Publications by authors named "Philip E Thompson"

95 Publications

A New Turn in Peptide-Based Imaging Agents: Foldamers Afford Improved Theranostics Targeting Cholecystokinin-2 Receptor-Positive Cancer.

J Med Chem 2021 Apr 7;64(8):4841-4856. Epub 2021 Apr 7.

Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, VIC 3010, Australia.

Proteins adopt unique folded secondary and tertiary structures that are responsible for their remarkable biological properties. This structural complexity is key in designing efficacious peptides that can mimic the three-dimensional structure needed for biological function. In this study, we employ different chemical strategies to induce and stabilize a β-hairpin fold of peptides targeting cholecystokinin-2 receptors for theranostic application (combination of a targeted therapeutic and a diagnostic companion). The newly developed peptides exhibited enhanced folding capacity as demonstrated by circular dichroism (CD) spectroscopy, ion-mobility spectrometry-mass spectrometry, and two-dimensional (2D) NMR experiments. Enhanced folding characteristics of the peptides led to increased biological potency, affording four optimal Ga-68 labeled radiotracers ([Ga]Ga-, [Ga]Ga-) targeting CCK-2R. In particular, [Ga]Ga- and [Ga]Ga- presented improved metabolic stability, enhanced cell internalization, and up to 6 fold increase in tumor uptake. These peptides hold great promise as next-generation theranostic radiopharmaceuticals.
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http://dx.doi.org/10.1021/acs.jmedchem.0c02213DOI Listing
April 2021

Design, Development, In Vitro and Preliminary In Vivo Evaluation of a Novel Photo-Angioplasty Device: Lumi-Solve.

Cardiovasc Eng Technol 2021 Mar 11. Epub 2021 Mar 11.

Eastern Health Clinical School, Monash University, Box Hill, Australia.

Purpose: Paclitaxel (PTX)-coated drug eluting balloon catheters (DEBc) used in the management of neointimal hyperplasia (NIH) have been associated with safety concerns. Alternative coating agents and targeted delivery systems may improve safety and DEBc efficacy. Utilizing a multi-platform approach we designed, developed and evaluated Lumi-Solve, a novel DEBc, coated with ultraviolet (UV) 365 nm-activated caged metacept-3 (c-MCT-3), an epigenetic agent from the histone deacetylase inhibitor (HDACi) class.

Methods: In vitro catheter and contrast media transmission of UV365nm was evaluated spectroscopically. UV365nm conversion of c-MCT-3 to MCT-3 was evaluated chromatographically. Cellular toxicity and HDACi activity of c-MCT-3 ∓UV365nm was evaluated in vitro. In vivo UV365nm conversion of c-MCT-3 to MCT-3 was evaluated in an ovine carotid artery model.

Results: Catheter material and dilute contrast media did not attenuate UV365nm transmission or c-MCT-3 activation. c-MCT-3 demonstrated less cellular toxicity than MCT-3 and PTX. UV365nm-activated c-MCT-3 demonstrated HDACi activity. In vivo activation of c-MCT-3 produced MCT-3.

Conclusions: Lumi-Solve, a novel DEBc device developed utilizing a combination of chemical, fibre-optic and catheter based technology platforms, demonstrated potential for targeted delivery of bioactive HDACi to the blood vessel wall supporting direct application to the management of NIH and warranting additional in vivo studies.
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http://dx.doi.org/10.1007/s13239-021-00525-yDOI Listing
March 2021

Macrocyclic peptidomimetics as inhibitors of insulin-regulated aminopeptidase (IRAP).

RSC Med Chem 2020 Feb 8;11(2):234-244. Epub 2020 Jan 8.

Medicinal Chemistry , Monash Institute of Pharmaceutical Sciences , Parkville , Victoria 3052 , Australia . Email:

Macrocyclic analogues of the linear hexapeptide, angiotensin IV (AngIV) have proved to be potent inhibitors of insulin-regulated aminopeptidase (IRAP, oxytocinase, EC 3.4.11.3). Along with higher affinity, macrocycles may also offer better metabolic stability, membrane permeability and selectivity, however predicting the outcome of particular cycle modifications is challenging. Here we describe the development of a series of macrocyclic IRAP inhibitors with either disulphide, olefin metathesis or lactam bridges and variations of ring size and other functionality. The binding mode of these compounds is proposed based on molecular dynamics analysis. Estimation of binding affinities (Δ) and relative binding free energies (ΔΔ) with the linear interaction energy (LIE) method and free energy perturbation (FEP) method showed good general agreement with the observed inhibitory potency. Experimental and calculated data highlight the cumulative importance of an intact N-terminal peptide, the specific nature of the macrocycle, the phenolic oxygen and the C-terminal functionality.
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http://dx.doi.org/10.1039/c9md00485hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7412577PMC
February 2020

IRAP Inhibitors: M1-Aminopeptidase Family Inspiration.

Front Pharmacol 2020 25;11:585930. Epub 2020 Sep 25.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.

The insulin regulated aminopeptidase (IRAP) has been proposed as an important therapeutic target for indications including Alzheimer's disease and immune disorders. To date, a number of IRAP inhibitor designs have been investigated but the total number of molecules investigated remains quite small. As a member the M1 aminopeptidase family, IRAP shares numerous structural features with the other M1 aminopeptidases. The study of those enzymes and the development of inhibitors provide key learnings and new approaches and are potential sources of inspiration for future IRAP inhibitors.
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http://dx.doi.org/10.3389/fphar.2020.585930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546331PMC
September 2020

Disrupting the platelet internal membrane via PI3KC2α inhibition impairs thrombosis independently of canonical platelet activation.

Sci Transl Med 2020 07;12(553)

Australian Centre for Blood Diseases, Monash University, Melbourne, VIC 3004, Australia.

Arterial thrombosis causes heart attacks and most strokes and is the most common cause of death in the world. Platelets are the cells that form arterial thrombi, and antiplatelet drugs are the mainstay of heart attack and stroke prevention. Yet, current drugs have limited efficacy, preventing fewer than 25% of lethal cardiovascular events without clinically relevant effects on bleeding. The key limitation on the ability of all current drugs to impair thrombosis without causing bleeding is that they block global platelet activation, thereby indiscriminately preventing platelet function in hemostasis and thrombosis. Here, we identify an approach with the potential to overcome this limitation by preventing platelet function independently of canonical platelet activation and in a manner that appears specifically relevant in the setting of thrombosis. Genetic or pharmacological targeting of the class II phosphoinositide 3-kinase (PI3KC2α) dilates the internal membrane reserve of platelets but does not affect activation-dependent platelet function in standard tests. Despite this, inhibition of PI3KC2α is potently antithrombotic in human blood ex vivo and mice in vivo and does not affect hemostasis. Mechanistic studies reveal this antithrombotic effect to be the result of impaired platelet adhesion driven by pronounced hemodynamic shear stress gradients. These findings demonstrate an important role for PI3KC2α in regulating platelet structure and function via a membrane-dependent mechanism and suggest that drugs targeting the platelet internal membrane may be a suitable approach for antithrombotic therapies with an improved therapeutic window.
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http://dx.doi.org/10.1126/scitranslmed.aar8430DOI Listing
July 2020

Improving Membrane Permeation in the Beyond Rule-of-Five Space by Using Prodrugs to Mask Hydrogen Bond Donors.

ACS Chem Biol 2020 08 20;15(8):2070-2078. Epub 2020 Jul 20.

Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.

A wide range of drug targets can be effectively modulated by peptides and macrocycles. Unfortunately, the size and polarity of these compounds prevents them from crossing the cell membrane to reach target sites in the cell cytosol. As such, these compounds do not conform to standard measures of drug-likeness and exist in beyond the rule-of-five space. In this work, we investigate whether prodrug moieties that mask hydrogen bond donors can be applied in the beyond rule-of-five domain to improve the permeation of macrocyclic compounds. Using a cyclic peptide model, we show that masking hydrogen bond donors in the natural polar amino acid residues (His, Ser, Gln, Asn, Glu, Asp, Lys, and Arg) imparts membrane permeability to the otherwise impermeable parent molecules, even though the addition of the masking group increases the overall compound molecular weight and the number of hydrogen bond acceptors. We demonstrate this strategy in PAMPA and Caco2 membrane permeability assays and show that masking with groups that reduce the number of hydrogen-bond donors at the cost of additional mass and hydrogen bond acceptors, a donor-acceptor swap, is effective.
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http://dx.doi.org/10.1021/acschembio.0c00218DOI Listing
August 2020

Structure-Interaction Relationship of Polymyxins with the Membrane of Human Kidney Proximal Tubular Cells.

ACS Infect Dis 2020 08 17;6(8):2110-2119. Epub 2020 Jul 17.

Biomedicine Discovery Institute, Infection & Immunity Program and Department of Microbiology, Monash University, Clayton, Melbourne, Victoria 3800, Australia.

Multidrug-resistant Gram-negative bacteria are a serious global threat to human health. Polymyxins are increasingly used in patients as a last-line therapy to treat infections caused by these life-threatening 'superbugs'. Unfortunately, polymyxin-induced nephrotoxicity is the major dose-limiting factor and understanding its mechanism is crucial for the development of novel, safer polymyxins. Here, we undertook the first all-atom molecular dynamics simulations of the interaction between four naturally occurring polymyxins A, B, M and colistin A (representative structural variations of the polymyxin core structure) and the membrane of human kidney proximal tubular cells. All polymyxins inserted spontaneously into the hydrophobic region of the membrane where they were retained, although their insertion abilities varied. Polymyxin A completely penetrated into the hydrophobic region of the membrane with a unique folded conformation, whereas the other three polymyxins only inserted their fatty acyl tails into this region. Furthermore, local membrane defects and increased water penetration were induced by each polymyxin, which may represent the initial stage of cellular membrane damage. Finally, the structure-interaction relationship of polymyxins was investigated based on atomic interactions at the cell membrane level. The hydrophobicity at positions 6/7 and stereochemistry at position 3 regulated the interactions of polymyxins with the cell membrane. Collectively, our results provide new mechanistic insights into polymyxin-induced nephrotoxicity at the atomic level and will facilitate the development of new-generation polymyxins.
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http://dx.doi.org/10.1021/acsinfecdis.0c00190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7485602PMC
August 2020

Heterodimeric Analogues of the Potent Y1R Antagonist 1229U91, Lacking One of the Pharmacophoric C-Terminal Structures, Retain Potent Y1R Affinity and Show Improved Selectivity over Y4R.

J Med Chem 2020 05 16;63(10):5274-5286. Epub 2020 May 16.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Victoria 3052, Australia.

The cyclic dimeric peptide 1229U91 (GR231118) has an unusual structure and displays potent, insurmountable antagonism of the Y receptor. To probe the structural basis for this activity, we have prepared ring size variants and heterodimeric compounds, identifying the specific residues underpinning the mechanism of 1229U91 binding. The homodimeric structure was shown to be dispensible, with analogues lacking key pharmacophoric residues in one dimer arm retaining high antagonist affinity. Compounds also showed enhanced Y1R selectivity over Y4R compared to 1229U91.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00027DOI Listing
May 2020

Application of a Sulfoxonium Ylide Electrophile to Generate Cathepsin X-Selective Activity-Based Probes.

ACS Chem Biol 2020 03 14;15(3):718-727. Epub 2020 Feb 14.

Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria 3052, Australia.

Cathepsin X/Z/P is cysteine cathepsin with unique carboxypeptidase activity. Its expression is associated with cancer and neurodegenerative diseases, although its roles during normal physiology are still poorly understood. Advances in our understanding of its function have been hindered by a lack of available tools that can specifically measure the proteolytic activity of cathepsin X. We present a series of activity-based probes that incorporate a sulfoxonium ylide warhead, which exhibit improved specificity for cathepsin X compared to previously reported probes. We apply these probes to detect cathepsin X activity in cell and tissue lysates, in live cells and in vivo, and to localize active cathepsin X in mouse tissues by microscopy. Finally, we utilize an improved method to generate chloromethylketones, necessary intermediates for synthesis of acyloxymethylketones probes, by way of sulfoxonium ylide intermediates. In conclusion, the probes presented in this study will be valuable for investigating cathepsin X pathophysiology.
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http://dx.doi.org/10.1021/acschembio.9b00961DOI Listing
March 2020

History, Chemistry and Antibacterial Spectrum.

Adv Exp Med Biol 2019 ;1145:15-36

Centre for Medicine Use and Safety, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Australia.

Polymyxins are naturally occurring cyclic lipopeptides that were discovered more than 60 years ago. They have a narrow antibacterial spectrum, which is mainly against Gram-negative pathogens. The dry antibiotic pipeline, together with the increasing incidence of bacterial resistance in the clinic, has been dubbed 'the perfect storm'. This has forced a re-evaluation of 'old' antibiotics, in particular the polymyxins, which retain activity against many multidrug-resistant (MDR) Gram-negative organisms. As a consequence, polymyxin B and colistin (polymyxin E) are now used as the last therapeutic option for infections caused by 'superbugs' such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. This chapter covers the history, chemistry and antibacterial spectrum of these very important last-line lipopeptide antibiotics.
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http://dx.doi.org/10.1007/978-3-030-16373-0_3DOI Listing
September 2019

An apically located hybrid guanylate cyclase-ATPase is critical for the initiation of Ca signaling and motility in .

J Biol Chem 2019 05 16;294(22):8959-8972. Epub 2019 Apr 16.

From the The Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Victoria 3052, Australia,

Protozoan parasites of the phylum Apicomplexa actively move through tissue to initiate and perpetuate infection. The regulation of parasite motility relies on cyclic nucleotide-dependent kinases, but how these kinases are activated remains unknown. Here, using an array of biochemical and cell biology approaches, we show that the apicomplexan parasite expresses a large guanylate cyclase (TgGC) protein, which contains several upstream ATPase transporter-like domains. We show that TgGC has a dynamic localization, being concentrated at the apical tip in extracellular parasites, which then relocates to a more cytosolic distribution during intracellular replication. Conditional TgGC knockdown revealed that this protein is essential for acute-stage tachyzoite growth, as TgGC-deficient parasites were defective in motility, host cell attachment, invasion, and subsequent host cell egress. We show that TgGC is critical for a rapid rise in cytosolic [Ca] and for secretion of microneme organelles upon stimulation with a cGMP agonist, but these deficiencies can be bypassed by direct activation of signaling by a Ca ionophore. Furthermore, we found that TgGC is required for transducing changes in extracellular pH and [K] to activate cytosolic [Ca] flux. Together, the results of our work implicate TgGC as a putative signal transducer that activates Ca signaling and motility in .
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http://dx.doi.org/10.1074/jbc.RA118.005491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6552420PMC
May 2019

Structural Determinants of Isoform Selectivity in PI3K Inhibitors.

Biomolecules 2019 02 26;9(3). Epub 2019 Feb 26.

Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Phosphatidylinositol 3-kinases (PI3Ks) are important therapeutic targets for the treatment of cancer, thrombosis, and inflammatory and immune diseases. The four highly homologous Class I isoforms, PI3K, PI3K, PI3K and PI3K have unique, non-redundant physiological roles and as such, isoform selectivity has been a key consideration driving inhibitor design and development. In this review, we discuss the structural biology of PI3Ks and how our growing knowledge of structure has influenced the medicinal chemistry of PI3K inhibitors. We present an analysis of the available structure-selectivity-activity relationship data to highlight key insights into how the various regions of the PI3K binding site influence isoform selectivity. The picture that emerges is one that is far from simple and emphasizes the complex nature of protein-inhibitor binding, involving protein flexibility, energetics, water networks and interactions with non-conserved residues.
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http://dx.doi.org/10.3390/biom9030082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468644PMC
February 2019

Controlled Construction of Cyclic d / l Peptide Nanorods.

Angew Chem Int Ed Engl 2019 01 5;58(2):596-601. Epub 2018 Dec 5.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria, 3052, Australia.

Cyclic d / l peptides (CPs) assemble spontaneously via backbone H-bonding to form extended nanostructures. These modular materials have great potential as versatile bionanomaterials. However, the useful development of CP nanomaterials requires practical methods to direct and control their assembly. In this work, we present novel, heterogeneous, covalently linked CP tetramers that achieve local control over the CP subunit order and composition through coupling of amino acid side-chains using copper-activated azide-alkyne cycloaddition and disulfide bond formation. Cryo-transmission electron microscopy revealed the formation of highly ordered, fibrous nanostructures, while NMR studies showed that these systems have strong intramolecular H-bonding in solution. The introduction of inter-CP tethers is expected to enable the development of complex nanomaterials with controllable chemical properties, facilitating the development of precisely functionalized or "decorated" peptide nanostructures.
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http://dx.doi.org/10.1002/anie.201811910DOI Listing
January 2019

A Cyclic Peptide Inhibitor of the iNOS-SPSB Protein-Protein Interaction as a Potential Anti-Infective Agent.

ACS Chem Biol 2018 10 18;13(10):2930-2938. Epub 2018 Sep 18.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences , Monash University , Parkville Victoria 3052 , Australia.

SPRY domain- and SOCS box-containing proteins SPSB1, SPSB2, and SPSB4 interact with inducible nitric oxide synthase (iNOS), causing the iNOS to be polyubiquitinated and targeted for degradation. Inhibition of this interaction increases iNOS levels, and consequently cellular nitric oxide (NO) concentrations, and has been proposed as a potential strategy for killing intracellular pathogens. We previously described two DINNN-containing cyclic peptides (CP1 and CP2) as potent inhibitors of the murine SPSB-iNOS interaction. In this study, we report the crystal structures of human SPSB4 bound to CP1 and CP2 and human SPSB2 bound to CP2. We then used these structures to design a new inhibitor in which an intramolecular hydrogen bond was replaced with a hydrocarbon linkage to form a smaller macrocycle while maintaining the bound geometry of CP2 observed in the crystal structures. This resulting pentapeptide SPSB-iNOS inhibitor (CP3) has a reduced macrocycle ring size, fewer nonbinding residues, and includes additional conformational constraints. CP3 has a greater affinity for SBSB2 ( K = 7 nM as determined by surface plasmon resonance) and strongly inhibits the SPSB2-iNOS interaction in macrophage cell lysates. We have also determined the crystal structure of CP3 in complex with human SPSB2, which reveals the structural basis for the increased potency of CP3 and validates the original design.
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http://dx.doi.org/10.1021/acschembio.8b00561DOI Listing
October 2018

Cyclic Hexapeptide Mimics of the LEDGF Integrase Recognition Loop in Complex with HIV-1 Integrase.

ChemMedChem 2018 08 6;13(15):1555-1565. Epub 2018 Jul 6.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Victoria, 3052, Australia.

The p75 splice variant of lens epithelium-derived growth factor (LEDGF) is a 75 kDa protein, which is recruited by the human immunodeficiency virus (HIV) to tether the pre-integration complex to the host chromatin and promote integration of proviral DNA into the host genome. We designed a series of small cyclic peptides that are structural mimics of the LEDGF binding domain, which interact with integrase as potential binding inhibitors. Herein we present the X-ray crystal structures, NMR studies, SPR analysis, and conformational studies of four cyclic peptides bound to the HIV-1 integrase core domain. Although the X-ray studies show that the peptides closely mimic the LEDGF binding loop, the measured affinities of the peptides are in the low millimolar range. Computational analysis using conformational searching and free energy calculations suggest that the low affinity of the peptides is due to mismatch between the low-energy solution and bound conformations.
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http://dx.doi.org/10.1002/cmdc.201800129DOI Listing
August 2018

Structure, Function, and Biosynthetic Origin of Octapeptin Antibiotics Active against Extensively Drug-Resistant Gram-Negative Bacteria.

Cell Chem Biol 2018 04 3;25(4):380-391.e5. Epub 2018 Feb 3.

Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia. Electronic address:

Resistance to the last-resort antibiotic colistin is now widespread and new therapeutics are urgently required. We report the first in toto chemical synthesis and pre-clinical evaluation of octapeptins, a class of lipopeptides structurally related to colistin. The octapeptin biosynthetic cluster consisted of three non-ribosomal peptide synthetases (OctA, OctB, and OctC) that produced an amphiphilic antibiotic, octapeptin C4, which was shown to bind to and depolarize membranes. While active against multi-drug resistant (MDR) strains in vitro, octapeptin C4 displayed poor in vivo efficacy, most likely due to high plasma protein binding. Nuclear magnetic resonance solution structures, empirical structure-activity and structure-toxicity models were used to design synthetic octapeptins active against MDR and extensively drug-resistant (XDR) bacteria. The scaffold was then subtly altered to reduce plasma protein binding, while maintaining activity against MDR and XDR bacteria. In vivo efficacy was demonstrated in a murine bacteremia model with a colistin-resistant P. aeruginosa clinical isolate.
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http://dx.doi.org/10.1016/j.chembiol.2018.01.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6560181PMC
April 2018

Design and Evaluation of Novel Polymyxin Fluorescent Probes.

Sensors (Basel) 2017 Nov 11;17(11). Epub 2017 Nov 11.

Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia.

Polymyxins (polymyxin B and colistin) are cyclic lipopeptide antibiotics that serve as a last-line defence against Gram-negative "superbugs". In the present study, two novel fluorescent polymyxin probes were designed through regio-selective modifications of the polymyxin B core structure at the -terminus and the hydrophobic motif at positions 6 and 7. The resulting probes, FADDI-285 and FADDI-286 demonstrated comparable antibacterial activity (MICs 2-8 mg/L) to polymyxin B and colistin (MICs 0.5-8 mg/L) against a panel of gram-negative clinical isolates of , and . These probes should prove to be of considerable utility for imaging cellular uptake and mechanistic investigations of these important last-line antibiotics.
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http://dx.doi.org/10.3390/s17112598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5713106PMC
November 2017

Methionine Ameliorates Polymyxin-Induced Nephrotoxicity by Attenuating Cellular Oxidative Stress.

Antimicrob Agents Chemother 2018 01 21;62(1). Epub 2017 Dec 21.

Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University, Melbourne, Victoria, Australia

Polymyxins are a last line of defense against multidrug-resistant Gram-negative pathogens. Recent pharmacological data show that intravenous polymyxins can cause nephrotoxicity in up to 60% of patients, and the plasma concentrations of polymyxins achieved with the currently recommended dosage regimens are suboptimal in a large proportion of patients. Simply increasing the daily dose of polymyxins is not possible due to nephrotoxicity. This study aimed to examine the protective effect of methionine against polymyxin-induced nephrotoxicity. Methionine (400 mg/kg of body weight), polymyxin B (35 mg/kg), a combination of methionine (100 or 400 mg/kg) and polymyxin B, and saline were administered to mice twice daily over 3.5 days. Kidneys were collected immediately at the end of the experiment for histological examination. The effect of methionine on the pharmacokinetics of polymyxin B was investigated in rats. The attenuation of polymyxin B (0.75 mM)-induced mitochondrial superoxide production by methionine (10.0 mM) was examined in rat kidney (NRK-52E) cells. Histological results revealed that the polymyxin-induced nephrotoxicity in mice was ameliorated by methionine in a dose-dependent manner. The methionine doses were well tolerated in the mice and rats, and the pharmacokinetics of polymyxin B in rats were not affected by methionine. In the group receiving polymyxin B-methionine, the total body clearance of polymyxin B was very similar to that in the group receiving polymyxin B alone (3.71 ± 0.57 versus 3.12 ± 1.66 ml/min/kg, > 0.05). A substantial attenuation of polymyxin-induced mitochondrial superoxide production in NRK-52E cells was observed following pretreatment with methionine. Our results demonstrate that coadministration of methionine significantly ameliorated polymyxin-induced nephrotoxicity and decreased mitochondrial superoxide production in renal tubular cells.
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http://dx.doi.org/10.1128/AAC.01254-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5740332PMC
January 2018

IP kinase Arg1 regulates cell wall homeostasis and surface architecture to promote clearance of Cryptococcus neoformans infection in a mouse model.

Virulence 2017 11 8;8(8):1833-1848. Epub 2017 Dec 8.

a Centre for Infectious Diseases and Microbiology, The Westmead Institute for Medical Research , 176 Hawkesbury road, Westmead NSW 2145 , Australia.

We previously identified a series of inositol polyphosphate kinases (IPKs), Arg1, Ipk1, Kcs1 and Asp1, in the opportunistic fungal pathogen Cryptococcus neoformans. Using gene deletion analysis, we characterized Arg1, Ipk1 and Kcs1 and showed that they act sequentially to convert IP to PP-IP (IP), a key metabolite promoting stress tolerance, metabolic adaptation and fungal dissemination to the brain. We have now directly characterized the enzymatic activity of Arg1, demonstrating that it is a dual specificity (IP/IP) kinase producing IP. We showed previously that IP is further phosphorylated by Ipk1 to produce IP, which is a substrate for the synthesis of PP-IP by Kcs1. Phenotypic comparison of the arg1Δ and kcs1Δ deletion mutants (both PP-IP-deficient) reveals that arg1Δ has the most deleterious phenotype: while PP-IP is essential for metabolic and stress adaptation in both mutant strains, PP-IP is dispensable for virulence-associated functions such as capsule production, cell wall organization, and normal N-linked mannosylation of the virulence factor, phospholipase B1, as these phenotypes were defective only in arg1Δ. The more deleterious arg1Δ phenotype correlated with a higher rate of arg1Δ phagocytosis by human peripheral blood monocytes and rapid arg1Δ clearance from lung in a mouse model. This observation is in contrast to kcs1Δ, which we previously reported establishes a chronic, confined lung infection. In summary, we show that Arg1 is the most crucial IPK for cryptococcal virulence, conveying PP-IP-dependent and novel PP-IP-independent functions.
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http://dx.doi.org/10.1080/21505594.2017.1385692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5810508PMC
November 2017

Synthesis and biological evaluation of 8-aryl-2-morpholino-7-O-substituted benzo[e][1,3]oxazin-4-ones against DNA-PK, PI3K, PDE3A enzymes and platelet aggregation.

Bioorg Med Chem 2017 10 15;25(20):5531-5536. Epub 2017 Aug 15.

Pharmacy and Applied Science, La Trobe Institute for Molecular Science, La Trobe University, P.O. Box 199 Bendigo, VIC 3552, Australia. Electronic address:

A series of 40 7-(O-substituted)-2-morpholino-8-aryl-4H-benzo[e][1,3]oxazin-4-one derivatives was synthesized. They were prepared via synthesis of a key precursor, 8-bromo-7-hydroxy-2-morpholino-4H-benzo[e][1,3]oxazin-4-one 13 which was amenable to ether synthesis at the 7-position and Suzuki coupling at the 8-position. The 2 protons of 7-OCH in compounds 18g, 18h, 18i, 18l and 18m prove to be magnetically non-equivalent, atropisomerism (axial chirality), as result of sterically hindered rotation of the bulky 8-aryl-substituent. The products were evaluated for their activities against PI3K isoforms, DNA-PK and PDE3. The results showed that this substitution pattern has a deleterious effect on PI3K activities, which may arise from steric hindrance in the active site. PI3Kδ was somewhat more tolerant of this substitution particularly where 8-(4-methoxylphenyl) substituents were present (ICs∼2-3μM). Good activities against PDE3 were also obtained for compounds, with particular members of the 7-(2-pyridinyl) methoxy series 19 showing good inhibition (ICs∼2-3μM), comparable to previously described analogues. A piperazinyl derivative 26a effectively inhibited ADP-induced platelet aggregation with an IC of 8μM.
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http://dx.doi.org/10.1016/j.bmc.2017.08.022DOI Listing
October 2017

Investigating the Interaction of Octapeptin A3 with Model Bacterial Membranes.

ACS Infect Dis 2017 08 11;3(8):606-619. Epub 2017 Jul 11.

Drug Development and Innovation, Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University , Parkville 3052, Victoria Australia.

Octapeptins are cyclic lipopeptides with a broader spectrum of activity against fungi and polymyxin-resistant Gram-negative and Gram-positive bacteria. In the present study, we investigated the interaction of octapeptin A3 with asymmetric outer membrane models of Gram-negative pathogen Pseudomonas aeruginosa using neutron reflectometry, together with fluorimetric and calorimetry methods. For the first time, our neutron reflectometry results reveal that the interaction of octapeptin A3 with the Gram-negative outer membrane involves an initial transient polar interaction with the phospholipid and lipid A headgroups, followed by the penetration of the entire octapeptin molecule into the fatty acyl core of the outer membrane. This mechanism contrasts with that of polymyxin B, which specifically targets lipid A, whereas octapeptins appear to target both lipid A and phospholipids. Furthermore, the mechanism of octapeptins does not appear to be highly dependent on an initial complementary electrostatic interaction with lipid A, which accounts for their ability to bind to lipid A of polymyxin-resistant Gram-negative bacteria that is modified with cationic moieties that act to electrostatically repel the cationic polymyxin molecule. The presented findings shed new light on the mechanism whereby octapeptins penetrate the outer membrane of polymyxin-resistant Gram-negative pathogens and highlight their potential as candidates for development as new antibiotics against problematic multi-drug-resistant pathogens.
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http://dx.doi.org/10.1021/acsinfecdis.7b00065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955700PMC
August 2017

Parallel and antiparallel cyclic d/l peptide nanotubes.

Chem Commun (Camb) 2017 Jun 5;53(49):6613-6616. Epub 2017 Jun 5.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia.

Nanotubes made from H-bonded cyclic d/l peptide (CP) subunits have great potential for the construction of nanomaterials of wide chemical and structural diversity but, to date, difficulties in structural characterisation have restricted development of these materials. We present the first crystal structures of continuous CP nanotubes with antiparallel and parallel stacking arrangements, assembled separately from two peptides; cyclo[(Asp-d-Leu-Lys-d-Leu)] and cyclo[(Asp-d-Ala-Lys-d-Ala)].
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http://dx.doi.org/10.1039/c7cc00846eDOI Listing
June 2017

Characterization of the Polymyxin D Synthetase Biosynthetic Cluster and Product Profile of Paenibacillus polymyxa ATCC 10401.

J Nat Prod 2017 05 2;80(5):1264-1274. Epub 2017 May 2.

Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University , Clayton, Victoria 3800, Australia.

The increasing prevalence of polymyxin-resistant bacteria has stimulated the search for improved polymyxin lipopeptides. Here we describe the sequence and product profile for polymyxin D nonribosomal peptide synthetase from Paenibacillus polymyxa ATCC 10401. The polymyxin D synthase gene cluster comprised five genes that encoded ABC transporters (pmxC and pmxD) and enzymes responsible for the biosynthesis of polymyxin D (pmxA, pmxB, and pmxE). Unlike polymyxins B and E, polymyxin D contains d-Ser at position 3 as opposed to l-α,γ-diaminobutyric acid and has an l-Thr at position 7 rather than l-Leu. Module 3 of pmxE harbored an auxiliary epimerization domain that catalyzes the conversion of l-Ser to the d-form. Structural modeling suggested that the adenylation domains of module 3 in PmxE and modules 6 and 7 in PmxA could bind amino acids with larger side chains than their preferred substrate. Feeding individual amino acids into the culture media not only affected production of polymyxins D and D but also led to the incorporation of different amino acids at positions 3, 6, and 7 of polymyxin D. Interestingly, the unnatural polymyxin analogues did not show antibiotic activity against a panel of Gram-negative clinical isolates, while the natural polymyxins D and D exhibited excellent in vitro antibacterial activity and were efficacious against Klebsiella pneumoniae and Acinetobacter baumannii in a mouse blood infection model. The results demonstrate the excellent antibacterial activity of these unusual d-Ser polymxyins and underscore the possibility of incorporating alternate amino acids at positions 3, 6, and 7 of polymyxin D via manipulation of the polymyxin nonribosomal biosynthetic machinery.
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http://dx.doi.org/10.1021/acs.jnatprod.6b00807DOI Listing
May 2017

Pharmacokinetics of the Individual Major Components of Polymyxin B and Colistin in Rats.

J Nat Prod 2017 01 12;80(1):225-229. Epub 2017 Jan 12.

Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University , Clayton, Victoria 3800, Australia.

The pharmacokinetics of polymyxin B, polymyxin B, colistin A, and colistin B were investigated in a rat model following intravenous administration (0.8 mg/kg) of each individual component. Plasma and urine concentrations were determined by LC-MS/MS, and plasma protein binding was measured by ultracentrifugation. Total and unbound pharmacokinetic parameters for each component were calculated using noncompartmental analysis. All of the polymyxin components had a similar clearance, volume of distribution, elimination half-life, and urinary recovery. The area under the concentration-time curve for polymyxins B and B was greater than those of colistins A and B. Colistin A (56.6 ± 9.25%) and colistin B (41.7 ± 12.4%) displayed lower plasma protein binding in rat plasma compared to polymyxin B (82.3 ± 4.30%) and polymyxin B (68.4 ± 3.50%). These differences in plasma protein binding potentially equate to significant differences in unbound pharmacokinetics, highlighting the need for more stringent standardization of the composition of commercial products currently available for clinical use.
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http://dx.doi.org/10.1021/acs.jnatprod.6b01176DOI Listing
January 2017

Functional Characterization of the Unique Terminal Thioesterase Domain from Polymyxin Synthetase.

Biochemistry 2017 01 17;56(4):657-668. Epub 2017 Jan 17.

Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University , Parkville, Victoria 3800, Australia.

Polymyxins remain one of the few antibiotics available for treating antibiotic resistant bacteria. Here we describe polymyxin B thioesterase which performs the final step in polymyxin B biosynthesis. Isolated thioesterase catalyzed cyclization of an N-acetylcystamine polymyxin B analogue to form polymyxin B. The thioesterase contained a catalytic cysteine unlike most thioesterases which possess a serine. Supporting this, incubation of polymyxin B thioesterase with reducing agents abolished enzymatic activity, while mutation of the catalytic cysteine to serine significantly decreased activity. NMR spectroscopy demonstrated that uncyclized polymyxin B was disordered in solution, unlike other thioesterase substrates which adopt a transient structure similar to their product. Modeling showed the thioesterase substrate-binding cleft was highly negatively charged, suggesting a mechanism for the cyclization of the substrate. These studies provide new insights into the role of polymyxin thioesterase in polymyxin biosynthesis and highlight its potential use for the chemoenzymatic synthesis of polymyxin lipopeptides.
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http://dx.doi.org/10.1021/acs.biochem.6b01139DOI Listing
January 2017

Synthesis of linear and angular aryl-morpholino-naphth-oxazines, their DNA-PK, PI3K, PDE3A and antiplatelet activity.

Bioorg Med Chem Lett 2016 11 5;26(22):5534-5538. Epub 2016 Oct 5.

Pharmacy and Applied Science, La Trobe Institute for Molecular Science, La Trobe University, PO Box 199, Bendigo, VIC 3552, Australia. Electronic address:

To continue our study of 2-morpholino-benzoxazine based compounds, which show useful activity against PI3K family enzymes or antiplatelet activity, we designed and synthesized a series of linear 6.7-fused, 5,6-angular fused and 7,8-angular fused-aryl-morpholino-naphth-oxazines. The compounds were prepared from substituted 2-hydroxynaphthoic acid to give the corresponding thioxo analogues 8, 9, 15 and 19. The thioxo products were then converted to the morpholino substituted analogue. The aryl group was introduced by Suzuki coupling of bromo precursors. The products were evaluated for activity at PI3K family enzymes and as platelet aggregation inhibitors and compared to reported unsubstituted analogues. The linear 6.7-fused product 13a and 13b were moderated potent but selective PI3Kδ isoform inhibitors (IC=7.7 and 5.61μM). Good antiplatelet activity was noticed for the angular 7,8-fused compounds 22a, b, k and l with IC=3.0,14.0, 2.0 and 5.0μM respectively. The antiplatelet activity is independent of PDE3.
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http://dx.doi.org/10.1016/j.bmcl.2016.10.003DOI Listing
November 2016

Class II Phosphoinositide 3-Kinases as Novel Drug Targets.

J Med Chem 2017 01 21;60(1):47-65. Epub 2016 Oct 21.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus) , 381 Royal Parade, Parkville, Victoria 3052, Australia.

The phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases central to regulating a wide range of important intracellular processes. Despite the vast knowledge around class I PI3Ks, the class II PI3Ks have been neglected, seemingly only due to the chronology of their discovery. Here we focus on the cellular functions of the three class II PI3K isoforms, PI3KC2α, PI3KC2β, and PI3KC2γ, in different cell systems and underline the emerging importance of these enzymes in different physiological and pathological contexts. We provide an overview on the current development of class II PI3 kinase inhibitors and outline the potential use for such inhibitors. The field is in its infancy as compared to their class I counterparts. Nevertheless, recent advances in understanding the roles of class II PI3 kinases in different pathological contexts is leading to an increased interest in the development of specific inhibitors that can provide potential novel pharmacological tools.
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http://dx.doi.org/10.1021/acs.jmedchem.6b00963DOI Listing
January 2017

A Novel Chemical Biology Approach for Mapping of Polymyxin Lipopeptide Antibody Binding Epitopes.

ACS Infect Dis 2016 05 29;2(5):341-51. Epub 2016 Mar 29.

CSIRO Manufacturing , Parkville, Victoria 3052, Australia.

Polymyxins B and E (i.e., colistin) are a family of naturally occurring lipopeptide antibiotics that are our last line of defense against multidrug resistant (MDR) Gram-negative pathogens. Unfortunately, nephrotoxicity is a dose-limiting factor for polymyxins that limits their clinical utility. Our recent studies demonstrate that polymyxin-induced nephrotoxicity is a result of their extensive accumulation in renal tubular cells. The design and development of safer, novel polymyxin lipopeptides is hampered by our limited understanding of their complex structure-nephrotoxicity relationships. This is the first study to employ a novel targeted chemical biology approach to map the polymyxin recognition epitope of a commercially available polymyxin mAb and demonstrate its utility for mapping the kidney distribution of a novel, less nephrotoxic polymyxin lipopeptide. Eighteen novel polymyxin lipopeptide analogues were synthesized with modifications in the polymyxin core domains, namely, the N-terminal fatty acyl region, tripeptide linear segment, and cyclic heptapeptide. Surface plasmon resonance epitope mapping revealed that the monoclonal antibody (mAb) recognition epitope consisted of the hydrophobic domain (N-terminal fatty acyl and position 6/7) and diaminobutyric acid (Dab) residues at positions 3, 5, 8, and 9 of the polymyxin molecule. Structural diversity within the hydrophobic domains and Dab 3 position are tolerated. Enlightened with an understating of the structure-binding relationships between the polymyxin mAb and the core polymyxin scaffold, we can now rationally employ the mAb to probe the kidney distribution of novel polymyxin lipopeptides. This information will be vital in the design of novel, safer polymyxins through chemical tailoring of the core scaffold and exploration of the elusive/complex polymyxin structure-nephrotoxicity relationships.
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http://dx.doi.org/10.1021/acsinfecdis.6b00031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5321607PMC
May 2016

Development of single and mixed isoform selectivity PI3Kδ inhibitors by targeting Asn836 of PI3Kδ.

Bioorg Med Chem Lett 2016 10 11;26(19):4790-4794. Epub 2016 Aug 11.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.

A series of PI3Kδ inhibitors derived from the pan-PI3K inhibitor ZSTK474 was prepared that target a non-conserved region of the catalytic site. Dependent upon the substituents present, these analogues show different levels of isoform selectivity and sensitivity to the mutation N836D in PI3Kδ. As a marker of 'on-target' activity and permeability, a selection of the most potent PI3Kδ inhibitors were shown to inhibit pAkt production in the Nawalma Burkitt lymphoma cell line.
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http://dx.doi.org/10.1016/j.bmcl.2016.08.028DOI Listing
October 2016

Antimicrobial Activity and Toxicity of the Major Lipopeptide Components of Polymyxin B and Colistin: Last-line Antibiotics against Multidrug-Resistant Gram-negative Bacteria.

ACS Infect Dis 2015 Nov 4;1(11):568-575. Epub 2015 Sep 4.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville 3052, Victoria, Australia.

Polymyxin B and colistin are currently used as a 'last-line' treatment for multidrug-resistant Gram-negative bacteria. However very little is known about the pharmacological differences between polymyxin B, polymyxin B, colistin A, colistin B, the major cyclic lipopeptides components present in polymyxin B and colistin products. Here, we report on the and antimicrobial activity and toxicity of these major lipopeptide components. All four lipopeptides had comparable MICs (<0.125-4 mg/L) against a panel of clinical Gram-negative isolates. They also had comparable antimicrobial activity (Δlog CFU/mL >-3) and nephrotoxicity (mild to moderate histological damage) in mouse models. However, polymyxin B and colistin A showed significantly higher (> 3-fold) apoptotic effect on human kidney proximal tubular HK-2 cells than polymyxin B2 and colistin B, respectively. Compared to the commercial polymyxin and colistin products, the individual lipopeptide components had slightly more antimicrobial activity. Our results highlight the need to re-assess pharmacopoeial standards for polymyxins B and colistin and to standardize the composition of the different commercial products of polymyxin antibiotics.
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http://dx.doi.org/10.1021/acsinfecdis.5b00085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4980087PMC
November 2015