31 research outputs found
Neutrophil responses to Vibrio cholerae autoinducer-1 and structural analogues
No full textVibrio cholerae is a pathogen responsible for cholera, an infectious disease that usually manifests as severe diarrhea. V. cholerae cells can regulate population-wide gene expression changes in a density-dependent manner, in a process known as quorum sensing (QS). QS involves communication between bacterial cells using secreted signalling molecules. V. cholerae autoinducer-1 (CAI-1) is the dominant signalling molecule in the V. cholerae QS circuit and has roles in regulating biofilm formation/degradation and expression of virulence genes.
Interactions between bacterial-produced QS molecules and eukaryotic cells have been documented. This is known as interkingdom or cross-kingdom signalling. CAI-1 has been reported to act as a chemoattractant for the nematode, Caenorhabditis elegans which feeds on V. cholerae cells as a food source. Legionella autoinducer-1 (LAI-1), which is structurally similar to CAI-1, is a signalling molecule produced by Legionella pneumophila. LAI-1 has been reported to impede the migration of Dictyostelium discoideum amoebae and macrophage-like RAW 264.7 cells, and has also been shown to destabilise the cytoskeleton of RAW 264.7 cells. Structural analogues of CAI-1 with more potent activity within the V. cholerae QS circuit have been developed as potential novel therapeutics against cholera. These QS agonists would force the bacterial cells to express high cell density behaviours, impairing colonisation and promoting detachment, therefore reducing pathogenesis.
These previous findings led to the hypothesis that CAI-1 and structural analogues may have immunomodulatory effects on host cells during V. cholerae infection, particularly recruited immune cells which may be exposed to CAI-1 during cholera. There are several lines of evidence the neutrophil recruitment is prominent during cholera and that these granular leukocytes play a role in controlling infection.
Thus, the key aims of this study were to characterise interactions between CAI-1, as well as structural analogues of CAI-1, and neutrophils. In vitro HL-60 cell culture revealed an upregulation of CD11b expression when cells were differentiated with DMSO in the presence of CAI-1. This increased differentiation marker expression was at the expense of both cell viability and total cell count. Additionally, two 3-acyl pyrrole analogues of CAI-1 also increased CD11b expression greater than CAI-1, when cells were differentiated with DMSO in the presence of either analogue. Again, this resulted in significantly reduced cell viability and total cell count, although at similar levels to CAI-1. HL-60 cells differentiated in the presence of CAI-1 or either analogue were generally more granular than cells differentiated with DMSO alone.
The effects of CAI-1 and structural analogues on neutrophil effector functions were assessed, namely chemotaxis and oxidative burst. CAI-1 did not act as a chemoattractant for DMSO-differentiated HL-60 cells, nor did it reduce or enhance migration towards fMLP, a known chemoattractant. Pre-treatment of differentiated HL-60 cells with CAI-1 or one of the 3-acyl pyrrole analogues for 3 h resulted in decreased reactive oxygen species production. However, concomitant reduction in cell viability was observed over 3 h.
Preliminary experiments assessed the effect of CAI-1 on primary human neutrophils. Isolated neutrophils appeared larger and less round with CAI-1 treatment. In contrast, CAI-1 treatment of whole blood resulted in apparent reductions in cell size as assessed by flow cytometry. Expression of activation markers (CD11b, CD64, CD66b) on granulocytes in whole blood appeared unaffected by CAI-1.
Overall, the results within this study shed light onto the cross-kingdom interactions that may exist between host cells and bacterial signalling molecules. Identifying these interactions may lead to a deeper understanding of additional mechanisms that may be involved in V. cholerae pathogenesis. Additionally, these interactions may be important in revealing off-target effects that developing novel therapies, which interfere with bacterial QS, may have on host cells.
These data highlight an advance that reveals many opportunities for further investigations surrounding host-microbe interactions.Thesis (Masters)Master of Medical Research (MMedRes)School of Medical ScienceGriffith HealthFull Tex
The relationship between single nucleotide polymorphisms in ARRB2, KCNJ6 and BDNF genes and methadone response for pain management in palliative care.
No full textBackground: Pain has a negative impact on cancer patients’ quality of life. It is highly prevalent within this vulnerable population, with an estimated 70 to 90% of patients with advanced cancer experiencing pain. It is the most feared symptom of advanced cancer. Opioids are recommended for moderate to severe pain in palliative care. Methadone has advantages over other opioids, but it is associated with significant interindividual variability and complex pharmacokinetic and pharmacodynamic parameters, which makes dosing challenging in practice. There is limited pharmacogenetic research on cancer pain. However, recent research on single nucleotide polymorphisms (SNPs) and pharmacodynamics has shown that SNPs contribute to interindividual variability in response to opioids. The aim of this study was to investigate the relationship between SNPs in the three genes, namely KCNJ6, BDNF and ARRB2 and their influence on interindividual variability in methadone dosing requirements for pain management in advanced cancer.
Methods: Fifty-five participants were recruited from the palliative and supportive care services at Mater Adults Hospital and St Vincent’s Private Hospital, Brisbane, in a prospective multi-centre, open labelled, dose individualisation study. Patients were prescribed varying doses of oral methadone by specialist palliative care clinicians for the management of pain. Patient characteristics were collected at baseline, with pain scores recorded using the Brief Pain Inventory, on a numerical rating scale of 0 to 10. Genotyping was conducted using pyrosequencing for both BDNF and KCNJ6 and TaqMan assays were used for ARRB2.
Results: Forty-six participants were included in the final study and received an average methadone dose of 17.7 mg. The mean pain score was 4.2 out of 10. The mean age of the population was 60.7 years. The patient characteristics measured in this study were not found to be covariates affecting methadone dose, response or pain scores. A patient was considered to be experiencing high pain if they had a pain score of > 4/10. There was a significant association between high pain scores and the following SNPs in BDNF and ARRB2: rs1491850 (p = 0.033), rs3786047 (p = 0.011), rs1045280 (p = 0.004) and rs2036657 (p = 0.05). SNPs in KCNJ6, BDNF and ARRB2 did not show significant associations with methadone dose.
Conclusion: These findings suggest that specific SNPs in BDNF and ARRB2 may play a role in methadone response and that genetics may be an important factor in interindividual variability. In the future, the SNPs in these genes could be factored into a multimodal treatment algorithm for cancer pain.Thesis (Masters)Master of Medical Research (MMedRes)School of Medical ScienceGriffith HealthFull Tex
Stereoselective Approaches toward the Synthesis of Nucleoside Antibiotic Core Aminoribosyl Glycyluridine
No full textAntibiotics that have a novel mechanism of action are urgently required for treatment of drug-resistant microorganisms. Naturally occurring nucleoside antibiotics have shown promising antibacterial activity by inhibiting bacterial translocase MraY, a key enzyme involved in catalysis of the first step of bacterial peptidoglycan biosynthesis. Despite having promising antibiotic properties, a major challenge toward development of this important class of compounds as drug candidates is their complex multistep synthesis. Specifically, efficient synthetic methodologies toward producing the aminoribosylated uridine-derived core unit in a stereo-controlled manner is seen as an essential prerequisite for detailed structure-activity relationship (SAR) studies. This review summarizes approaches available for the stereoselective synthesis of nucleoside core pharmacophores, including both 5′-C-glycyluridine (GlyU) as well as it's β-selective ribosylation.No Full Tex
Association of genetic variation in KCNJ6, ARRB2 and BDNF and fentanyl response for pain management in advanced cancer
No full textBackground:
Pain is common in cancer patients, particularly in the advanced stage of the disease when the prevalence is estimated to be more than 70%, contributing to poor physical and emotional well‐being. Despite the presence of increased attention regarding cancer research and pain management the ability of clinicians to predict and manage cancer patients’ pain has not changed significantly. A recent evidence‐based review into the pain associated polymorphisms and opioids stated that the clinical benefits of opioid analgesics are dependent on substantial variations in the responses to opioids, insufficient drug dosing and/or a high rate of adverse events. This wide interindividual variability in sensitivity to opioids leads to unpredictable clinical responses to opioid treatment and adverse events. This is troublesome to clinicians given the narrow therapeutic window in which treatment may be beneficial or harmful to the patient.
Method:
The aim of this study was to investigate how selected SNPs in KCNJ6, ARRB2 and BDNF affect fentanyl dose requirements and response. This was a sub‐study of a prospective, open label, dose individualisation study on the use of fentanyl for pain management in advanced cancer. Adult in‐patients or outpatients of the oncology and palliative care services of Mater Adults Hospital in Brisbane were eligible for inclusion. Fentanyl was administered via the transdermal route with the dose titrated according to clinical need by the palliative care specialists. Participant characteristics including age, height, weight, type of cancer, liver and renal function, and fentanyl doses were recorded. Participants were required to rate their pain on a numerical rating scale from 0 to 10, with a score of 0 representing “no pain” and 10 representing “pain as bad as you can imagine” using the Brief Pain Inventory questionnaire. Pain scores were recorded each time blood and saliva was collected for the dose individualisation study. Genotyping was conducted using pyrosequencing for KCNJ6 and BDNF, and TaqMan assays were used for ARRB2. Regression analysis was used to examine whether outcomes were dependent on nongenetic patient characteristics. Deviation of Hardy‐Weinberg equilibrium was determined by comparing the observed genotype frequencies with the expected values using the chisquare test. The Kruskal‐Wallis H test was used to determine whether genotypes were associated with fentanyl dose or pain score. 2 analysis was used to determine significant associations for low (3.0/10) pain score and low (50 mcg/hr), when outcomes were categorised.
Results:
Fifty‐six adults with advanced cancer receiving transdermal fentanyl were recruited to the study. The median fentanyl dose was 50 mcg/hr, with a range of 12 to 300 mcg/hr, and the mean pain score was 3.0/10.0 with a standard deviation of 2.3. No association was found between patient characteristics, fentanyl dose, and pain score. As each of the P values for age, gender, cancer diagnosis, BMI, BSA, weight, liver function, kidney function, and blood cell counts were above that of 0.05. KCNJ6 rs2070995, ARRB2 (rs34230287, rs3786047, rs1045280, rs2036657), and BDNF (rs7934165, rs10835210, rs1491850) were determined to have no association in relation to dose and pain score.
Conclusion:
There is so little understanding in the effect of genetics on the pharmacodynamic effect of medications in advanced cancer pain. While clinical studies in palliative care are challenging, we encourage continued research in the area to provide evidence to support clinicians in achieving better treatment outcomes and quality of life for their patients. In the case of this study, no association of SNPs in KCNJ6, ARRB2 and BDNF on fentanyl dose requirements or response was identified. However, any contribution to the evidence base will allow for continued development of gene‐drug dosing guidelines provided by the Clinical Pharmacogenetics Implementation Consortium (CPIC), thereby expediting the translation of research findings to clinical practice. This coupled with increasing technological advancement of low‐cost genetic mapping that is accessible to the general public, and the accessibility of patient electronic records, will allow gene‐based prescription of medications to become a reality.Thesis (Masters)Master of Medical Research (MMedRes)School of Pharmacy & Med SciGriffith HealthFull Tex
Investigating the Bioactivity of Pigments from the Native Australian Cortinarius Mushroom
No full textThis study investigated the bioactivity of six pigments from the Australian Cortinarius mushroom genus, specifically targeting their influence on various fungi, viruses, and an inflammatory stimulus. It was discovered that when tested between 25-26.3 [microns], (1S,3S)-Austrocortirubin [M1], (1R,3R)-Austrocortilutein [M2], (1S,3S)-Austrocortilutein [M3] and (1S,3R)-Austrocortilutein [M4] were all equally effective at inhibiting C. albicans growth. At these same concentrations, M1 and M3 effectively prevented A. fumigatus growth. It was theorised that the identical S-configured hydroxyls on these compounds' 1st and 3rd carbons were advantageous against mould growth, specifically A. fumigatus. It was also confirmed that (3R/S)-1-deoxy-Austrocortirubin [M5] and Torosachrysone [T] were significantly less effective at preventing either yeast or mould growth compared to the other Cortinarius pigments. The compounds were deemed to have no antiviral effect against either HPIV3 or RSV. When the compounds were exposed to human peripheral macrophages, their anti-inflammatory activity was shown to be minimal. Additionally, across all in vitro assays, these compounds were highly cytotoxic against LLC-MK2, HEp-2 and Human macrophage cells. Although, during cytotoxicity screening, the Compounds M2-M5 were identified as selectively toxic to the cancerous cell line (HEp-2) over a normal cell line (LLC-MK2), which could indicate a beneficial future research path.Thesis (Masters)Master of Medical ResearchSchool of Pharmacy & Med SciGriffith HealthFull Tex
An in vitro evaluation of novel strategies and compounds to target Pseudomonas aeruginosa
No full textBackground. Antimicrobial resistance is a significant global threat associated with increased mortality. Pseudomonas aeruginosa is a critically prioritised organism needing new antimicrobials with unique mechanisms. Multidrug-resistant P. aeruginosa strains are becoming increasingly burdensome and justify the need for developing new antipseudomonal antibiotics. Traditional antibiotic drug discovery strategies have yet to deliver a pipeline of novel antipseudomonal antibiotics. It has become imperative to adopt new drug discovery strategies to develop novel lead compounds before these pathogens become resistant to all clinically available antibiotics. This study investigates two important drug targets to identify novel antipseudomonal antibiotics.
The research aims and hypothesis. This project aimed to evaluate a series of novel compounds that could interfere with new drug targets in P. aeruginosa and investigate their ability to potentiate the activities of existing antipseudomonal antibiotics. The P. aeruginosa drug targets of interest in this project included the enzyme MraY and the resistance-nodulation-division efflux pumps. This study included a series of synthesised and commercially available compounds belonging to muraymycins and phenazine compound classes. It was hypothesised that inhibiting these drug targets with these compounds would significantly enhance the activities of existing antipseudomonal antibiotics irrespective of P. aeruginosa strain. [...]Thesis (PhD Doctorate)Doctor of Philosophy (PhD)School of Pharmacy & Med SciGriffith HealthFull Tex
An Investigation Toward the Synthesis of Quinolinone Analogues as 1,4-Dihydroxy-2-Napthoate Prenyltransferase (MenA) Inhibitors
No full textBacterial drug resistance is a major concern to the World Health Organisation and the future of human
health. Increasing resistance is consistently being observed to all commonly used antibiotic agents, and
more recently to the last-line antibiotic agents used when all other drugs fail. This is of major concern in
all bacterial species, but it is of special concern in deadly and infectious bacteria such as Mycobacterium
tuberculosis. This projected aimed at synthesising chemical agents which would act as probes
investigating their antibiotic activity via a new mechanism of action previously unseen in current
antibiotic agents in use today.
Target compounds were derived from a structure mimicking that of a natural product, aurachin RE,
which shows some antibiotic activity in a new mechanism of action. This mechanism of action is based
upon the inhibition of the bacterial production of menaquinone, as menaquinone is used to shuttle
electrons within its electron transport chain. This structure shows inhibitory action at a particular
enzyme step, MenA, in the biosynthetic pathway of menaquinone.
These target compounds were derived from retrosynthetic analysis, whereby the final target structures
were based on preserving the key structure activity relationships present in aurachin RE. These target
compounds will serve as probes in future research to further the understanding of the structure activity
relationships with respect to MenA inhibition to provide the groundwork for further generations of
compounds with increased inhibition and bactericidal activity compared to that of aurachin RE.Thesis (Masters)Master of Medical Research (MMedRes)School of Medical ScienceGriffith HealthFull Tex
Echiumin E, an Aryl Dihydronaphthalene Lignan from the Australian Invasive Plant Patersons Curse (Echium plantagineum)
No full textA new aryl dihydronaphthalene lignan, echiumin E (1), and four known compounds, echiumin A, globoidnan A, (-)-rabdosiin, and rosmarinic acid (2-5), were isolated from the Australian invasive plant Echium plantagineum (Paterson's curse) for the first time. Echiumin E (1) was characterized by 1D/2D NMR spectroscopy and MS spectrometry, with its absolute configuration assigned through comparison of experimental and TDDFT-calculated ECD data. Echiumin E (1) along with compounds 3-5 were screened in vitro against three cancer cell lines (SH-SY5Y, HeLa, and PC-3) and a prostate stromal (normal) cell line (WPMY-1) using a resazurin reduction assay. Echiumin E (1) was found to be active toward HeLa cells (IC50 0.21 μM).No Full Tex
Design, synthesis and biological evaluation of novel simplified muraymycins analogues
No full textBacterial resistance against clinically used antibiotics is an emerging health concern in contemporary healthcare.1 The suggested long-term solution to tackle the globally prevalent multidrug resistance is to explore new classes of antibiotics.2 Bacterial cell-wall peptidoglycan layer biosynthesis has been promising antibacterial target for decades -lactam (penicillin) and glycopeptide (vancomycin) classes of antibiotics.3 However, current cell wall inhibitors target the late extracellular steps of peptidoglycan synthesis. The early intracellular steps of peptidoglycan synthesis are not well explored clinically, therefore provide an exciting opportunity to explore the current need for novel targets.4 The transmembrane enzyme MraY (phospho-Nacetylmuramoyl-pentapeptide-transferase) is one such intracellular enzyme, which fulfils the requirement of a novel target.5 MraY catalyses the first membrane-associated step of peptidoglycan formation which involves transfer of an UDP-N-acetylmuramoyl (UDP-MurNAc) pentapeptide (-L-Ala1-D--Glu2-Lys/DAP3-D-Ala4-D-Ala5-COOH) (Park’s nucleotide) to the membrane-soluble C55 isoprenoid carrier lipid known as bactoprenol-phosphate (bactoprenol-P), resulting in the formation of lipid I (undecaprenyl-pyrophosphoryl-MurNAc-pentapeptide).6 MraY enzyme (translocase I) is the target of nucleoside antibiotics, class of natural products containing a nucleoside core structure.7 The muraymycins, belongs to the family of ribosamino-uridines class of nucleoside antibiotics, were first discovered as promising structures acting against MraY enzyme in year 2002.8 Though naturally occurring muraymycins quenched the contemporary need of novel structures acting against clinically unexplored target MraY, the challenge faced, to move forward in drug discovery, was their complex and synthetically challenging structures. To address this issue, we are investigating a bioactive, structurally simplified muraymycin analogues acting against a range of bacterial strains.No Full Tex
Design, synthesis and bioactivity evaluation of novel pyrazole linked phenylthiazole derivatives in context of antibacterial activity
No full textMethicillin-resistant Staphylococcus aureus (MRSA) infections are a significant burden both clinically and economically worldwide. Increasing resistance to current antibiotics requires an urgent investigation into novel classes of antimicrobial agents. This study presents a structure-activity relationship (SAR) rationale for pyrazole linked phenylthiazole analogues as new antibacterial agents. A library of 23 novel pyrazole linked phenylthiazole compounds were synthesised, followed by screening for antimicrobial activity against five bacterial species and two fungi. The most active compound 14b has shown promising antibacterial activity against the Gram-positive methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300) strain (MIC 4 μg/mL). Furthermore, the active pyrazole linked phenylthiazole compound exhibited a better toxicity profile than standard antibiotics. In summary, these results demonstrate that a pyrazole linked phenylthiazole scaffold has potential as a lead for further investigation to afford novel antibacterial agents.No Full Tex
