Small Molecule Inhibitors of Staphylococcus aureus RnpA Alter Cellular mRNA Turnover, Exhibit Antimicrobial Activity, and Attenuate Pathogenesis

Methicillin-resistant Staphylococcus aureus is estimated to cause more U.S. deaths annually than HIV/AIDS. The emergence of hypervirulent and multidrug-resistant strains has further amplified public health concern and accentuated the need for new classes of antibiotics. RNA degradation is a required cellular process that could be exploited for novel antimicrobial drug development. However, such discovery efforts have been hindered because components of the Gram-positive RNA turnover machinery are incompletely defined. In the current study we found that the essential S. aureus protein, RnpA, catalyzes rRNA and mRNA digestion in vitro. Exploiting this activity, high through-put and secondary screening assays identified a small molecule inhibitor of RnpA-mediated in vitro RNA degradation. This agent was shown to limit cellular mRNA degradation and exhibited antimicrobial activity against predominant methicillin-resistant S. aureus (MRSA) lineages circulating throughout the U.S., vancomycin intermediate susceptible S. aureus (VISA), vancomycin resistant S. aureus (VRSA) and other Gram-positive bacterial pathogens with high RnpA amino acid conservation. We also found that this RnpA-inhibitor ameliorates disease in a systemic mouse infection model and has antimicrobial activity against biofilm-associated S. aureus. Taken together, these findings indicate that RnpA, either alone, as a component of the RNase P holoenzyme, and/or as a member of a more elaborate complex, may play a role in S. aureus RNA degradation and provide proof of principle for RNA catabolism-based antimicrobial therapy

Evaluation of Ceftazidime and NXL104 in Two Murine Models of Infection Due to KPC-Producing Klebsiella pneumoniae▿

We evaluated the efficacy of NXL104, a novel β-lactamase inhibitor, in combination with ceftazidime (CAZ) in two murine infection models (septicemia and thigh infection). We chose two KPC-producing Klebsiella pneumoniae strains (VA-361 and VA-406) showing MICs of CAZ of ≥256 μg/ml. Septicemia was induced by the intraperitoneal injection of KPC-producing K. pneumoniae followed 30 min later by a single subcutaneous treatment with CAZ alone or CAZ-NXL104 in ratios of 2:1, 4:1, 8:1, and 16:1. In this model, the median effective doses for 50% (ED50) of the animals for CAZ alone versus VA-361 and VA-406 were 1,578 and 709 mg/kg of body weight, respectively. When combined with NXL104 at 2:1, 4:1, 8:1, and 16:1 ratios, the CAZ ED50s for VA-361 and VA-406 were reduced to 8.1 and 3.5 mg/kg, 15.1 and 3.8 mg/kg, 16.9 and 7.2 mg/kg, and 29.5 and 12.1 mg/kg, respectively. For thigh infection, neutropenia was induced by the intraperitoneal injection of cyclophosphamide at days −4 and −1 preinfection. Infection was established by the intramuscular injection of KPC-producing K. pneumoniae into the right thigh. Mice were treated 1.5 h postinfection with either CAZ alone or CAZ-NXL104 at constant ratios of 4:1. When thighs were removed at 24 h postinfection, a >2-log CFU reduction was observed for mice treated with CAZ-NXL104 at doses of ≥128:32 mg/kg. In contrast, CAZ doses of ≥1,024 mg/kg were unable to reduce the numbers of CFU. Despite resistance to CAZ and possessing a complex β-lactamase background, NXL104 combined with CAZ proved to be very effective in murine models of infection due to contemporary highly resistant KPC-producing K. pneumoniae isolates

Mutacin 1140 Lantibiotic Variants Are Efficacious Against Clostridium difficile Infection

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 44TMin 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

Novel Chitohexaose Analog Protects Young and Aged mice from CLP Induced Polymicrobial Sepsis

Abstract In Gram-negative bacterial sepsis, production of excess pro-inflammatory cytokines results in hyperinflammation and tissue injury. Anti-inflammatory cytokines such as IL-10 inhibit inflammation and enhance tissue healing. Here, we report a novel approach to treat septicemia associated with intra-abdominal infection in a murine model by delicately balancing pro- and anti-inflammatory cytokines. A novel oligosaccharide compound AVR-25 selectively binds to the TLR4 protein (IC50 = 0.15 µM) in human peripheral blood monocytes and stimulates IL-10 production. Following the cecal ligation and puncture (CLP) procedure, intravenous dosing of AVR-25 (10 mg/kg, 6–12 h post-CLP) alone and in combination with antibiotic imipenem protected both young adult (10–12 week old) and aged (16–18 month old) mice against polymicrobial infection, organ dysfunction, and death. Proinflammatory cytokines (TNF-α, MIP-1, i-NOS) were decreased significantly and restoration of tissue damage was observed in all organs. A decrease in serum C-reactive protein (CRP) and bacterial colony forming unit (CFU) confirmed improved bacterial clearance. Together, these findings demonstrate the therapeutic ability of AVR-25 to mitigate the storm of inflammation and minimize tissue injury with high potential for adjunctive therapy in intra-abdominal sepsis

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

Small Molecule Inhibitors of \u3ci\u3eStaphylococcus aureus\u3c/i\u3e RnpA Alter Cellular mRNA Turnover, Exhibit Antimicrobial Activity, and Attenuate Pathogenesis

Methicillin-resistant Staphylococcus aureus is estimated to cause more U.S. deaths annually than HIV/AIDS. The emergence of hypervirulent and multidrug-resistant strains has further amplified public health concern and accentuated the need for new classes of antibiotics. RNA degradation is a required cellular process that could be exploited for novel antimicrobial drug development. However, such discovery efforts have been hindered because components of the Gram-positive RNA turnover machinery are incompletely defined. In the current study we found that the essential S. aureus protein, RnpA, catalyzes rRNA and mRNA digestion in vitro. Exploiting this activity, high through-put and secondary screening assays identified a small molecule inhibitor of RnpA-mediated in vitro RNA degradation. This agent was shown to limit cellular mRNA degradation and exhibited antimicrobial activity against predominant methicillin-resistant S. aureus (MRSA) lineages circulating throughout the U.S., vancomycin intermediate susceptible S. aureus (VISA), vancomycin resistant S. aureus (VRSA) and other Gram-positive bacterial pathogens with high RnpA amino acid conservation. We also found that this RnpA-inhibitor ameliorates disease in a systemic mouse infection model and has antimicrobial activity against biofilm-associated S. aureus. Taken together, these findings indicate that RnpA, either alone, as a component of the RNase P holoenzyme, and/ or as a member of a more elaborate complex, may play a role in S. aureus RNA degradation and provide proof of principle for RNA catabolism-based antimicrobial therapy

Hamster CDAD study design.

<p>Hamster CDAD study design.</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

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