biologically-relevant substrates. <i>In vivo</i> efficacy performed in the Golden Syrian hamster model of CDAD. See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197467#sec002" target="_blank">Materials and Methods</a></b> section for details. Legend: FaSSGF, fasted-state simulated gastric fluid; FaSSIF, fasted-state simulated intestinal fluid.</p

Solubility, stability under forced degradation, and half-life in bio-relevant matrices.

<p>Solubility, stability under forced degradation, and half-life in bio-relevant matrices.</p

Spore titers at death or at the outcome of the treatment phase.

<p>The cecal contents from all hamsters that died on study or from hamsters euthanized at the end of the observation period (Day 21, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197467#pone.0197467.g004" target="_blank">Fig 4</a>) were collected and tested for spore counts (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197467#sec002" target="_blank">Materials and Methods</a></b> section for details).</p

Data_Sheet_2.PDF

<p>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 44^TM, 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 44^TMin 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.</p