Phase 1 trial of the safety, pharmacokinetics, and antiviral activity of EDP-514 in untreated viremic chronic hepatitis B patients.

BACKGROUND/AIMS: Oral EDP-514 is a potent core protein inhibitor of hepatitis B virus (HBV) replication, which produced a >4-log viral load reduction in HBV-infected chimeric mice with human liver cells. This study evaluated the safety, pharmacokinetics, and antiviral activity of three doses of EDP-514 in treatment-naive viremic patients with HBeAgpositive or -negative chronic HBV infection. METHODS: Patients with HBsAg detectable at screening and at least 6 months previously were eligible. HBeAg-positive and -negative patients had a serum/plasma HBV DNA level ≥20,000 and ≥2,000 IU/mL, respectively. Twenty-five patients were randomized to EDP-514 200 (n=6), 400 (n=6) or 800 mg (n=7) or placebo (n=6) once daily for 28 days. RESULTS: A dose-related increase in EDP-514 exposure (AUClast and Cmax) was observed across doses. At Day 28, mean reductions in HBV DNA were -2.9, -3.3, -3.5 and -0.2 log10 IU/mL with EDP-514 200 mg, 400 mg, 800 mg, and placebo groups, respectively. The corresponding mean change from baseline for HBV RNA levels was -2.9, -2.4, -2.0, and -0.02 log10 U/mL. No virologic failures were observed. No clinically meaningful changes from baseline were observed for HBsAg, HBeAg or HBcrAg. Nine patients reported treatment emergent adverse events of mild or moderate severity with no discontinuations, serious AEs or deaths. CONCLUSION: In treatment-naïve viremic patients, oral EDP-514 was generally safe and well-tolerated, displayed PK profile supportive of once-daily dosing, and markedly reduced HBV DNA and HBV RNA.

Bexagliflozin as an adjunct to metformin for the treatment of type 2 diabetes in adults: A 24-week, randomized, double-blind, placebo-controlled trial.

AIM: To evaluate the relative safety and effectiveness of bexagliflozin as an adjunct to metformin for the treatment of type 2 diabetes mellitus. METHODS: In total, 317 participants were randomized to receive bexagliflozin or placebo plus metformin. The primary endpoint was the change in glycated haemoglobin (HbA1c) from baseline to week 24, with secondary endpoints for systolic blood pressure (SBP), fasting plasma glucose and weight loss. An open label arm enrolled participants with HbA1c >10.5% and was analysed separately. RESULTS: The mean change in HbA1c was -1.09% (95% CI -1.24%, -0.94%) in the bexagliflozin arm and -0.56% (-0.71%, -0.41%) in the placebo arm, a difference of -0.53% (-0.74%, -0.32%; p < .0001). Excluding observations after rescue medication, the intergroup difference was -0.70% (-0.92, -0.48; p < .0001). The open label group change in HbA1c was -2.82% (-3.23%, -2.41%). Placebo-adjusted changes from baseline SBP, fasting plasma glucose and body mass were -7.07 mmHg (-9.83, -4.32; p < .0001), -1.35 mmol/L (-1.83, -0.86; p < .0001) and -2.51 kg (-3.45, -1.57; p < .0001). Adverse events affected 42.4% and 47.2% of subjects in the bexagliflozin and placebo arms, respectively; fewer subjects in the bexagliflozin arm experienced serious adverse events. CONCLUSIONS: Bexagliflozin produced clinically meaningful improvement in glycaemic control, estimated glomerular filtration rate and SBP when added to metformin in a population of adults with diabetes.

A 96-week, double-blind, randomized controlled trial comparing bexagliflozin to glimepiride as an adjunct to metformin for the treatment of type 2 diabetes in adults.

AIM: To compare the effects of bexagliflozin tablets 20 mg, with those of optimally titrated glimepiride when used to treat adults with type 2 diabetes mellitus (T2DM) inadequately controlled by metformin. METHODS: Adults with type 2 diabetes (n = 426) taking metformin, and with a glycated haemoglobin (HbA1c) level between 53 and 91 mmol/mol [7.0% and 10.5%], were randomized to receive bexagliflozin tablets 20 mg or titrated glimepiride. The primary endpoint was the intergroup difference in the change from baseline to Week 60 in percent HbA1c. Secondary endpoints included changes from baseline in body mass and systolic blood pressure (SBP), and proportion of subjects experiencing severe or documented symptomatic hypoglycaemia. RESULTS: The intergroup difference in percent HbA1c (bexagliflozin minus glimepiride) from baseline to Week 60 was -0.55 mmol/mol (95% confidence interval [CI] -2.30, 1.20)-[-0.05% (-0.21, 0.11)], establishing noninferiority of bexagliflozin to glimepiride by the prespecified margin of 3.83 mmol/mol [0.35%]. Prespecified tests gave, in order, a difference in body mass of -4.31 kg (95% CI -5.10, -3.52; P < 0.0001), a difference in SBP of -6.53 mm Hg (95% CI -10.56, -2.51; P = 0.0008), and an odds ratio of 0.12 (95% CI 0.05, 0.28; P < 0.0001) for severe or documented symptomatic hypoglycaemia. At the follow-up visit the mean difference in estimated glomerular filtration rate (eGFR) between arms was 6.05 mL min-1 per 1.73 m2 (95% CI, 3.24, 8.87; P < 0.0001). CONCLUSIONS: Bexagliflozin was noninferior to glimepiride in lowering HbA1c, was superior to glimepiride for decreases in body mass and SBP, and was associated with significantly fewer hypoglycaemic events than glimepiride. A favourable effect on eGFR was observed.

EDP-514 in healthy subjects and nucleos(t)ide reverse transcriptase inhibitor-suppressed patients with chronic hepatitis B.

BACKGROUND: Chronic hepatitis B (CHB) remains a major cause of morbidity and mortality. EDP-514 is a potent core inhibitor of hepatitis B virus (HBV) that reduces viral load reduction in HBV-infected chimeric mice. This first-in-human study evaluated the safety, tolerability, and pharmacokinetics (PK) of EDP-514 in healthy subjects and antiviral activity in patients with CHB. METHODS: In Part 1, 82 subjects received placebo or EDP-514 in fed or fasted state as single ascending doses of 50-800 mg and multiple ascending doses of 200-800 mg for 14 days. In Part 2, 24 HBV DNA-suppressed, nucleos(t)ide (NUC)-treated (i.e., NUC-suppressed) CHB patients received EDP-514 200-800 mg or placebo for 28 days. RESULTS: EDP-514 was well tolerated in healthy subjects and CHB patients with most adverse events of mild intensity. In Part 1, EDP-514 exposure increased in an approximately dose proportional manner up to 600 mg after single doses and up to 400 mg after 14-day dosing. In Part 2, EDP-514 exposure increased linearly with dose on Day 1 and Day 28, with some accumulation for Day 28 and median trough concentrations (Ctrough) approximately 20-fold above the protein-adjusted 50% effective concentration (EC50) for the dose range. Mean change in HBV RNA from baseline to Day 28 was -2.03, -1.67, -1.87, and -0.58 log U/mL in the 200 mg, 400 mg, 800 mg, and placebo CHB groups, respectively. CONCLUSIONS: EDP-514 was well tolerated, had a PK profile supporting once daily dosing, and reduced HBV RNA levels in NUC-suppressed CHB patients.

Discovery and Optimization of nTZDpa as an Antibiotic Effective Against Bacterial Persisters.

Conventional antibiotics are not effective in treating infections caused by drug-resistant or persistent nongrowing bacteria, creating a dire need for the development of new antibiotics. We report that the small molecule nTZDpa, previously characterized as a nonthiazolidinedione peroxisome proliferator-activated receptor gamma partial agonist, kills both growing and persistent Staphylococcus aureus cells by lipid bilayer disruption. S. aureus exhibited no detectable development of resistance to nTZDpa, and the compound acted synergistically with aminoglycosides. We improved both the potency and selectivity of nTZDpa against MRSA membranes compared to mammalian membranes by leveraging synthetic chemistry guided by molecular dynamics simulations. These studies provide key insights into the design of selective and potent membrane-active antibiotics effective against bacterial persisters.

A new class of synthetic retinoid antibiotics effective against bacterial persisters.

A challenge in the treatment of Staphylococcus aureus infections is the high prevalence of methicillin-resistant S. aureus (MRSA) strains and the formation of non-growing, dormant 'persister' subpopulations that exhibit high levels of tolerance to antibiotics and have a role in chronic or recurrent infections. As conventional antibiotics are not effective in the treatment of infections caused by such bacteria, novel antibacterial therapeutics are urgently required. Here we used a Caenorhabditis elegans-MRSA infection screen to identify two synthetic retinoids, CD437 and CD1530, which kill both growing and persister MRSA cells by disrupting lipid bilayers. CD437 and CD1530 exhibit high killing rates, synergism with gentamicin, and a low probability of resistance selection. All-atom molecular dynamics simulations demonstrated that the ability of retinoids to penetrate and embed in lipid bilayers correlates with their bactericidal ability. An analogue of CD437 was found to retain anti-persister activity and show an improved cytotoxicity profile. Both CD437 and this analogue, alone or in combination with gentamicin, exhibit considerable efficacy in a mouse model of chronic MRSA infection. With further development and optimization, synthetic retinoids have the potential to become a new class of antimicrobials for the treatment of Gram-positive bacterial infections that are currently difficult to cure.

Tribbles ortholog NIPI-3 and bZIP transcription factor CEBP-1 regulate a Caenorhabditis elegans intestinal immune surveillance pathway.

BACKGROUND: Many pathogens secrete toxins that target key host processes resulting in the activation of immune pathways. The secreted Pseudomonas aeruginosa toxin Exotoxin A (ToxA) disrupts intestinal protein synthesis, which triggers the induction of a subset of P. aeruginosa-response genes in the nematode Caenorhabditis elegans. RESULTS: We show here that one ToxA-induced C. elegans gene, the Tribbles pseudokinase ortholog nipi-3, is essential for host survival following exposure to P. aeruginosa or ToxA. We find that NIPI-3 mediates the post-developmental expression of intestinal immune genes and proteins and primarily functions in parallel to known immune pathways, including p38 MAPK signaling. Through mutagenesis screening, we identify mutants of the bZIP C/EBP transcription factor cebp-1 that suppress the hypersusceptibility defects of nipi-3 mutants. CONCLUSIONS: NIPI-3 is a negative regulator of CEBP-1, which in turn negatively regulates protective immune mechanisms. This pathway represents a previously unknown innate immune signaling pathway in intestinal epithelial cells that is involved in the surveillance of cellular homeostasis. Because NIPI-3 and CEBP-1 are also essential for C. elegans development, NIPI-3 is analogous to other key innate immune signaling molecules such as the Toll receptors in Drosophila that have an independent role during development.

A microRNA program in the C. elegans hypodermis couples to intestinal mTORC2/PQM-1 signaling to modulate fat transport.

Animals integrate metabolic, developmental, and environmental information before committing key resources to reproduction. In Caenorhabditis elegans, adult animals transport fat from intestinal cells to the germline to promote reproduction. We identified a microRNA (miRNA)-regulated developmental timing pathway that functions in the hypodermis to nonautonomously coordinate the mobilization of intestinal fat stores to the germline upon initiation of adulthood. This developmental timing pathway, which is controlled by the lin-4 and let-7 miRNAs, engages mTOR signaling in the intestine. The intestinal signaling component is specific to mTORC2 and functions in parallel to the insulin pathway to modulate the activity of the serum/glucocorticoid-regulated kinase (SGK-1). Surprisingly, SGK-1 functions independently of DAF-16/FoxO; instead, SGK-1 promotes the cytoplasmic localization of the PQM-1 transcription factor, which antagonizes intestinal fat mobilization at the transcriptional level when localized to the nucleus. These results revealed that a non-cell-autonomous developmental input regulates intestinal fat metabolism by engaging mTORC2 signaling to promote the intertissue transport of fat reserves from the soma to the germline.

NH125 kills methicillin-resistant Staphylococcus aureus persisters by lipid bilayer disruption.

BACKGROUND: NH125, a known WalK inhibitor kills MRSA persisters. However, its precise mode of action is still unknown. METHODS & RESULTS: The mode of action of NH125 was investigated by comparing its spectrum of antimicrobial activity and its effects on membrane permeability and giant unilamellar vesicles (GUVs) with walrycin B, a WalR inhibitor and benzyldimethylhexadecylammonium chloride (16-BAC), a cationic surfactant. NH125 killed persister cells of a variety of Staphylococcus aureus strains. Similar to 16-BAC, NH125 killed MRSA persisters by inducing rapid membrane permeabilization and caused the rupture of GUVs, whereas walrycin B did not kill MRSA persisters or induce membrane permeabilization and did not affect GUVs. CONCLUSION: NH125 kills MRSA persisters by interacting with and disrupting membranes in a detergent-like manner.

Aberrant Activation of p38 MAP Kinase-Dependent Innate Immune Responses Is Toxic to Caenorhabditis elegans.

Inappropriate activation of innate immune responses in intestinal epithelial cells underlies the pathophysiology of inflammatory disorders of the intestine. Here we examine the physiological effects of immune hyperactivation in the intestine of the nematode Caenorhabditis elegans. We previously identified an immunostimulatory xenobiotic that protects C. elegans from bacterial infection by inducing immune effector expression via the conserved p38 MAP kinase pathway, but was toxic to nematodes developing in the absence of pathogen. To investigate a possible connection between the toxicity and immunostimulatory properties of this xenobiotic, we conducted a forward genetic screen for C. elegans mutants that are resistant to the deleterious effects of the compound, and identified five toxicity suppressors. These strains contained hypomorphic mutations in each of the known components of the p38 MAP kinase cassette (tir-1, nsy-1, sek-1, and pmk-1), demonstrating that hyperstimulation of the p38 MAPK pathway is toxic to animals. To explore mechanisms of immune pathway regulation in C. elegans, we conducted another genetic screen for dominant activators of the p38 MAPK pathway, and identified a single allele that had a gain-of-function (gf) mutation in nsy-1, the MAP kinase kinase kinase that acts upstream of p38 MAPK pmk-1. The nsy-1(gf) allele caused hyperinduction of p38 MAPK PMK-1-dependent immune effectors, had greater levels of phosphorylated p38 MAPK, and was more resistant to killing by the bacterial pathogen Pseudomonas aeruginosa compared to wild-type controls. In addition, the nsy-1(gf) mutation was toxic to developing animals. Together, these data suggest that the activity of the MAPKKK NSY-1 is tightly regulated as part of a physiological mechanism to control p38 MAPK-mediated innate immune hyperactivation, and ensure cellular homeostasis in C. elegans.

Antibacterial properties of 3-(phenylsulfonyl)-2-pyrazinecarbonitrile.

The emergence of multidrug-resistant bacterial strains has heightened the need for new antimicrobial agents based on novel chemical scaffolds that are able to circumvent current modes of resistance. We recently developed a whole-animal drug-screening methodology in pursuit of this goal and now report the discovery of 3-(phenylsulfonyl)-2-pyrazinecarbonitrile (PSPC) as a novel antibacterial effective against resistant nosocomial pathogens. The minimum inhibitory concentrations (MIC) of PSPC against Staphylococcus aureus and Enterococcus faecium were 4 µg/mL and 8 µg/mL, respectively, whereas the MICs were higher against the Gram-negative bacteria Klebsiella pneumoniae (64 µg/mL), Acinetobacter baumannii (32 µg/mL), Pseudomonas aeruginosa (>64 µg/mL), and Enterobacter spp. (>64 µg/mL). However, co-treatment of PSPC with the efflux pump inhibitor phenylalanine arginyl ß-naphthylamide (PAßN) or with sub-inhibitory concentrations of the lipopeptide antibiotic polymyxin B reduced the MICs of PSPC against the Gram-negative strains by >4-fold. A sulfide analog of PSPC (PSPC-1S) showed no antibacterial activity, whereas the sulfoxide analog (PSPC-6S) showed identical activity as PSPC across all strains, confirming structure-dependent activity for PSPC and suggesting a target-based mechanism of action. PSPC displayed dose dependent toxicity to both Caenorhabditis elegans and HEK-293 mammalian cells, culminating with a survival rate of 16% (100 µg/mL) and 8.5% (64 µg/mL), respectively, at the maximum tested concentration. However, PSPC did not result in hemolysis of erythrocytes, even at a concentration of 64 µg/mL. Together these results support PSPC as a new chemotype suitable for further development of new antibiotics against Gram-positive and Gram-negative bacteria.

A Defensin from the Model Beetle Tribolium castaneum Acts Synergistically with Telavancin and Daptomycin against Multidrug Resistant Staphylococcus aureus.

The red flour beetle Tribolium castaneum is a common insect pest and has been established as a model beetle to study insect development and immunity. This study demonstrates that defensin 1 from T. castaneum displays in vitro and in vivo antimicrobial activity against drug resistant Staphylococcus aureus strains. The minimum inhibitory concentration (MIC) of defensin 1 against 11 reference and clinical staphylococcal isolates was between 16-64 µg/ml. The putative mode of action of the defensin peptide is disruption of the bacterial cell membrane. The antibacterial activity of defensin 1 was attenuated by salt concentrations of 1.56 mM and 25 mM for NaCl and CaCl2 respectively. Treatment of defensin 1 with the reducing agent dithiothreitol (DTT) at concentrations 1.56 to 3.13 mM abolished the antimicrobial activity of the peptide. In the presence of subinhibitory concentrations of antibiotics that also target the bacterial cell envelope such as telavancin and daptomycin, the MIC of the peptide was as low as 1 µg/ml. Moreover, when tested against an S. aureus strain that was defective in D-alanylation of the cell wall, the MIC of the peptide was 0.5 µg/ml. Defensin 1 exhibited no toxicity against human erythrocytes even at 400 µg/ml. The in vivo activity of the peptide was validated in a Caenorhabditis elegans-MRSA liquid infection assay. These results suggest that defensin 1 behaves similarly to other cationic AMPs in its mode of action against S. aureus and that the activity of the peptide can be enhanced in combination with other antibiotics with similar modes of action or with compounds that have the ability to decrease D-alanylation of the bacterial cell wall.

Identification of an Antimicrobial Agent Effective against Methicillin-Resistant Staphylococcus aureus Persisters Using a Fluorescence-Based Screening Strategy.

Persisters are a subpopulation of normal bacterial cells that show tolerance to conventional antibiotics. Persister cells are responsible for recalcitrant chronic infections and new antibiotics effective against persisters would be a major development in the treatment of these infections. Using the reporter dye SYTOX Green that only stains cells with permeabilized membranes, we developed a fluorescence-based screening assay in a 384-well format for identifying compounds that can kill methicillin-resistant Staphylococcus aureus (MRSA) persisters. The assay proved robust and suitable for high throughput screening (Z`-factor: >0.7). In screening a library of hits from a previous screen, which identified compounds that had the ability to block killing of the nematode Caenorhabditis by MRSA, we discovered that the low molecular weight compound NH125, a bacterial histidine kinase inhibitor, kills MRSA persisters by causing cell membrane permeabilization, and that 5 µg/mL of the compound can kill all cells to the limit of detection in a 108 CFU/mL culture of MRSA persisters within 3h. Furthermore, NH125 disrupts 50% of established MRSA biofilms at 20 µg/mL and completely eradicates biofilms at 160 µg/mL. Our results suggest that the SYTOX Green screening assay is suitable for large-scale projects to identify small molecules effective against MRSA persisters and should be easily adaptable to a broad range of pathogens that form persisters. Since NH125 has strong bactericidal properties against MRSA persisters and high selectivity to bacteria, we believe NH125 is a good anti-MRSA candidate drug that should be further evaluated.

Repurposing salicylanilide anthelmintic drugs to combat drug resistant Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive bacterium that has become the leading cause of hospital acquired infections in the US. Repurposing Food and Drug Administration (FDA) approved drugs for antimicrobial therapy involves lower risks and costs compared to de novo development of novel antimicrobial agents. In this study, we examined the antimicrobial properties of two commercially available anthelmintic drugs. The FDA approved drug niclosamide and the veterinary drug oxyclozanide displayed strong in vivo and in vitro activity against methicillin resistant S. aureus (minimum inhibitory concentration (MIC): 0.125 and 0.5 µg/ml respectively; minimum effective concentration: ≤ 0.78 µg/ml for both drugs). The two drugs were also effective against another Gram-positive bacteria Enterococcus faecium (MIC 0.25 and 2 µg/ml respectively), but not against the Gram-negative species Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter aerogenes. The in vitro antimicrobial activity of niclosamide and oxyclozanide were determined against methicillin, vancomycin, linezolid or daptomycin resistant S. aureus clinical isolates, with MICs at 0.0625-0.5 and 0.125-2 µg/ml for niclosamide and oxyclozanide respectively. A time-kill study demonstrated that niclosamide is bacteriostatic, whereas oxyclozanide is bactericidal. Interestingly, oxyclozanide permeabilized the bacterial membrane but neither of the anthelmintic drugs exhibited demonstrable toxicity to sheep erythrocytes. Oxyclozanide was non-toxic to HepG2 human liver carcinoma cells within the range of its in vitro MICs but niclosamide displayed toxicity even at low concentrations. These data show that the salicylanilide anthelmintic drugs niclosamide and oxyclozanide are suitable candidates for mechanism of action studies and further clinical evaluation for treatment of staphylococcal infections.

A new antibiotic with potent activity targets MscL.

The growing problem of antibiotic-resistant bacteria is a major threat to human health. Paradoxically, new antibiotic discovery is declining, with most of the recently approved antibiotics corresponding to new uses for old antibiotics or structurally similar derivatives of known antibiotics. We used an in silico approach to design a new class of nontoxic antimicrobials for the bacteria-specific mechanosensitive ion channel of large conductance, MscL. One antimicrobial of this class, compound 10, is effective against methicillin-resistant Staphylococcus aureus with no cytotoxicity in human cell lines at the therapeutic concentrations. As predicted from in silico modeling, we show that the mechanism of action of compound 10 is at least partly dependent on interactions with MscL. Moreover we show that compound 10 cured a methicillin-resistant S. aureus infection in the model nematode Caenorhabditis elegans. Our work shows that compound 10, and other drugs that target MscL, are potentially important therapeutics against antibiotic-resistant bacterial infections.

Insect-derived cecropins display activity against Acinetobacter baumannii in a whole-animal high-throughput Caenorhabditis elegans model.

The rise of multidrug-resistant Acinetobacter baumannii and a concomitant decrease in antibiotic treatment options warrants a search for new classes of antibacterial agents. We have found that A. baumannii is pathogenic and lethal to the model host organism Caenorhabditis elegans and have exploited this phenomenon to develop an automated, high-throughput, high-content screening assay in liquid culture that can be used to identify novel antibiotics effective against A. baumannii. The screening assay involves coincubating C. elegans with A. baumannii in 384-well plates containing potential antibacterial compounds. At the end of the incubation period, worms are stained with a dye that stains only dead animals, and images are acquired using automated microscopy and then analyzed using an automated image analysis program. This robust assay yields a Z' factor consistently greater than 0.7. In a pilot experiment to test the efficacy of the assay, we screened a small custom library of synthetic antimicrobial peptides (AMPs) that were synthesized using publicly available sequence data and/or transcriptomic data from immune-challenged insects. We identified cecropin A and 14 other cecropin or cecropin-like peptides that were able to enhance C. elegans survival in the presence of A. baumannii. Interestingly, one particular hit, BR003-cecropin A, a cationic peptide synthesized by the mosquito Aedes aegypti, showed antibiotic activity against a panel of Gram-negative bacteria and exhibited a low MIC (5 µg/ml) against A. baumannii. BR003-cecropin A causes membrane permeability in A. baumannii, which could be the underlying mechanism of its lethality.

On the Mechanism of Berberine-INF55 (5-Nitro-2-phenylindole) Hybrid Antibacterials.

Berberine-INF55 hybrids are a promising class of antibacterials that combine berberine and the NorA multidrug resistance pump inhibitor INF55 (5-nitro-2-phenylindole) together in one molecule via a chemically stable linkage. Previous studies demonstrated the potential of these compounds for countering efflux-mediated antibacterial drug resistance but they didn't establish whether the compounds function as originally intended, i.e. with the berberine moiety providing antibacterial activity and the attached INF55 component independently blocking multidrug resistance pumps, thereby enhancing the activity of berberine by reducing its efflux. We hypothesised that if the proposed mechanism is correct, then hybrids carrying more potent INF55 pump inhibitor structures should show enhanced antibacterial effects relative to those bearing weaker inhibitors. Two INF55 analogues showing graded reductions in NorA inhibitory activity compared with INF55 were identified and their corresponding berberine-INF55 hybrids carrying equivalent INF55 inhibitor structures synthesised. Multiple assays comparing the antibacterial effects of the hybrids and their corresponding berberine-INF55 analogue combinations showed that the three hybrids all show very similar activities, leading us to conclude that the antibacterial mechanism(s) of berberine-INF55 hybrids is different from berberine-INF55 combinations.

The evolutionarily conserved mediator subunit MDT-15/MED15 links protective innate immune responses and xenobiotic detoxification.

Metazoans protect themselves from environmental toxins and virulent pathogens through detoxification and immune responses. We previously identified a small molecule xenobiotic toxin that extends survival of Caenorhabditis elegans infected with human bacterial pathogens by activating the conserved p38 MAP kinase PMK-1 host defense pathway. Here we investigate the cellular mechanisms that couple activation of a detoxification response to innate immunity. From an RNAi screen of 1,420 genes expressed in the C. elegans intestine, we identified the conserved Mediator subunit MDT-15/MED15 and 28 other gene inactivations that abrogate the induction of PMK-1-dependent immune effectors by this small molecule. We demonstrate that MDT-15/MED15 is required for the xenobiotic-induced expression of p38 MAP kinase PMK-1-dependent immune genes and protection from Pseudomonas aeruginosa infection. We also show that MDT-15 controls the induction of detoxification genes and functions to protect the host from bacteria-derived phenazine toxins. These data define a central role for MDT-15/MED15 in the coordination of xenobiotic detoxification and innate immune responses.

High-throughput screening for novel anti-infectives using a C. elegans pathogenesis model.

In recent history, the nematode Caenorhabditis elegans has provided a compelling platform for the discovery of novel antimicrobial drugs. In this protocol, we present an automated, high-throughput C. elegans pathogenesis assay, which can be used to screen for anti-infective compounds that prevent nematodes from dying due to Pseudomonas aeruginosa. New antibiotics identified from such screens would be promising candidates for treatment of human infections, and also can be used as probe compounds to identify novel targets in microbial pathogenesis or host immunity.

Stimulation of host immune defenses by a small molecule protects C. elegans from bacterial infection.

The nematode Caenorhabditis elegans offers currently untapped potential for carrying out high-throughput, live-animal screens of low molecular weight compound libraries to identify molecules that target a variety of cellular processes. We previously used a bacterial infection assay in C. elegans to identify 119 compounds that affect host-microbe interactions among 37,214 tested. Here we show that one of these small molecules, RPW-24, protects C. elegans from bacterial infection by stimulating the host immune response of the nematode. Using transcriptome profiling, epistasis pathway analyses with C. elegans mutants, and an RNAi screen, we show that RPW-24 promotes resistance to Pseudomonas aeruginosa infection by inducing the transcription of a remarkably small number of C. elegans genes (∼1.3% of all genes) in a manner that partially depends on the evolutionarily-conserved p38 MAP kinase pathway and the transcription factor ATF-7. These data show that the immunostimulatory activity of RPW-24 is required for its efficacy and define a novel C. elegans-based strategy to identify compounds with activity against antibiotic-resistant bacterial pathogens.