High throughput and targeted screens for prepilin peptidase inhibitors do not identify common inhibitors of eukaryotic gamma-secretase.

INTRODUCTION: Prepilin peptidases (PPP) are essential enzymes for the biogenesis of important virulence factors, such as type IV pili (T4P), type II secretion systems, and other T4P-related systems of bacteria and archaea. PPP inhibitors could be valuable pharmaceuticals, but only a few have been reported. Interestingly, PPP share similarities with presenilin enzymes from the gamma-secretase protease complex, which are linked to Alzheimer's disease. Numerous gamma-secretase inhibitors have been reported, and some have entered clinical trials, but none has been tested against PPP. OBJECTIVE: The objective of this study is to develop a high-throughput screening (HTS) method to search for inhibitors of PPP from various chemical libraries and reported gamma-secretase inhibitors. METHOD: More than 15,000 diverse compounds, including 13 reported gamma-secretase inhibitors and other reported peptidase inhibitors, were screened to identify potential PPP inhibitors. RESULTS: The authors developed a novel screening method and screened 15,869 compounds. However, the screening did not identify a PPP inhibitor. Nevertheless, the study suggests that gamma-secretase is sufficiently different from PPP that specific inhibitors may exist in a larger chemical space. CONCLUSION: The authors believe that the HTS method that they describe has numerous advantages and encourage others to consider its application in the search for PPP inhibitors.

Investigation of the biofilm formation in extra-intestinal pathogenic Escherichia coli ST131 strains and its correlation with the presence of fimH, afa, and kpsMSTII genes.

Escherichia coli sequence type 131 (ST131) is a multidrug-resistant strain with the global dissemination. Biofilm formation-related factors include the most important virulence factors in extra-intestinal pathogenic E. coli (ExPEC) ST131 strains causing infections with treatment-limited subjects. This study aims to investigate the biofilm formation ability and its correlation with the presence of fimH, afa, and kpsMSTII genes in clinical isolates of ExPEC ST131. In this regard, the prevalence and characteristics of these strains collected and evaluated. The results revealed strong, moderate, and weak attachment abilities related to biofilm formation attributes in 45%, 20%, and 35% of strains, respectively. In the meantime, the frequency of the fimH, afa, and kpsMSTII genes among the isolates was observed as follows: fimH positive: 65%; afa positive: 55%; and kpsMSTII positive: 85%. The results convey a significant different of biofilm formation ability between clinical E. coli ST131 and non-ST131 isolates. Furthermore, while 45% of ST131 isolates produced strong biofilms, only 2% of non-ST131 isolates showed the ability to form strong biofilms. The attending of fimH, afa, and kpsMSTII genes in the majority of ST131 strains demonstrated a key role leading to biofilm formation. These findings suggested the application of fimH, afa, and kpsMSTII gene suppressors for treating biofilm infections caused by drug-resistant ST131 strains.

Elucidating novel antibacterial compounds from the NPASS database against the FimH lectin domain for the treatment of urinary tract infections: an in-silico study.

The increase in multidrug-resistant pathogens in urinary tract infections (UTIs) among communities and hospitals threatens our ability to treat these common pathogens. Uropathogenic Escherichia coli (UPEC) strains are the most frequent uropathies linked to the development of UTIs. This work aims to introduce bioactive natural products via virtual screening of small molecules from a public database to prevent biofilm formation by inhibiting FimH, a type 1 fimbriae that plays a crucial role in UPEC pathogenicity. A total of 30926 small molecules from the NPASS database were subjected to screening via molecular docking. Followed by performing in silico ADME studies, seven molecules showed promising docking results ranging from -6.8 to -8.7 kcal/mol. As a result of the docking score findings, 100 ns Molecular dynamics (MD) simulations were performed. Based on MM-PBSA analysis, NPC313334 ligand showed high binding affinity -42 and stability with the binding pocket of FimH protein during molecular dynamic simulations. DFT calculations were also performed on the ligands to calculate the HOMO-LUMO energies of the compounds in order to an idea about their structure and reactivity. This research suggests that NPC313334 may be a possible antibacterial drug candidate that targets FimH to reduce the number of UPEC-related urinary tract infections.Communicated by Ramaswamy H. Sarma.

Safety, pharmacokinetic, and pharmacodynamic study of sibofimloc, a novel FimH blocker in patients with active Crohn's disease.

BACKGROUND AND AIM: Expression of FimH adhesin by invasive Escherichia coli in the gastrointestinal tract of patients with Crohn's disease (CD) facilitates binding to epithelial glycoproteins and release of pro-inflammatory cytokines. Sibofimloc is a first-in-class FimH blocker that showed little systemic absorption in healthy volunteers. The current study evaluated systemic absorption, safety, and effect on inflammatory biomarkers of sibofimloc in patients with CD. METHODS: This was an open-label, multicenter phase 1b study in adults with active CD. In part 1, two patients received a single oral dose of 3000-mg sibofimloc followed by 1500 mg b.i.d. for 13 days. In part 2, six patients received 1500-mg sibofimloc b.i.d. for 13 days. Blood was drawn for pharmacokinetic and biomarker analysis, and stool was collected for biomarker and microbiome analysis. RESULTS: Eight patients with active ileal or ileocolonic CD were enrolled into the study. Systemic sibofimloc exposure was low. Sibofimloc was well tolerated with only grade 1-2 events observed. Several pro-inflammatory biomarkers, including IL-1ß, IL-6, IL-8, TNF-α, IFN-γ, and calprotectin, were decreased in stool by end of study. CONCLUSIONS: This first study of the novel FimH blocker, sibofimloc, in patients with active CD demonstrated minimal systemic exposure with good tolerance, while decreasing several inflammatory biomarkers. EudraCT number: 2017-003279-70.

Oral therapy with colonization factor antigen I prevents development of type 1 diabetes in Non-obese Diabetic mice.

Antigen (Ag)-specific tolerization prevents type 1 diabetes (T1D) in non-obese diabetic (NOD) mice but proved less effective in humans. Several auto-Ags are fundamental to disease development, suggesting T1D etiology is heterogeneous and may limit the effectiveness of Ag-specific therapies to distinct disease endotypes. Colonization factor antigen I (CFA/I) fimbriae from Escherichia coli can inhibit autoimmune diseases in murine models by inducing bystander tolerance. To test if Ag-independent stimulation of regulatory T cells (Tregs) can prevent T1D onset, groups of NOD mice were orally treated with Lactococcus lactis (LL) expressing CFA/I. LL-CFA/I treatment beginning at 6 weeks of age reduced disease incidence by 50% (p < 0.05) and increased splenic Tregs producing both IL-10 and IFN-γ 8-fold (p < 0.005) compared to LL-vehicle treated controls. To further describe the role of these Tregs in preventing T1D, protective phenotypes were examined at different time-points. LL-CFA/I treatment suppressed splenic TNF-α+CD8+ T cells 6-fold at 11 weeks (p < 0.005) and promoted a distinct microbiome. At 17 weeks, IFN-γ+CD4+ T cells were suppressed 10-fold (p < 0.005), and at 30 weeks, pancreatic Tbet+CD4+ T cells were suppressed (p < 0.05). These results show oral delivery of modified commensal organisms, such as LL-CFA/I, may be harnessed to restrict Th1 cell-mediated immunity and protect against T1D.

Subcutaneous Injections of the Mannose-Sensitive Hemagglutination Pilus Strain of Pseudomonas aeruginosa Stimulate Host Immunity, Reduce Bladder Cancer Size and Improve Tumor Survival in Mice.

We wished to evaluate the effects of Pseudomonas aeruginosa (mannose-sensitive hemagglutination pilus strain, PA-MSHA) as an immunostimulating and anti-tumor agent for treatment of bladder cancer. Immunostimulating effects were assessed by the in vitro proliferation assay of murine splenic lymphocytes. Anti-tumor effects were studied in a subcutaneous tumor model established in female C57BL/6 mice using the MB49 bladder cell line. These mice received subcutaneous injections of normal saline (control group) or PA-MSHA (high, medium, or low dose, respectively, 1.6-2.0 × 10(9), 3.2- .0 × 10(8), 6.4-8.0 × 10(7) CFU/ml) twice a week for 3 weeks. Mice survival, tumor volume, vascular endothelial growth factor (VEGF) expression, microvessel density (MVD), serum levels of TNF-α and IFN-γ, and blood CD4(+) /CD8(+) counts were the study outcomes. We observed that PA-MSHA promoted the growth of splenic lymphocytes in vitro. In the murine tumor model, PA-MSHA prolonged mice survival and reduced tumor growth. Furthermore, VEGF and MVD were also diminished by PA-MSHA. Mice that received high and medium dose of PA-MSHA had significantly higher serum levels of IFN-γ and TNF-α (days 21 and 28), and higher levels of CD4(+) /CD8(+) cells (days 21 and 28). In conclusion, PA-MSHA exerts beneficial effects on increasing proliferation of murine splenic lymphocytes in vitro and inhibits the growth of bladder tumor in a murine model. Therefore, PA-MSHA may be useful an immunostimulating and anti-tumor agent for bladder cancer therapy.

Autotransporters and virulenceof enteroaggregative E. coli

EAEC, enteroaggregative E. coli; EPEC, enteropathogenic E. coli; ETEC, enterotoxigenic E. coli; EIEC,enteroinvasive E. coli; STEC, shiga-toxin producing E. coli; DAEC, diffusely adhering E. coli; AA, agregative adherence; OMP, outer membrane protein; AAF, aggregative adherence fimbriae; ECP, E. coli common pilus; T1SS, type I secretion system; EAST-1,enteroaggregative E. coli heat stable enterotoxin 1; ORF, open reading frame; Pet, plasmid-encoded toxin; Pic, protein involved in colonization; ShET1, Shigella enterotoxin 1; HlyE, hemolysin E; OM, outer membrane; T5SS, type V secretion system; TPS, twopartner secretion; SPATE, serine protease autotransporter of the Enterobacteriaceae; ER, endoplasmic reticulum; Shmu, Shigellamucinase; cAMP, cyclic adhenosin monophosphate; cGMP, cyclic guanosine monophosphate; STa, heat-stable toxin a; GC-C, guanylyl cyclase C; IL, interleukin; IEC, intestinal epithelial cells; MAPK, mitogen-activating protein kinase; TLR5, toll-like receptor 5; TNFá, tumour necrosis factor á; GRO, growth-related gene product; ICAM-1, intercellular adhesion molecule 1;GM-CSF, granulocyte-macrophage colony-stimulating factor.