Pharmacology and Therapeutics - Research Publications
Now showing items 1-12 of 277
Generation of MicroRNA-34 Sponges and Tough Decoys for the Heart: Developments and Challenges
(FRONTIERS MEDIA SA, 2018-09-21)
Heart failure (HF) is a debilitating and deadly chronic disease, with almost 50% of patients with HF dying within 5 years of diagnosis. With limited effective therapies to treat or cure HF, new therapies are greatly needed. microRNAs (miRNAs) are small non-coding RNA molecules that are powerful regulators of gene expression and play a key role in almost every biological process. Disruptions in miRNA gene expression has been functionally linked to numerous diseases, including cardiovascular disease. Molecular tools for manipulating miRNA activity have been developed, and there is evidence from preclinical studies demonstrating the potential of miRNAs to be therapeutic targets for cardiovascular disease. For clinical application, miRNA sponges and tough decoys have been developed for more stable suppression and targeted delivery of the miRNA of choice. The aim of this study was to generate miRNA sponges and tough decoys to target miR-34 in the mouse heart. We present data to show that using both approaches we were unable to get significant knockdown of miR-34 or regulate miR-34 target genes in the heart in vivo. We also review recent applications of this method in the heart and discuss further considerations for optimisation in construct design and testing, and the obstacles to be overcome before they enter the clinic.
Role of the Sympathetic Nervous System and Its Modulation in Renal Hypertension
(FRONTIERS MEDIA SA, 2018-03-29)
The kidneys are densely innervated with renal efferent and afferent nerves to communicate with the central nervous system. Innervation of major structural components of the kidneys, such as blood vessels, tubules, the pelvis, and glomeruli, forms a bidirectional neural network to relay sensory and sympathetic signals to and from the brain. Renal efferent nerves regulate renal blood flow, glomerular filtration rate, tubular reabsorption of sodium and water, as well as release of renin and prostaglandins, all of which contribute to cardiovascular and renal regulation. Renal afferent nerves complete the feedback loop via central autonomic nuclei where the signals are integrated and modulate central sympathetic outflow; thus both types of nerves form integral parts of the self-regulated renorenal reflex loop. Renal sympathetic nerve activity (RSNA) is commonly increased in pathophysiological conditions such as hypertension and chronic- and end-stage renal disease. Increased RSNA raises blood pressure and can contribute to the deterioration of renal function. Attempts have been made to eliminate or interfere with this important link between the brain and the kidneys as a neuromodulatory treatment for these conditions. Catheter-based renal sympathetic denervation has been successfully applied in patients with resistant hypertension and was associated with significant falls in blood pressure and renal protection in most studies performed. The focus of this review is the neural contribution to the control of renal and cardiovascular hemodynamics and renal function in the setting of hypertension and chronic kidney disease, as well as the specific roles of renal efferent and afferent nerves in this scenario and their utility as a therapeutic target.
Polymyxins–Curcumin Combination Antimicrobial Therapy: Safety Implications and Efficacy for Infection Treatment
(Molecular Diversity Preservation International (MDPI) AG, 2020-06-09)
The emergence of antimicrobial resistance in Gram‐negative bacteria poses a huge health challenge. The therapeutic use of polymyxins (i.e., colistin and polymyxin B) is commonplace due to high efficacy and limiting treatment options for multidrug‐resistant Gram‐negative bacterial infections. Nephrotoxicity and neurotoxicity are the major dose‐limiting factors that limit the therapeutic window of polymyxins; nephrotoxicity is a complication in up to ~60% of patients. The emergence of polymyxin‐resistant strains or polymyxin heteroresistance is also a limiting factor. These caveats have catalyzed the search for polymyxin combinations that synergistically kill polymyxin‐susceptible and resistant organisms and/or minimize the unwanted side effects. Curcumin—an FDA‐approved natural product—exerts many pharmacological activities. Recent studies showed that polymyxins–curcumin combinations showed a synergistically inhibitory effect on the growth of bacteria (e.g., Gram‐positive and Gram‐negative bacteria) in vitro. Moreover, curcumin co‐administration ameliorated colistin‐induced nephrotoxicity and neurotoxicity by inhibiting oxidative stress, mitochondrial dysfunction, inflammation and apoptosis. In this review, we summarize the current knowledge‐base of polymyxins–curcumin combination therapy and discuss the underlying mechanisms. For the clinical translation of this combination to become a reality, further research is required to develop novel polymyxins–curcumin formulations with optimized pharmacokinetics and dosage regimens.
Regulating polymyxin resistance in Gram-negative bacteria: roles of two-component systems PhoPQ and PmrAB
(Future Medicine Ltd, 2020-04)
Polymyxins (polymyxin B and colistin) are last-line antibiotics against multidrug-resistant Gram-negative pathogens. Polymyxin resistance is increasing worldwide, with resistance most commonly regulated by two-component systems such as PmrAB and PhoPQ. This review discusses the regulatory mechanisms of PhoPQ and PmrAB in mediating polymyxin resistance, from receiving an external stimulus through to activation of genes responsible for lipid A modifications. By analyzing the reported nonsynonymous substitutions in each two-component system, we identified the domains that are critical for polymyxin resistance. Notably, for PmrB 71% of resistance-conferring nonsynonymous mutations occurred in the HAMP (present in histidine kinases, adenylate cyclases, methyl accepting proteins and phosphatase) linker and DHp (dimerization and histidine phosphotransfer) domains. These results enhance our understanding of the regulatory mechanisms underpinning polymyxin resistance and may assist with the development of new strategies to minimize resistance emergence.
Polymyxin triple combinations against polymyxin-resistant, multidrug-resistant KPC-producing Klebsiella pneumoniae
(American Society for Microbiology, 2020)
Resistance to polymyxin antibiotics is increasing. Without new antibiotic classes, combination therapy is often required. We systematically investigated bacterial killing with polymyxin-based combinations against multidrug-resistant (including polymyxin-resistant), carbapenemase-producing Klebsiella pneumoniae. Mono, double and triple combination therapies were compared to identify the most efficacious treatment using static time-kill studies (24h, six isolates), an in vitro pharmacokinetic/pharmacodynamic model (IVM; 48h, two isolates), and the mouse thigh infection model (24h, six isolates). In static time-kill studies, all monotherapies (polymyxin B/rifampicin/amikacin/meropenem/minocycline) were ineffective. Initial bacterial killing was enhanced with various polymyxin B-containing double combinations, however substantial regrowth occurred in most cases by 24h. Most polymyxin B-containing triple combinations provided greater and more sustained killing than double combinations. Standard dosage regimens of polymyxin B (2.5 mg/kg/d), rifampicin (600 mg/12-hourly) and amikacin (7.5 mg/kg/12-hourly) were simulated in the IVM. Against isolate ATH 16, no viable bacteria were detected across 5-25h with triple therapy, with regrowth to ∼2-log10 CFU/mL occurring at 48h. Against isolate BD 32, rapid initial killing of ∼3.5-log10 CFU/mL at 5h was followed by a slow decline to ∼2-log10 CFU/mL at 48h. In infected mice, polymyxin B monotherapy (60 mg/kg/d) was generally ineffective. With triple therapy (polymyxin B 60 mg/kg/d, rifampicin 120 mg/kg/d, and amikacin 300 mg/kg/d), at 24h there was an ∼1.7-log10 CFU/thigh reduction compared to the starting inoculum for all six isolates. Our results demonstrate that the polymyxin B/rifampicin/amikacin combination significantly enhanced in vitro and in vivo bacterial killing, providing important information for the optimization of polymyxin-based combinations in patients
Pan-transcriptomic analysis identified common differentially expressed genes of Acinetobacter baumannii in response to polymyxin treatments
(Royal Society of Chemistry (RSC), 2020)
Multidrug-resistant Acinetobacter baumannii is a top-priority Gram-negative pathogen and polymyxins are a last-line therapeutic option. Previous systems pharmacological studies examining polymyxin killing and resistance usually focused on individual strains, and the derived knowledge could be limited by strain-specific genomic context. In this study, we examined the gene expression of five A. baumannii strains (34654, 1207552, 1428368, 1457504 and ATCC 19606) to determine the common differentially expressed genes in response to polymyxin treatments. A pan-genome containing 6061 genes was identified for 89 A. baumannii genomes from RefSeq database which included the five strains examined in this study; 2822 of the 6061 genes constituted the core genome. After 2 mg L−1 or 0.75 × MIC polymyxin treatments for 15 min, 41 genes were commonly up-regulated, including those involved in membrane biogenesis and homeostasis, lipoprotein and phospholipid trafficking, efflux pump and poly-N-acetylglucosamine biosynthesis; six genes were commonly down-regulated, three of which were related to fatty acid biosynthesis. Additionally, comparison of the gene expression at 15 and 60 min in ATCC 19606 revealed that polymyxin treatment resulted in a rapid change in amino acid metabolism at 15 min and perturbations on envelope biogenesis at both time points. This is the first pan-transcriptomic study for polymyxin-treated A. baumannii and our results identified that the remodelled outer membrane, up-regulated efflux pumps and down-regulated fatty acid biosynthesis might be essential for early responses to polymyxins in A. baumannii. Our findings provide important mechanistic insights into bacterial responses to polymyxin killing and may facilitate the optimisation of polymyxin therapy against this problematic ‘superbug’.
The Killing Mechanism of Teixobactin against Methicillin-Resistant Staphylococcus aureus: an Untargeted Metabolomics Study
(American Society for Microbiology, 2020)
Antibiotics have served humankind through their use in modern medicine as effective treatments for otherwise fatal bacterial infections. Teixobactin is a first member of newly discovered natural antibiotics that was recently identified from a hitherto-unculturable soil bacterium, Eleftheria terrae, and recognized as a potent antibacterial agent against various Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci. The most distinctive characteristic of teixobactin as an effective antibiotic is that teixobactin resistance could not be evolved in a laboratory setting. It is purported that teixobactin’s “resistance-resistant” mechanism of action includes binding to the essential bacterial cell wall synthesis building blocks lipid II and lipid III. In the present study, metabolomics was used to investigate the potential metabolic pathways involved in the mechanisms of antibacterial activity of the synthetic teixobactin analogue Leu10-teixobactin against a MRSA strain, S. aureus ATCC 700699. The metabolomes of S. aureus ATCC 700699 cells 1, 3, and 6 h following treatment with Leu10-teixobactin (0.5 μg/ml, i.e., 0.5× MIC) were compared to those of the untreated controls. Leu10-teixobactin significantly perturbed bacterial membrane lipids (glycerophospholipids and fatty acids), peptidoglycan (lipid I and II) metabolism, and cell wall teichoic acid (lipid III) biosynthesis as early as after 1 h of treatment, reflecting an initial activity on the cell envelope. Concordant with its time-dependent antibacterial killing action, Leu10-teixobactin caused more perturbations in the levels of key intermediates in pathways of amino-sugar and nucleotide-sugar metabolism and their downstream peptidoglycan and teichoic acid biosynthesis at 3 and 6 h. Significant perturbations in arginine metabolism and the interrelated tricarboxylic acid cycle, histidine metabolism, pantothenate, and coenzyme A biosynthesis were also observed at 3 and 6 h. To conclude, this is the first study to provide novel metabolomics mechanistic information, which lends support to the development of teixobactin as an antibacterial drug for the treatment of multidrug-resistant Gram-positive infections
A Portrait of the Sialyl Glycan Receptor Specificity of the H10 Influenza Virus Hemagglutinin-A Picture of an Avian Virus on the Verge of Becoming a Pandemic?
Pandemic influenza is a constant global threat to human health. In particular, the pandemic potential of novel avian influenza viruses such as the H10N7 and H10N8 avian strains, which recently managed to cross the species barrier from birds to humans, are always of great concern as we are unlikely to have any prior immunity. Human and avian isolates of H10 influenza display the ability to rapidly adapt to replication in mammalian hosts. Fortunately, so far there is no evidence of efficient human-to-human transmission of any avian influenza virus. This review examines all of the available clinical and biological data for H10 influenza viruses with an emphasis on hemagglutinin as it is a major viral antigen that determines host range and immunity. The available glycan binding data on the influenza H10 hemagglutinin are discussed in a structure-recognition perspective. Importantly, this review raises the question of whether the emerging novel avian H10 influenza viruses truly represents a threat to global health that warrants close monitoring.
On-chip surface acoustic wave and micropipette aspiration techniques to assess cell elastic properties.
(A I P Publishing LLC, 2020-01)
The cytoskeletal mechanics and cell mechanical properties play an important role in cellular behaviors. In this study, in order to provide comprehensive insights into the relationship between different cytoskeletal components and cellular elastic moduli, we built a phase-modulated surface acoustic wave microfluidic device to measure cellular compressibility and a microfluidic micropipette-aspiration device to measure cellular Young's modulus. The microfluidic devices were validated based on experimental data and computational simulations. The contributions of structural cytoskeletal actin filament and microtubule to cellular compressibility and Young's modulus were examined in MCF-7 cells. The compressibility of MCF-7 cells was increased after microtubule disruption, whereas actin disruption had no effect. In contrast, Young's modulus of MCF-7 cells was reduced after actin disruption but unaffected by microtubule disruption. The actin filaments and microtubules were stained to confirm the structural alteration in cytoskeleton. Our findings suggest the dissimilarity in the structural roles of actin filaments and microtubules in terms of cellular compressibility and Young's modulus. Based on the differences in location and structure, actin filaments mainly contribute to tensile Young's modulus and microtubules mainly contribute to compressibility. In addition, different responses to cytoskeletal alterations between acoustophoresis and micropipette aspiration demonstrated that micropipette aspiration was better at detecting the change from actin cortex, while the response to acoustophoresis was governed by microtubule networks.
Synergistic Combination of Polymyxin B and Enrofloxacin Induced Metabolic Perturbations in Extensive Drug-Resistant Pseudomonas aeruginosa
(FRONTIERS MEDIA SA, 2019-10-03)
Polymyxins are used as a last-resort class of antibiotics against multidrug-resistant (MDR) Gram-negative Pseudomonas aeruginosa. As polymyxin monotherapy is associated with potential development of resistance, combination therapy is highly recommended. This study investigated the mechanism underlying the synergistic killing of polymyxin B and enrofloxacin against extensive drug-resistant (XDR) P. aeruginosa. An XDR isolate P. aeruginosa 12196 was treated with clinically relevant concentrations of polymyxin B (2 mg/L) and enrofloxacin (1 mg/L) alone or in combination. Metabolome profiles were investigated from bacterial samples collected at 1-and 4-h posttreatment using liquid chromatography with tandem mass spectrometry (LC-MS/MS), and data were analyzed using univariate and multivariate statistics. Significantly perturbed metabolites (q < 0.05, fold change ≥ 2) were subjected to pathway analysis. The synergistic killing by polymyxin B-enrofloxacin combination was initially driven by polymyxin B as indicated by the perturbation of lipid metabolites at 1 h in particular. The killing was subsequently driven by enrofloxacin via the inhibition of DNA replication, resulting in the accumulation of nucleotides at 4 h. Furthermore, the combination uniquely altered levels of metabolites in energy metabolism and cell envelope biogenesis. Most importantly, the combination significantly minimized polymyxin resistance via the inhibition of lipid A modification pathway, which was most evident at 4 h. This is the first study to elucidate the synergistic mechanism of polymyxin B-enrofloxacin combination against XDR P. aeruginosa. The metabolomics approach taken in this study highlights its power to elucidate the mechanism of synergistic killing by antibiotic combinations at the systems level.
Global metabolic analyses identify key differences in metabolite levels between polymyxin-susceptible and polymyxin-resistant Acinetobacter baumannii
(NATURE PUBLISHING GROUP, 2016-02-29)
Multidrug-resistant Acinetobacter baumannii presents a global medical crisis and polymyxins are used as the last-line therapy. This study aimed to identify metabolic differences between polymyxin-susceptible and polymyxin-resistant A. baumannii using untargeted metabolomics. The metabolome of each A. baumannii strain was measured using liquid chromatography-mass spectrometry. Multivariate and univariate statistics and pathway analyses were employed to elucidate metabolic differences between the polymyxin-susceptible and -resistant A. baumannii strains. Significant differences were identified between the metabolic profiles of the polymyxin-susceptible and -resistant A. baumannii strains. The lipopolysaccharide (LPS) deficient, polymyxin-resistant 19606R showed perturbation in specific amino acid and carbohydrate metabolites, particularly pentose phosphate pathway (PPP) and tricarboxylic acid (TCA) cycle intermediates. Levels of nucleotides were lower in the LPS-deficient 19606R. Furthermore, 19606R exhibited a shift in its glycerophospholipid profile towards increased abundance of short-chain lipids compared to the parent polymyxin-susceptible ATCC 19606. In contrast, in a pair of clinical isolates 03-149.1 (polymyxin-susceptible) and 03-149.2 (polymyxin-resistant, due to modification of lipid A), minor metabolic differences were identified. Notably, peptidoglycan biosynthesis metabolites were significantly depleted in both of the aforementioned polymyxin-resistant strains. This is the first comparative untargeted metabolomics study to show substantial differences in the metabolic profiles of the polymyxin-susceptible and -resistant A. baumannii.
Probing the Penetration of Antimicrobial Polymyxin Lipopeptides into Gram-Negative Bacteria
(AMER CHEMICAL SOC, 2014-04-01)
The dry antibiotic development pipeline coupled with the emergence of multidrug resistant Gram-negative 'superbugs' has driven the revival of the polymyxin lipopeptide antibiotics. Polymyxin resistance implies a total lack of antibiotics for the treatment of life-threatening infections. The lack of molecular imaging probes that possess native polymyxin-like antibacterial activity is a barrier to understanding the resistance mechanisms and the development of a new generation of polymyxin lipopeptides. Here we report the regioselective modification of the polymyxin B core scaffold at the N-terminus with the dansyl fluorophore to generate an active probe that mimics polymyxin B pharmacologically. Time-lapse laser scanning confocal microscopy imaging of the penetration of probe (1) into Gram-negative bacterial cells revealed that the probe initially accumulates in the outer membrane and subsequently penetrates into the inner membrane and finally the cytoplasm. The implementation of this polymyxin-mimetic probe will advance the development of platforms for the discovery of novel polymyxin lipopeptides with efficacy against polymyxin-resistant strains.