Design, synthesis, and biological evaluation of PqsR antagonists guided by classic hit-to- lead optimisation process and fragment- based methods for the treatment of Pseudomonas aeruginosa infections

Pseudomonas aeruginosa (P. aeruginosa) a nosocomial pathogen, has become a serious public health threat due to its high mortality rates and serious antibiotic resistance issue. The Pseudomonas quinolone signal (pqs) system of P. aeruginosa is essential in regulating the biosynthesis of virulence factors. The transcriptional regulator of pqs system PqsR has been regarded as an interesting research topic for the treatment of P. aeruginosa infections. This thesis is focused on using multiple hit-to-lead optimization methods to find novel PqsR antagonists to overcome P. aeruginosa infections. Chapter 1 provides background information about P. aeruginosa pathogenicity, the pqs system and current progress towards finding PqsR antagonists. An overview of fragment-based lead discovery (FBLD) including hit identification, fragment library construction, biophysical methods and hit-to-lead evolution methods is also provided. Chapter 2 describes a classic hit-to-lead optimisation process starting from the virtual screening of an in-house compound library against PqsR protein to obtain 19. Compound 19 displayed good hit likeness and was subjected to hit-to-lead optimization to achieve a potent drug sized PqsR antagonist 69 with IC50 values of 0.25 μM and 0.34 μM in PAO1-LmCTX::PpqsA-lux and PA14mCTX::PpqsA-lux reporter assays respectively. The X-ray crystal structure of the 69-PqsR LBD complex was also obtained, which provides insights into specific ligand-target interactions. Chapter 3 focuses on fragment-based methods in the discovery of PqsR antagonists. Assisted by in silico methods, five fragment libraries were screened against PqsR protein and the high scoring fragments were subjected to a thermal shift assay (TSA) to give fragment hits 106, 107. Through hit exploration study, fragments 106, 107 were optimised and led to the identification of fragments 145a, 145c and 146b displaying improved biophysical profiles and these fragments can act as good starting points for the identification drug-sized PqsR antagonists (350 < MWt < 500). Chapter 4 demonstrates the evolution of fragment hits 106, 149, 145a, 145c and 146b to drug-sized molecules through fragment linking, merging, and growing methods. Applying a fragment growing method on 106 led to the discovery of 148b and 148c displaying pqs inhibition observed as remaining activity (RA%) values of 60% and 63% at 50 μM screening concentration in PAO1-LmCTX::PpqsA-lux reporter assays, respectively. Linking fragment 146b and 152a led to the discovery of compound 154b showing a RA% value of 34% at 10 μM screening concentration. It was hypothesized that two fragments bound to the PqsR LBD in different sub-pockets can functionalize as synergistic combinations observed as the fragment cocktails displaying a greater effect in bioreporter assay and biophysical experiments than the single fragments. A synergistic exploration experiment was designed assisted by TSA and mCTX::PpqsA-lux based bioreporter assay and led to the identification of two pairs of synergistic combinations (81 and 108, 81 and 105) showing improved in vitro or biophysical profiles in combination than in single fragments

Design, synthesis, and biological evaluation of PqsR antagonists guided by classic hit-to- lead optimisation process and fragment- based methods for the treatment of Pseudomonas aeruginosa infections

Pseudomonas aeruginosa (P. aeruginosa) a nosocomial pathogen, has become a serious public health threat due to its high mortality rates and serious antibiotic resistance issue. The Pseudomonas quinolone signal (pqs) system of P. aeruginosa is essential in regulating the biosynthesis of virulence factors. The transcriptional regulator of pqs system PqsR has been regarded as an interesting research topic for the treatment of P. aeruginosa infections. This thesis is focused on using multiple hit-to-lead optimization methods to find novel PqsR antagonists to overcome P. aeruginosa infections. Chapter 1 provides background information about P. aeruginosa pathogenicity, the pqs system and current progress towards finding PqsR antagonists. An overview of fragment-based lead discovery (FBLD) including hit identification, fragment library construction, biophysical methods and hit-to-lead evolution methods is also provided. Chapter 2 describes a classic hit-to-lead optimisation process starting from the virtual screening of an in-house compound library against PqsR protein to obtain 19. Compound 19 displayed good hit likeness and was subjected to hit-to-lead optimization to achieve a potent drug sized PqsR antagonist 69 with IC50 values of 0.25 μM and 0.34 μM in PAO1-LmCTX::PpqsA-lux and PA14mCTX::PpqsA-lux reporter assays respectively. The X-ray crystal structure of the 69-PqsR LBD complex was also obtained, which provides insights into specific ligand-target interactions. Chapter 3 focuses on fragment-based methods in the discovery of PqsR antagonists. Assisted by in silico methods, five fragment libraries were screened against PqsR protein and the high scoring fragments were subjected to a thermal shift assay (TSA) to give fragment hits 106, 107. Through hit exploration study, fragments 106, 107 were optimised and led to the identification of fragments 145a, 145c and 146b displaying improved biophysical profiles and these fragments can act as good starting points for the identification drug-sized PqsR antagonists (350 < MWt < 500). Chapter 4 demonstrates the evolution of fragment hits 106, 149, 145a, 145c and 146b to drug-sized molecules through fragment linking, merging, and growing methods. Applying a fragment growing method on 106 led to the discovery of 148b and 148c displaying pqs inhibition observed as remaining activity (RA%) values of 60% and 63% at 50 μM screening concentration in PAO1-LmCTX::PpqsA-lux reporter assays, respectively. Linking fragment 146b and 152a led to the discovery of compound 154b showing a RA% value of 34% at 10 μM screening concentration. It was hypothesized that two fragments bound to the PqsR LBD in different sub-pockets can functionalize as synergistic combinations observed as the fragment cocktails displaying a greater effect in bioreporter assay and biophysical experiments than the single fragments. A synergistic exploration experiment was designed assisted by TSA and mCTX::PpqsA-lux based bioreporter assay and led to the identification of two pairs of synergistic combinations (81 and 108, 81 and 105) showing improved in vitro or biophysical profiles in combination than in single fragments

Paternal Smoking and Risk of Childhood Acute Lymphoblastic Leukemia: Systematic Review and Meta-Analysis

Objective. To investigate the association between paternal smoking and childhood acute lymphoblastic leukemia (ALL). Method. We identified 18 published epidemiologic studies that reported data on both paternal smoking and childhood ALL risk. We performed a meta-analysis and analyzed dose-response relationships on ALL risk for smoking during preconception, during pregnancy, after birth, and ever smoking. Results. The summary odds ratio (OR) of childhood ALL associated with paternal smoking was 1.11 (95% Confidence Interval (CI): 1.05–1.18, I2 = 18%) during any time period, 1.25 (95% CI: 1.08–1.46, I2 = 53%) preconception; 1.24 (95% CI: 1.07–1.43, I2 = 54%) during pregnancy, and 1.24 (95% CI: 0.96–1.60, I2 = 64%) after birth, with a dose-response relationship between childhood ALL and paternal smoking preconception or after birth. Conclusion. The evidence supports a positive association between childhood ALL and paternal ever smoking and at each exposure time period examined. Future epidemiologic studies should assess paternal smoking during well-defined exposure windows and should include biomarkers to assess smoking exposure and toxicological mechanisms

Elongated Physiological Structure Segmentation via Spatial and Scale Uncertainty-aware Network

Robust and accurate segmentation for elongated physiological structures is challenging, especially in the ambiguous region, such as the corneal endothelium microscope image with uneven illumination or the fundus image with disease interference. In this paper, we present a spatial and scale uncertainty-aware network (SSU-Net) that fully uses both spatial and scale uncertainty to highlight ambiguous regions and integrate hierarchical structure contexts. First, we estimate epistemic and aleatoric spatial uncertainty maps using Monte Carlo dropout to approximate Bayesian networks. Based on these spatial uncertainty maps, we propose the gated soft uncertainty-aware (GSUA) module to guide the model to focus on ambiguous regions. Second, we extract the uncertainty under different scales and propose the multi-scale uncertainty-aware (MSUA) fusion module to integrate structure contexts from hierarchical predictions, strengthening the final prediction. Finally, we visualize the uncertainty map of final prediction, providing interpretability for segmentation results. Experiment results show that the SSU-Net performs best on cornea endothelial cell and retinal vessel segmentation tasks. Moreover, compared with counterpart uncertainty-based methods, SSU-Net is more accurate and robust

Preparation of modified whey protein isolate with gum acacia by ultrasound maillard reaction

peer-reviewedEffect of ultrasound treatment on whey protein isolate (WPI)-gum Acacia (GA) conjugation via Maillard reaction was investigated. And the physicochemical properties of the conjugates obtained by ultrasound treatment were compared with those obtained by classical heating. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-performance size exclusion chromatography and fourier transform infrared spectroscopy provided evidence on the formation of the Maillard type conjugation. Compared with classical heating, ultrasound treatment could accelerate the glycation reaction between WPI and GA. A degree of graft of 11.20% was reached by classical heating for 48 h, whereas only 20 min was required by ultrasound treatment. Structural analyses suggested that the conjugates obtained by ultrasound treatment had less α-helix content, higher surface hydrophobicity and fluorescence intensity than those obtained by classical heating. Significantly lower level of browning intensity and significantly higher (p < 0.05) level of solubility (under alkaline conditions), thermal stability, emulsifying activity and emulsifying stability were observed for the conjugates obtained by ultrasound treatment as compared with those obtained by classical heating

Novel quinazolinone inhibitors of the Pseudomonas aeruginosa quorum sensing transcriptional regulator PqsR

© 2020 The Authors Rising numbers of cases of multidrug- and extensively drug-resistant Pseudomonas aeruginosa over recent years have created an urgent need for novel therapeutic approaches to cure potentially fatal infections. One such approach is virulence attenuation where anti-virulence compounds, designed to reduce pathogenicity without affording bactericidal effects, are employed to treat infections. P. aeruginosa uses the pqs quorum sensing (QS) system, to coordinate the expression of a large number of virulence determinants as well as bacterial-host interactions and hence represents an excellent anti-virulence target. We report the synthesis and identification of a new series of thiazole-containing quinazolinones capable of inhibiting PqsR, the transcriptional regulator of the pqs QS system. The compounds demonstrated high potency (IC50 < 300 nM) in a whole-cell assay, using a mCTX:PpqsA-lux-based bioreporter for the P. aeruginosa PAO1-L and PA14 strains. Structural evaluation defined the binding modes of four analogues in the ligand-binding domain of PqsR through X-ray crystallography. Further work showed the ability of 6-chloro-3((2-pentylthiazol-4-yl)methyl)quinazolin-4(3H)-one (18) and 6-chloro-3((2-hexylthiazol-4-yl)methyl)quinazolin-4(3H)-one (19) to attenuate production of the PqsR-regulated virulence factor pyocyanin. Compounds 18 and 19 showed a low cytotoxic profile in the A549 human epithelial lung cell line making them suitable candidates for further pre-clinical evaluation

Restaurant and Bar Owners’ Exposure to Secondhand Smoke and Attitudes Regarding Smoking Bans in Five Chinese Cities

Despite the great progress made towards smoke-free environments, only 9% of countries worldwide mandate smoke-free restaurants and bars. Smoking was generally not regulated in restaurants and bars in China before 2008. This study was designed to examine the public attitudes towards banning smoking in these places in China. A convenience sample of 814 restaurants and bars was selected in five Chinese cities and all owners of these venues were interviewed in person by questionnaire in 2007. Eighty six percent of current nonsmoking subjects had at least one-day exposure to secondhand smoke (SHS) at work in the past week. Only 51% of subjects knew SHS could cause heart disease. Only 17% and 11% of subjects supported prohibiting smoking completely in restaurants and in bars, respectively, while their support for restricting smoking to designated areas was much higher. Fifty three percent of subjects were willing to prohibit or restrict smoking in their own venues. Of those unwilling to do so, 82% thought smoking bans would reduce revenue, and 63% thought indoor air quality depended on ventilation rather than smoking bans. These results showed that there was support for smoking bans among restaurant or bar owners in China despite some knowledge gaps. To facilitate smoking bans in restaurants and bars, it is important to promote health education on specific hazards of SHS, provide country-specific evidence on smoking bans and hospitality revenues, and disseminate information that restricting smoking and ventilation alone cannot eliminate SHS hazards

Analysis of Astringent Components and Differential Gene Expression of Related Key Enzymes in Different Tissues of Fresh Lotus Seeds

This study was performed to explore the differences in the composition of astringent substances in different tissues of fresh lotus seeds and the expression of key enzyme genes involved in the synthesis of astringent compounds. The contents of soluble tannins, insoluble tannins and proanthocyanidins in lotus seed coat, lotus seed pulp without coat and lotus seed plumule were determined. Meanwhile, the activity and gene expression of key enzymes in the tannin synthesis pathway were detected. Sensory evaluation and electronic tongue analysis were performed on lotus seed samples. The data obtained was analyzed by orthogonal partial least squares discriminant analysis (OPLS-DA) and correlation analysis. Results showed that the major substance contributing to the astringency of lotus seeds was soluble tannins. Tannins had a positive effect on its excellent flavor at the waxy ripeness stage. Anthocyanin reductase was a key enzyme affecting the astringency intensity of lotus seeds. SnANR9 played a role in regulating the astringency of different tissues of lotus seeds