Pharmacology and Therapeutics - Research Publications

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    Synthesis and structure-activity relationships of teixobactin
    Karas, JA ; Chen, F ; Schneider-Futschik, EK ; Kang, Z ; Hussein, M ; Swarbrick, J ; Hoyer, D ; Giltrap, AM ; Payne, RJ ; Li, J ; Velkov, T (WILEY, 2019-12-02)
    The discovery of antibiotics has led to the effective treatment of bacterial infections that were otherwise fatal and has had a transformative effect on modern medicine. Teixobactin is an unusual depsipeptide natural product that was recently discovered from a previously unculturable soil bacterium and found to possess potent antibacterial activity against several Gram positive pathogens, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci. One of the key features of teixobactin as an antibiotic lead is that resistance could not be generated in a laboratory setting. This is proposed to be a result of a mechanism of action that involves binding to essential cell wall synthesis building blocks, lipid II and lipid III. Since the initial isolation report in 2015, significant efforts have been made to understand its unique mechanism of action, develop efficient synthetic routes for its production, and thus enable the generation of analogues for structure-activity relationship studies and optimization of its pharmacological properties. Our review provides a comprehensive treatise on the progress in understanding teixobactin chemistry, structure-activity relationships, and mechanisms of antibacterial activity. Teixobactin represents an exciting starting point for the development of new antibiotics that can be used to combat multidrug-resistant bacterial ("superbug") infections.
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    The impact of backbone N-methylation on the structure-activity relationship of Leu(10)-teixobactin
    Velkov, T ; Swarbrick, JD ; Hussein, MH ; Schneider-Futschik, EK ; Hoyer, D ; Li, J ; Karas, JA (WILEY, 2019-08-06)
    Antimicrobial resistance is a serious threat to global human health; therefore, new anti-infective therapeutics are required. The cyclic depsi-peptide teixobactin exhibits potent antimicrobial activity against several Gram-positive pathogens. To study the natural product's mechanism of action and improve its pharmacological properties, efficient chemical methods for preparing teixobactin analogues are required to expedite structure-activity relationship studies. Described herein is a synthetic route that enables rapid access to analogues. Furthermore, our new N-methylated analogues highlight that hydrogen bonding along the N-terminal tail is likely to be important for antimicrobial activity.
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    Structure of micelle bound cationic peptides by NMR spectroscopy using a lanthanide shift reagent
    Swarbrick, JD ; Karas, JA ; Li, J ; Velkov, T (Royal Society of Chemistry, 2020-03-07)
    [Tm(DPA)3]3- was used to generate multiple, paramagnetic nuclear Overhauser effect NMR spectra of cationic peptides when weakly bound to a lipopolysaccharide micelle. Increased spectral resolution combined with a marked increase in the number of distance restraints yielded high resolution structures of polymyxin and MSI-594 in the liposaccharide bound state.
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    Genome-Scale Metabolic Modeling Reveals Metabolic Alterations of Multidrug-Resistant Acinetobacter baumannii in a Murine Bloodstream Infection Model
    Zhao, J ; Zhu, Y ; Han, J ; Lin, Y-W ; Aichem, M ; Wang, J ; Chen, K ; Velkov, T ; Schreiber, F ; Li, J (MDPI, 2020-11-01)
    Multidrug-resistant (MDR) Acinetobacter baumannii is a critical threat to human health globally. We constructed a genome-scale metabolic model iAB5075 for the hypervirulent, MDR A. baumannii strain AB5075. Predictions of nutrient utilization and gene essentiality were validated using Biolog assay and a transposon mutant library. In vivo transcriptomics data were integrated with iAB5075 to elucidate bacterial metabolic responses to the host environment. iAB5075 contains 1530 metabolites, 2229 reactions, and 1015 genes, and demonstrated high accuracies in predicting nutrient utilization and gene essentiality. At 4 h post-infection, a total of 146 metabolic fluxes were increased and 52 were decreased compared to 2 h post-infection; these included enhanced fluxes through peptidoglycan and lipopolysaccharide biosynthesis, tricarboxylic cycle, gluconeogenesis, nucleotide and fatty acid biosynthesis, and altered fluxes in amino acid metabolism. These flux changes indicate that the induced central metabolism, energy production, and cell membrane biogenesis played key roles in establishing and enhancing A. baumannii bloodstream infection. This study is the first to employ genome-scale metabolic modeling to investigate A. baumannii infection in vivo. Our findings provide important mechanistic insights into the adaption of A. baumannii to the host environment and thus will contribute to the development of new therapeutic agents against this problematic pathogen.
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    Allosteric folding correction of F508del and rare CFTR mutants by elexacaftor-tezacaftor-ivacaftor (Trikafta) combination
    Veit, G ; Roldan, A ; Hancock, MA ; Da Fonte, DF ; Xu, H ; Hussein, M ; Frenkiel, S ; Matouk, E ; Velkov, T ; Lukacs, GL (AMER SOC CLINICAL INVESTIGATION INC, 2020-09-17)
    Based on its clinical benefits, Trikafta - the combination of folding correctors VX-661 (tezacaftor), VX-445 (elexacaftor), and the gating potentiator VX-770 (ivacaftor) - was FDA approved for treatment of patients with cystic fibrosis (CF) carrying deletion of phenylalanine at position 508 (F508del) of the CF transmembrane conductance regulator (CFTR) on at least 1 allele. Neither the mechanism of action of VX-445 nor the susceptibility of rare CF folding mutants to Trikafta are known. Here, we show that, in human bronchial epithelial cells, VX-445 synergistically restores F508del-CFTR processing in combination with type I or II correctors that target the nucleotide binding domain 1 (NBD1) membrane spanning domains (MSDs) interface and NBD2, respectively, consistent with a type III corrector mechanism. This inference was supported by the VX-445 binding to and unfolding suppression of the isolated F508del-NBD1 of CFTR. The VX-661 plus VX-445 treatment restored F508del-CFTR chloride channel function in the presence of VX-770 to approximately 62% of WT CFTR in homozygous nasal epithelia. Substantial rescue of rare misprocessing mutations (S13F, R31C, G85E, E92K, V520F, M1101K, and N1303K), confined to MSD1, MSD2, NBD1, and NBD2 of CFTR, was also observed in airway epithelia, suggesting an allosteric correction mechanism and the possible application of Trikafta for patients with rare misfolding mutants of CFTR.
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    Polymyxin B combinations with FDA-approved non-antibiotic phenothiazine drugs targeting multi-drug resistance of Gram-negative pathogens
    Hussein, M ; Hu, X ; Paulin, OKA ; Crawford, S ; Zhou, QT ; Baker, M ; Schneider-Futschik, EK ; Zhu, Y ; Li, J ; Velkov, T (ELSEVIER, 2020-01-01)
    The status quo for combating uprising antibacterial resistance is to employ synergistic combinations of antibiotics. Nevertheless, the currently available combination therapies are fast becoming untenable. Combining antibiotics with various FDA-approved non-antibiotic drugs has emerged as a novel strategy against otherwise untreatable multi-drug resistant (MDR) pathogens. The apex of this study was to investigate the mechanisms of antibacterial synergy of the combination of polymyxin B with the phenothiazines against the MDR Gram-negative pathogens Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. The synergistic antibacterial effects were tested using checkerboard and static time-kill assays. Electron microscopy (EM) and untargeted metabolomics were used to ascertain the mechanism(s) of the antibacterial synergy. The combination of polymyxin B and the phenothiazines showed synergistic antibacterial activity in checkerboard and static time-kill assays at clinically relevant concentrations against both polymyxin-susceptible and polymyxin-resistant isolates. EM revealed that the polymyxin B-prochlorperazine combination resulted in greater damage to the bacterial cell compared to each drug monotherapy. In metabolomics, at 0.5 h, polymyxin B monotherapy and the combination (to a greatest extent) disorganised the bacterial cell envelope as manifested by a major perturbation in bacterial membrane lipids (glycerophospholipids and fatty acids), peptidoglycan and lipopolysaccharide (LPS) biosynthesis. At the late time exposure (4 h), the aforementioned effects (except LPS biosynthesis) perpetuated mainly with the combination therapy, indicating the disorganising bacterial membrane biogenesis is potentially behind the mechanisms of antibacterial synergy. In conclusion, the study highlights the potential usefulness of the combination of polymyxin B with phenothiazines for the treatment of polymyxin-resistant Gram-negative infections (e.g. CNS infections).
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    Polymyxins Bind to the Cell Surface of Unculturable Acinetobacter baumannii and Cause Unique Dependent Resistance
    Zhu, Y ; Lu, J ; Han, M-L ; Jiang, X ; Azad, MAK ; Patil, NA ; Lin, Y-W ; Zhao, J ; Hu, Y ; Yu, HH ; Chen, K ; Boyce, JD ; Dunstan, RA ; Lithgow, T ; Barlow, CK ; Li, W ; Schneider-Futschik, EK ; Wang, J ; Gong, B ; Sommer, B ; Creek, DJ ; Fu, J ; Wang, L ; Schreiber, F ; Velkov, T ; Li, J (WILEY, 2020-06-08)
    Multidrug-resistant Acinetobacter baumannii is a top-priority pathogen globally and polymyxins are a last-line therapy. Polymyxin dependence in A. baumannii (i.e., nonculturable on agar without polymyxins) is a unique and highly-resistant phenotype with a significant potential to cause treatment failure in patients. The present study discovers that a polymyxin-dependent A. baumannii strain possesses mutations in both lpxC (lipopolysaccharide biosynthesis) and katG (reactive oxygen species scavenging) genes. Correlative multiomics analyses show a significantly remodeled cell envelope and remarkably abundant phosphatidylglycerol in the outer membrane (OM). Molecular dynamics simulations and quantitative membrane lipidomics reveal that polymyxin-dependent growth emerges only when the lipopolysaccharide-deficient OM distinctively remodels with ≥ 35% phosphatidylglycerol, and with "patch" binding on the OM by the rigid polymyxin molecules containing strong intramolecular hydrogen bonding. Rather than damaging the OM, polymyxins bind to the phosphatidylglycerol-rich OM and strengthen the membrane integrity, thereby protecting bacteria from external reactive oxygen species. Dependent growth is observed exclusively with polymyxin analogues, indicating a critical role of the specific amino acid sequence of polymyxins in forming unique structures for patch-binding to bacterial OM. Polymyxin dependence is a novel antibiotic resistance mechanism and the current findings highlight the risk of 'invisible' polymyxin-dependent isolates in the evolution of resistance.
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    The Antimicrobial Activity of Cannabinoids
    Karas, JA ; Wong, LJM ; Paulin, OKA ; Mazeh, AC ; Hussein, MH ; Li, J ; Velkov, T (MDPI AG, 2020-07-13)
    A post-antibiotic world is fast becoming a reality, given the rapid emergence of pathogens that are resistant to current drugs. Therefore, there is an urgent need to discover new classes of potent antimicrobial agents with novel modes of action. Cannabis sativa is an herbaceous plant that has been used for millennia for medicinal and recreational purposes. Its bioactivity is largely due to a class of compounds known as cannabinoids. Recently, these natural products and their analogs have been screened for their antimicrobial properties, in the quest to discover new anti-infective agents. This paper seeks to review the research to date on cannabinoids in this context, including an analysis of structure–activity relationships. It is hoped that it will stimulate further interest in this important issue.
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    Polymyxins–Curcumin Combination Antimicrobial Therapy: Safety Implications and Efficacy for Infection Treatment
    Velkov, T (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.
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    Regulating polymyxin resistance in Gram-negative bacteria: roles of two-component systems PhoPQ and PmrAB
    Huang, J ; Li, C ; Song, J ; Velkov, T ; Wang, L ; Zhu, Y ; Li, J (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.