A new series of short pyrrole tetraamides are described whose submicromolar DNA binding affinity is an essential component for their strong antibacterial activity. This class of compounds is related to the linked bis-netropsins and bis-distamycins, but here, only one amino-pyrrole-carboxamide unit and an amidine tail is connected to either side of a central dicarboxylic acid linker. The highest degree of DNA binding, measured by compound-induced changes in UV melting temperatures of an AT-rich DNA oligomer, was observed for flat, aromatic linkers with no inherent bent, i.e., terephthalic acid or 1,4-pyridine-dicarboxylic acid. However, the antibacterial activity is critically linked to the size of the N-alkyl substiutent of the pyrrole unit. None of the tetraamides with the commonly used methyl-pyrrole showed antibacterial activity. Isoamyl- or cyclopropylmethylene-substituted dipyrrole derivatives have the minimum inhibitory concentrations in the submicromolar range. In vitro toxicity against human T-cells was studied for all compounds. The degree to which compounds inhibited cell growth was neither directly correlated to DNA binding affinity nor directly correlated to antibacterial activity but seemed to depend strongly on the nature of the N-alkyl pyrrole substituents.

We reported previously that priming of the respiratory tract with immunobiotic Lactobacillus prior to virus challenge protects mice against subsequent lethal infection with pneumonia virus of mice (PVM). We present here the results of gene microarray which document differential expression of proinflammatory mediators in response to PVM infection alone and those suppressed in response to Lactobacillus plantarum. We also demonstrate for the first time that intranasal inoculation with live or heat-inactivated L. plantarum or Lactobacillus reuteri promotes full survival from PVM infection when administered within 24 h after virus challenge. Survival in response to L. plantarum administered after virus challenge is associated with suppression of proinflammatory cytokines, limited virus recovery, and diminished neutrophil recruitment to lung tissue and airways. Utilizing this post-virus challenge protocol, we found that protective responses elicited by L. plantarum at the respiratory tract were distinct from those at the gastrointestinal mucosa, as mice devoid of the anti-inflammatory cytokine, interleukin (IL)-10, exhibit survival and inflammatory responses that are indistinguishable from those of their wild-type counterparts. Finally, although L. plantarum interacts specifically with pattern recognition receptors TLR2 and NOD2, the respective gene-deleted mice were fully protected against lethal PVM infection by L. plantarum, as are mice devoid of type I interferon receptors. Taken together, L. plantarum is a versatile and flexible agent that is capable of averting the lethal sequelae of severe respiratory infection both prior to and post-virus challenge via complex and potentially redundant mechanisms.

Pattern recognition receptors (PRRs) engage microbial components in the lung, although their role in providing primary host defense against respiratory virus infection is not fully understood. We have previously shown that Gram-positive Lactobacillus plantarum (Lp) administered to the respiratory tract promotes full and sustained protection in response to an otherwise lethal mouse pneumovirus (PVM) infection, a robust example of heterologous immunity. While Lp engages PRRs TLR2 and NOD2 in ex vivo signaling assays, we found that Lp-mediated protection was unimpaired in single gene-deleted TLR2−/− and NOD2−/− mice. Here we demonstrate substantial loss of Lp-mediated protection in a double gene-deleted NOD2−/−TLR2−/− strain. Furthermore, we demonstrate protection against PVM infection by administration of the bi-functional NOD2-TLR2 agonist, CL-429. The bi-functional NOD2-TLR2 ligand CL-429 not only suppresses virus-induced inflammation, it is significantly more effective at preventing lethal infection than equivalent amounts of mono-molecular TLR2 and NOD2 agonists. Interestingly, and in contrast to biochemical NOD2 and/or TLR2 agonists, Lp remained capable of eliciting primary proinflammatory responses from NOD2−/−TLR2−/− mice in vivo and from alveolar macrophages challenged ex vivo. Taken together, we conclude that coordinate engagement of NOD2 and TLR2 constitutes a key step in the genesis of Lp-mediated protection from a lethal respiratory virus infection, and represents a critical target for modulation of virus-induced inflammatory pathology.

To identify novel antivirals to the hepatitis C virus (HCV) NS4B protein, we utilized encoded library technology (ELT), which enables purified proteins not amenable to standard biochemical screening methods to be tested against large combinatorial libraries in a short period of time. We tested NS4B against several DNA-encoded combinatorial libraries (DEL) and identified a single DEL feature that was subsequently progressed to off-DNA synthesis. The most active of the initial synthesized compounds had 50% inhibitory concentrations (IC 50 s) of 50 to 130 nM in a NS4B radioligand binding assay and 300 to 500 nM in an HCV replicon assay. Chemical optimization yielded compounds with potencies as low as 20 nM in an HCV genotype 1b replicon assay, 500 nM against genotype 1a, and 5 M against genotype 2a. Through testing against other genotypes and genotype 2a-1b chimeric replicons and from resistance passage using the genotype 1b replicon, we confirmed that these compounds were acting on the proposed first transmembrane region of NS4B. A single sequence change (F98L) was identified as responsible for resistance, and it was thought to largely explain the relative lack of potency of this series against genotype 2a. Unlike other published series that appear to interact with this region, we did not observe sensitivity to amino acid substitutions at positions 94 and 105. The discovery of this novel compound series highlights ELT as a valuable approach for identifying direct-acting antivirals to nonenzymatic targets. Significant progress has been made in recent years in the discovery and development of novel direct-acting antivirals (DAAs) to treat hepatitis C virus (HCV) infection. However, the ability of HCV to rapidly evolve and develop resistance is such that there is a continued need to discover DAAs that act through novel mechanisms. The standard of care for chronic HCV infection was a combination of pegylated alpha interferon and ribavirin up until the approval of the NS3 protease inhibitors telaprevir and boceprevir in 2011 (1, 2). Other classes of DAAs showing clinical efficacy target the NS5A protein (3, 4) or viral polymerase (NS5B; reviewed in reference 5). Of the other viral proteins, several smallmolecule DAAs to nonstructural protein NS4B have been identified (6-14), but to date, there are no examples of clinically active NS4B-targeting compounds.NS4B is a small (27-kDa), hydrophobic, membrane-associated protein that is derived through processing of the HCV polyprotein by the viral protease NS3/4A (reviewed in references 15, 16, and 17). It complexes with other HCV nonstructural and possibly a host cell factor(s) to form the viral replicase (18-22), which serves to replicate the viral RNA and is likely coupled to the assembly process (20, 23). Besides its structural role in the replicase, NS4B is thought to be involved in the induction of membranous vesicles that provide a platform for HCV RNA replication (24, 25), and it may also participate in virion assembly and release (26,27). NS4B is also reported to hydr...