DNA Targeting as a Likely Mechanism Underlying the Antibacterial Activity of Synthetic Bis-Indole Antibiotics.

We previously reported the synthesis and biological activity of a series of cationic bis-indoles with potent, broad-spectrum antibacterial properties. Here, we describe mechanism of action studies to test the hypothesis that these compounds bind to DNA and that this target plays an important role in their antibacterial outcome. The results reported here indicate that the bis-indoles bind selectively to DNA at A/T-rich sites, which is correlated with the inhibition of DNA and RNA synthesis in representative Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) organisms. Further, exposure of E. coli and S. aureus to representative bis-indoles resulted in induction of the DNA damage-inducible SOS response. In addition, the bis-indoles were found to be potent inhibitors of cell wall biosynthesis; however, they do not induce the cell wall stress stimulon in S. aureus, suggesting that this pathway is inhibited by an indirect mechanism. In light of these findings, the most likely basis for the observed activities of these compounds is their ability to bind to the minor groove of DNA, resulting in the inhibition of DNA and RNA synthesis and other secondary effects.

Cutting Edge: A Natural Antisense Transcript, AS-IL1α, Controls Inducible Transcription of the Proinflammatory Cytokine IL-1α.

Natural antisense transcripts (NATs) are a class of long noncoding RNAs (lncRNAs) that are complementary to other protein-coding genes. Although thousands of NATs are encoded by mammalian genomes, their functions in innate immunity are unknown. In this study, we identified and characterized a novel NAT, AS-IL1α, which is partially complementary to IL-1α. Similar to IL-1α, AS-IL1α is expressed at low levels in resting macrophages and is induced following infection with Listeria monocytogenes or stimulation with TLR ligands (Pam3CSK4, LPS, polyinosinic-polycytidylic acid). Inducible expression of IL-1α mRNA and protein were significantly reduced in macrophages expressing shRNA that target AS-IL1α. AS-IL1α is located in the nucleus and did not alter the stability of IL-1α mRNA. Instead, AS-IL1α was required for the recruitment of RNA polymerase II to the IL-1α promoter. In summary, our studies identify AS-IL1α as an important regulator of IL-1α transcription during the innate immune response.

Synthesis and structure-activity relationships of novel phenoxyacetamide inhibitors of the Pseudomonas aeruginosa type III secretion system (T3SS).

The increasing prevalence of drug-resistant bacterial infections is driving the discovery and development not only of new antibiotics, but also of inhibitors of virulence factors that are crucial for in vivo pathogenicity. One such virulence factor is the type III secretion system (T3SS), which plays a critical role in the establishment and dissemination of Pseudomonas aeruginosa infections. We have recently described the discovery and characterization of a series of inhibitors of P. aeruginosa T3SS based on a phenoxyacetamide scaffold. To better characterize the factors involved in potent T3SS inhibition, we have conducted a systematic exploration of this structure, revealing several highly responsive structure-activity relationships indicative of interaction with a specific target. Most of the structural features contributing to potency were additive, and combination of those features produced optimized inhibitors with IC50 values <1µM.

Mutations in the Pseudomonas aeruginosa needle protein gene pscF confer resistance to phenoxyacetamide inhibitors of the type III secretion system.

The type III secretion system (T3SS) is a clinically important virulence mechanism in Pseudomonas aeruginosa that secretes and translocates effector toxins into host cells, impeding the host's rapid innate immune response to infection. Inhibitors of T3SS may be useful as prophylactic or adjunctive therapeutic agents to augment the activity of antibiotics in P. aeruginosa infections, such as pneumonia and bacteremia. One such inhibitor, the phenoxyacetamide MBX 1641, exhibits very responsive structure-activity relationships, including striking stereoselectivity, in its inhibition of P. aeruginosa T3SS. These features suggest interaction with a specific, but unknown, protein target. Here, we identify the apparent molecular target by isolating inhibitor-resistant mutants and mapping the mutation sites by deep sequencing. Selection and sequencing of four independent mutants resistant to the phenoxyacetamide inhibitor MBX 2359 identified the T3SS gene pscF, encoding the needle apparatus, as the only locus of mutations common to all four strains. Transfer of the wild-type and mutated alleles of pscF, together with its chaperone and cochaperone genes pscE and pscG, to a ΔpscF P. aeruginosa strain demonstrated that each of the single-codon mutations in pscF is necessary and sufficient to provide secretion and translocation that is resistant to a variety of phenoxyacetamide inhibitor analogs but not to T3SS inhibitors with different chemical scaffolds. These results implicate the PscF needle protein as an apparent new molecular target for T3SS inhibitor discovery and suggest that three other chemically distinct T3SS inhibitors interact with one or more different targets or a different region of PscF.

Evolution of the influenza A virus genome during development of oseltamivir resistance in vitro.

Influenza A virus (IAV) is a major cause of morbidity and mortality throughout the world. Current antiviral therapies include oseltamivir, a neuraminidase inhibitor that prevents the release of nascent viral particles from infected cells. However, the IAV genome can evolve rapidly, and oseltamivir resistance mutations have been detected in numerous clinical samples. Using an in vitro evolution platform and whole-genome population sequencing, we investigated the population genomics of IAV during the development of oseltamivir resistance. Strain A/Brisbane/59/2007 (H1N1) was grown in Madin-Darby canine kidney cells with or without escalating concentrations of oseltamivir over serial passages. Following drug treatment, the H274Y resistance mutation fixed reproducibly within the population. The presence of the H274Y mutation in the viral population, at either a low or a high frequency, led to measurable changes in the neuraminidase inhibition assay. Surprisingly, fixation of the resistance mutation was not accompanied by alterations of viral population diversity or differentiation, and oseltamivir did not alter the selective environment. While the neighboring K248E mutation was also a target of positive selection prior to H274Y fixation, H274Y was the primary beneficial mutation in the population. In addition, once evolved, the H274Y mutation persisted after the withdrawal of the drug, even when not fixed in viral populations. We conclude that only selection of H274Y is required for oseltamivir resistance and that H274Y is not deleterious in the absence of the drug. These collective results could offer an explanation for the recent reproducible rise in oseltamivir resistance in seasonal H1N1 IAV strains in humans.

Distinct cellular origins for serotonin-expressing and enterochromaffin-like cells in the gastric corpus.

BACKGROUND & AIMS: The alimentary tract contains a diffuse endocrine system comprising enteroendocrine cells that secrete peptides or biogenic amines to regulate digestion, insulin secretion, food intake, and energy homeostasis. Lineage analysis in the stomach revealed that a significant fraction of endocrine cells in the gastric corpus did not arise from Neurogenin3 (Neurog3)-expressing cells, unlike enteroendocrine cells elsewhere in the digestive tract. We aimed to isolate enriched serotonin-secreting and enterochromaffin-like (ECL) cells from the stomach and to clarify their cellular origin. METHODS: We used Neurogenic differentiation 1 (NeuroD1) and Neurog3 lineage analysis and examined the differentiation of serotonin-producing and ECL cells in stomach tissues of NeuroD1-cre;ROSA(tdTom), tryptophan hydroxylase 1 (Tph1)-cyan fluorescent protein (CFP), c-Kit(wsh/wsh), and Neurog3Cre;ROSA(tdTom) mice by immunohistochemistry. We used fluorescence-activated cell sorting to isolate each cell type for gene expression analysis. We also performed RNA sequencing analysis of ECL cells. RESULTS: Neither serotonin-secreting nor ECL cells of the corpus arose from cells expressing NeuroD1. Serotonin-secreting cells expressed a number of mast cell genes but not genes associated with endocrine differentiation; they did not develop in c-Kit(wsh/wsh) mice and were labeled with transplanted bone marrow cells. RNA sequencing analysis of ECL cells revealed high expression levels of many genes common to endocrine cells, including transcription factors, hormones, ion channels, and solute transporters but not markers of bone marrow cells. CONCLUSIONS: Serotonin-expressing cells of the gastric corpus of mice appear to be bone marrow-derived mucosal mast cells. Gene expression analysis of ECL cells indicated that they are endocrine cells of epithelial origin that do not express the same transcription factors as their intestinal enteroendocrine cell counterparts.

A long noncoding RNA mediates both activation and repression of immune response genes.

An inducible program of inflammatory gene expression is central to antimicrobial defenses. This response is controlled by a collaboration involving signal-dependent activation of transcription factors, transcriptional co-regulators, and chromatin-modifying factors. We have identified a long noncoding RNA (lncRNA) that acts as a key regulator of this inflammatory response. Pattern recognition receptors such as the Toll-like receptors induce the expression of numerous lncRNAs. One of these, lincRNA-Cox2, mediates both the activation and repression of distinct classes of immune genes. Transcriptional repression of target genes is dependent on interactions of lincRNA-Cox2 with heterogeneous nuclear ribonucleoprotein A/B and A2/B1. Collectively, these studies unveil a central role of lincRNA-Cox2 as a broad-acting regulatory component of the circuit that controls the inflammatory response.

Optimization of a novel potent and selective bacterial DNA helicase inhibitor scaffold from a high throughput screening hit.

Benzobisthiazole derivatives were identified as novel helicase inhibitors through high throughput screening against purified Staphylococcus aureus (Sa) and Bacillus anthracis (Ba) replicative helicases. Chemical optimization has produced compound 59 with nanomolar potency against the DNA duplex strand unwinding activities of both B. anthracis and S. aureus helicases. Selectivity index (SI=CC50/IC50) values for 59 were greater than 500. Kinetic studies demonstrated that the benzobisthiazole-based bacterial helicase inhibitors act competitively with the DNA substrate. Therefore, benzobisthiazole helicase inhibitors represent a promising new scaffold for evaluation as antibacterial agents.

Coumarin-based inhibitors of Bacillus anthracis and Staphylococcus aureus replicative DNA helicase: chemical optimization, biological evaluation, and antibacterial activities.

The increasing prevalence of drug-resistant bacterial infections demands the development of new antibacterials that are not subject to existing mechanisms of resistance. Previously, we described coumarin-based inhibitors of an underexploited bacterial target, namely the replicative helicase. Here we report the synthesis and evaluation of optimized coumarin-based inhibitors with 9-18-fold increased potency against Staphylococcus aureus (Sa) and Bacillus anthracis (Ba) helicases. Compounds 20 and 22 provided the best potency, with IC(50) values of 3 and 1 µM, respectively, against the DNA duplex strand-unwinding activities of both B. anthracis and S. aureus helicases without affecting the single strand DNA-stimulated ATPase activity. Selectivity index (SI = CC(50)/MIC) values against S. aureus and B. anthracis for compound 20 were 33 and 66 and for compound 22 were 20 and 40, respectively. In addition, compounds 20 and 22 demonstrated potent antibacterial activity against multiple ciprofloxacin-resistant MRSA strains, with MIC values ranging between 0.5 and 4.2 µg/mL.

Evolution of specific protein-protein interaction sites following gene duplication.

Gene duplication is a common evolutionary process that leads to the expansion and functional diversification of protein subfamilies. The evolutionary events that cause paralogous proteins to bind different protein ligands (functionally diverged interfaces) are investigated and compared to paralogous proteins that bind the same protein ligand (functionally preserved interfaces). We find that functionally diverged interfaces possess more subfamily-specific residues than functionally preserved interfaces. These subfamily-specific residues are usually partially buried at the interface rim and achieve specific binding through optimized hydrogen bond geometries. In addition to optimized hydrogen bond geometries, side-chain modeling experiments suggest that steric effects are also important for binding specificity. Residues that are completely buried at the interface hub are also less conserved in functionally diverged interfaces than in functionally preserved interfaces. Consistent with this finding, hub residues contribute less to free energy of binding in functionally diverged interfaces than in functionally preserved interfaces. Therefore, we propose that protein binding is a delicate balance between binding affinity that primarily occurs at the interface hub and binding specificity that primarily occurs at the interface rim.

Discovery and characterization of inhibitors of Pseudomonas aeruginosa type III secretion.

The type III secretion system (T3SS) is a clinically important virulence mechanism in Pseudomonas aeruginosa that secretes and translocates up to four protein toxin effectors into human cells, facilitating the establishment and dissemination of infections. To discover inhibitors of this important virulence mechanism, we developed two cellular reporter assays and applied them to a library of 80,000 compounds. The primary screen was based on the dependence of the transcription of T3SS operons on the T3SS-mediated secretion of a negative regulator and consisted of a transcriptional fusion of the Photorhabdus luminescens luxCDABE operon to the P. aeruginosa exoT effector gene. Secondary assays included direct measurements of the T3SS-mediated secretion of a P. aeruginosa ExoS effector-beta-lactamase fusion protein as well as the detection of the secretion of native ExoS by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of culture supernatants. Five inhibitors in three chemical classes were demonstrated to inhibit type III secretion selectively with minimal cytotoxicity and with no effects on bacterial growth or on the type II-mediated secretion of elastase. These inhibitors also block the T3SS-mediated secretion of a YopE effector-beta-lactamase fusion protein from an attenuated Yersinia pestis strain. The most promising of the inhibitors is a phenoxyacetamide that also blocks the T3SS-mediated translocation of effectors into mammalian cells in culture. Preliminary studies of structure-activity relationships in this phenoxyacetamide series demonstrated a strict requirement for the R-enantiomer at its stereocenter and indicated tolerance for a variety of substituents on one of its two aromatic rings.

Discovery, characterization and comparison of inhibitors of Bacillus anthracis and Staphylococcus aureus replicative DNA helicases.

Antibacterial compounds with new mechanisms of action are needed for effective therapy against drug-resistant pathogens in the clinic and in biodefense. Screens for inhibitors of the essential replicative helicases of Bacillus anthracis and Staphylococcus aureus yielded 18 confirmed hits (IC(50)25 microM). Several (5 of 18) of the inhibitors were also shown to inhibit DNA replication in permeabilized polA-deficient B. anthracis cells. One of the most potent inhibitors also displayed antibacterial activity (MIC approximately 5 microg/ml against a range of Gram-positive species including bacilli and staphylococci) together with good selectivity for bacterial versus mammalian cells (CC(50)/MIC>16) suitable for further optimization. This compound shares the bicyclic ring of the clinically proven aminocoumarin scaffold, but is not a gyrase inhibitor. It exhibits a mixed mode of helicase inhibition including a component of competitive inhibition with the DNA substrate (K(i)=8 microM) and is rapidly bactericidal at 4 x MIC.