Publications by authors named "Martin J Scanlon"

84 Publications

NMR fragment screening reveals a novel small molecule binding site near the catalytic surface of the disulfide-dithiol oxidoreductase enzyme DsbA from Burkholderia pseudomallei.

J Biomol NMR 2020 Nov 6;74(10-11):595-611. Epub 2020 Aug 6.

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

The presence of suitable cavities or pockets on protein structures is a general criterion for a therapeutic target protein to be classified as 'druggable'. Many disease-related proteins that function solely through protein-protein interactions lack such pockets, making development of inhibitors by traditional small-molecule structure-based design methods much more challenging. The 22 kDa bacterial thiol oxidoreductase enzyme, DsbA, from the gram-negative bacterium Burkholderia pseudomallei (BpsDsbA) is an example of one such target. The crystal structure of oxidized BpsDsbA lacks well-defined surface pockets. BpsDsbA is required for the correct folding of numerous virulence factors in B. pseudomallei, and genetic deletion of dsbA significantly attenuates B. pseudomallei virulence in murine infection models. Therefore, BpsDsbA is potentially an attractive drug target. Herein we report the identification of a small molecule binding site adjacent to the catalytic site of oxidized BpsDsbA. H CPMG relaxation dispersion NMR measurements suggest that the binding site is formed transiently through protein dynamics. Using fragment-based screening, we identified a small molecule that binds at this site with an estimated affinity of K ~ 500 µM. This fragment inhibits BpsDsbA enzymatic activity in vitro. The binding mode of this molecule has been characterized by NMR data-driven docking using HADDOCK. These data provide a starting point towards the design of more potent small molecule inhibitors of BpsDsbA.
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http://dx.doi.org/10.1007/s10858-020-00339-5DOI Listing
November 2020

Rapid Elaboration of Fragments into Leads by X-ray Crystallographic Screening of Parallel Chemical Libraries (REFiL).

J Med Chem 2020 07 24;63(13):6863-6875. Epub 2020 Jun 24.

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

A bottleneck in fragment-based lead development is the lack of systematic approaches to elaborate the initial fragment hits, which usually bind with low affinity to their target. Herein, we describe an analysis using X-ray crystallography of a diverse library of compounds prepared using microscale parallel synthesis. This approach yielded an 8-fold increase in affinity and detailed structural information for the resulting complex, providing an efficient and broadly applicable approach to early fragment development.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00111DOI Listing
July 2020

The Fragment-Based Development of a Benzofuran Hit as a New Class of DsbA Inhibitors.

Molecules 2019 Oct 18;24(20). Epub 2019 Oct 18.

Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne 3086, Australia.

A fragment-based drug discovery approach was taken to target the thiol-disulfide oxidoreductase enzyme DsbA from (DsbA). This enzyme is critical for the correct folding of virulence factors in many pathogenic Gram-negative bacteria, and small molecule inhibitors can potentially be developed as anti-virulence compounds. Biophysical screening of a library of fragments identified several classes of fragments with affinity to DsbA. One hit with high mM affinity, 2-(6-bromobenzofuran-3-yl)acetic acid (), was chemically elaborated at several positions around the scaffold. X-ray crystal structures of the elaborated analogues showed binding in the hydrophobic binding groove adjacent to the catalytic disulfide bond of DsbA. Binding affinity was calculated based on NMR studies and compounds and were identified as the highest affinity binders with dissociation constants () of 326 ± 25 and 341 ± 57 µM respectively. This work suggests the potential to develop benzofuran fragments into a novel class of DsbA inhibitors.
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http://dx.doi.org/10.3390/molecules24203756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832960PMC
October 2019

Identification of the Binding Site of Apical Membrane Antigen 1 (AMA1) Inhibitors Using a Paramagnetic Probe.

ChemMedChem 2019 03 13;14(5):603-612. Epub 2019 Feb 13.

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

Apical membrane antigen 1 (AMA1) is essential for the invasion of host cells by malaria parasites. Several small-molecule ligands have been shown to bind to a conserved hydrophobic cleft in Plasmodium falciparum AMA1. However, a lack of detailed structural information on the binding pose of these molecules has hindered their further optimisation as inhibitors. We have developed a spin-labelled peptide based on RON2, the native binding partner of AMA1, to probe the binding sites of compounds on PfAMA1. The crystal structure of this peptide bound to PfAMA1 shows that it binds at one end of the hydrophobic groove, leaving much of the binding site unoccupied and allowing fragment hits to bind without interference. In paramagnetic relaxation enhancement (PRE)-based NMR screening, the H relaxation rates of compounds binding close to the probe were enhanced. Compounds experienced different degrees of PRE as a result of their different orientations relative to the spin label while bound to AMA1. Thus, PRE-derived distance constraints can be used to identify binding sites and guide further hit optimisation.
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http://dx.doi.org/10.1002/cmdc.201800802DOI Listing
March 2019

A ligand-induced structural change in fatty acid-binding protein 1 is associated with potentiation of peroxisome proliferator-activated receptor α agonists.

J Biol Chem 2019 03 31;294(10):3720-3734. Epub 2018 Dec 31.

From Medicinal Chemistry,

Peroxisome proliferator-activated receptor α (PPARα) is a transcriptional regulator of lipid metabolism. GW7647 is a potent PPARα agonist that must reach the nucleus to activate this receptor. In cells expressing human fatty acid-binding protein 1 (FABP1), GW7647 treatment increases FABP1's nuclear localization and potentiates GW7647-mediated PPARα activation; GW7647 is less effective in cells that do not express FABP1. To elucidate the underlying mechanism, here we substituted residues in FABP1 known to dictate lipid signaling by other intracellular lipid-binding proteins. Substitutions of Lys-20 and Lys-31 to Ala in the FABP1 helical cap affected neither its nuclear localization nor PPARα activation. In contrast, Ala substitution of Lys-57, Glu-77, and Lys-96, located in the loops adjacent to the ligand-binding portal region, abolished both FABP1 nuclear localization and GW7647-induced PPARα activation but had little effect on GW7647-FABP1 binding affinity. Using solution NMR spectroscopy, we determined the WT FABP1 structure and analyzed the dynamics in the apo and GW7647-bound structures of both the WT and the K57A/E77A/K96A triple mutant. We found that GW7647 binding causes little change in the FABP1 backbone, but solvent exposes several residues in the loops around the portal region, including Lys-57, Glu-77, and Lys-96. These residues also become more solvent-exposed upon binding of FABP1 with the endogenous PPARα agonist oleic acid. Together with previous observations, our findings suggest that GW7647 binding stabilizes a FABP1 conformation that promotes its interaction with PPARα. We conclude that full PPARα agonist activity of GW7647 requires FABP1-dependent transport and nuclear localization processes.
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http://dx.doi.org/10.1074/jbc.RA118.006848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416440PMC
March 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

Design of a Fragment-Screening Library.

Methods Enzymol 2018 19;610:97-115. Epub 2018 Oct 19.

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

Herein we describe a method for the design, purchase, and assembly of a fragment-screening library from a list of commercially available compounds. The computational tools used in assessment of compound properties as well as the workflow for compound selection are provided for reference as implemented in commercially available software that is free and accessible to most academic users. The workflow can be modified as necessary to generate a fit-for-purpose fragment library with the desired compound property profiles. An analytical process for assessing the quality, identity, and suitability of a purchased fragment for inclusion in a screening collection is described. Results from our in-house library are presented as an example of compound progression through this quality control process.
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http://dx.doi.org/10.1016/bs.mie.2018.09.018DOI Listing
June 2019

Structural and biochemical insights into the disulfide reductase mechanism of DsbD, an essential enzyme for neisserial pathogens.

J Biol Chem 2018 10 4;293(43):16559-16571. Epub 2018 Sep 4.

From the Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne 3086, Victoria, Australia,

The worldwide incidence of neisserial infections, particularly gonococcal infections, is increasingly associated with antibiotic-resistant strains. In particular, extensively drug-resistant strains that are resistant to third-generation cephalosporins are a major public health concern. There is a pressing clinical need to identify new targets for the development of antibiotics effective against -specific processes. In this study, we report that the bacterial disulfide reductase DsbD is highly prevalent and conserved among spp. and that this enzyme is essential for survival of DsbD is a membrane-bound protein that consists of two periplasmic domains, n-DsbD and c-DsbD, which flank the transmembrane domain t-DsbD. In this work, we show that the two functionally essential periplasmic domains of DsbD catalyze electron transfer reactions through unidirectional interdomain interactions, from reduced c-DsbD to oxidized n-DsbD, and that this process is not dictated by their redox potentials. Structural characterization of the n- and c-DsbD domains in both redox states provides evidence that steric hindrance reduces interactions between the two periplasmic domains when n-DsbD is reduced, thereby preventing a futile redox cycle. Finally, we propose a conserved mechanism of electron transfer for DsbD and define the residues involved in domain-domain recognition. Inhibitors of the interaction of the two DsbD domains have the potential to be developed as anti-neisserial agents.
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http://dx.doi.org/10.1074/jbc.RA118.004847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204915PMC
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

Dietary docosahexaenoic acid supplementation enhances expression of fatty acid-binding protein 5 at the blood-brain barrier and brain docosahexaenoic acid levels.

J Neurochem 2018 07;146(2):186-197

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

The cytoplasmic trafficking of docosahexaenoic acid (DHA), a cognitively beneficial fatty acid, across the blood-brain barrier (BBB) is governed by fatty acid-binding protein 5 (FABP5). Lower levels of brain DHA have been observed in Alzheimer's disease (AD), which is associated with diminished BBB expression of FABP5. Therefore, up-regulating FABP5 expression at the BBB may be a novel approach for enhancing BBB transport of DHA in AD. DHA supplementation has been shown to be beneficial in various mouse models of AD, and therefore, the aim of this study was to determine whether DHA has the potential to up-regulate the BBB expression of FABP5, thereby enhancing its own uptake into the brain. Treating human brain microvascular brain endothelial (hCMEC/D3) cells with the maximum tolerable concentration of DHA (12.5 μM) for 72 h resulted in a 1.4-fold increase in FABP5 protein expression. Associated with this was increased expression of fatty acid transport proteins 1 and 4. To study the impact of dietary DHA supplementation, 6- to 8-week-old C57BL/6 mice were fed with a control diet or a DHA-enriched diet for 21 days. Brain microvascular FABP5 protein expression was up-regulated 1.7-fold in mice fed the DHA-enriched diet, and this was associated with increased brain DHA levels (1.3-fold). Despite an increase in brain DHA levels, reduced BBB transport of C-DHA was observed over a 1 min perfusion, possibly as a result of competitive binding to FABP5 between dietary DHA and C-DHA. This study has demonstrated that DHA can increase BBB expression of FABP5, as well as fatty acid transporters, overall increasing brain DHA levels.
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http://dx.doi.org/10.1111/jnc.14342DOI Listing
July 2018

Fragment-Based Discovery of Inhibitors of the Bacterial DnaG-SSB Interaction.

Antibiotics (Basel) 2018 Feb 22;7(1). Epub 2018 Feb 22.

Molecular Horizons and School of Chemistry, University of Wollongong, and Illawarra Health and Medical Research Institute, Wollongong, NSW 2522, Australia.

In bacteria, the DnaG primase is responsible for synthesis of short RNA primers used to initiate chain extension by replicative DNA polymerase(s) during chromosomal replication. Among the proteins with which DnaG interacts is the single-stranded DNA-binding protein, SSB. The C-terminal hexapeptide motif of SSB (DDDIPF; SSB-Ct) is highly conserved and is known to engage in essential interactions with many proteins in nucleic acid metabolism, including primase. Here, fragment-based screening by saturation-transfer difference nuclear magnetic resonance (STD-NMR) and surface plasmon resonance assays identified inhibitors of the primase/SSB-Ct interaction. Hits were shown to bind to the SSB-Ct-binding site using N-¹H HSQC spectra. STD-NMR was used to demonstrate binding of one hit to other SSB-Ct binding partners, confirming the possibility of simultaneous inhibition of multiple protein/SSB interactions. The fragment molecules represent promising scaffolds on which to build to discover new antibacterial compounds.
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http://dx.doi.org/10.3390/antibiotics7010014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5872125PMC
February 2018

Production, biophysical characterization and initial crystallization studies of the N- and C-terminal domains of DsbD, an essential enzyme in Neisseria meningitidis.

Acta Crystallogr F Struct Biol Commun 2018 01 1;74(Pt 1):31-38. Epub 2018 Jan 1.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria 3086, Australia.

The membrane protein DsbD is a reductase that acts as an electron hub, translocating reducing equivalents from cytoplasmic thioredoxin to a number of periplasmic substrates involved in oxidative protein folding, cytochrome c maturation and oxidative stress defence. DsbD is a multi-domain protein consisting of a transmembrane domain (t-DsbD) flanked by two periplasmic domains (n-DsbD and c-DsbD). Previous studies have shown that DsbD is required for the survival of the obligate human pathogen Neisseria meningitidis. To help understand the structural and functional aspects of N. meningitidis DsbD, the two periplasmic domains which are required for electron transfer are being studied. Here, the expression, purification and biophysical properties of n-NmDsbD and c-NmDsbD are described. The crystallization and crystallographic analysis of n-NmDsbD and c-NmDsbD are also described in both redox states, which differ only in the presence or absence of a disulfide bond but which crystallized in completely different conditions. Crystals of n-NmDsbD, n-NmDsbD, c-NmDsbD and c-NmDsbD diffracted to 2.3, 1.6, 2.3 and 1.7 Å resolution and belonged to space groups P23, P321, P4 and P121, respectively.
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http://dx.doi.org/10.1107/S2053230X17017800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947690PMC
January 2018

Fatty Acid-Binding Protein 5 Mediates the Uptake of Fatty Acids, but not Drugs, Into Human Brain Endothelial Cells.

J Pharm Sci 2018 04 14;107(4):1185-1193. Epub 2017 Dec 14.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia. Electronic address:

The purpose of this study was to examine the involvement of fatty acid-binding protein 5 (FABP5), a lipid-binding protein expressed at the blood-brain barrier (BBB), in fatty acid and drug uptake into human brain endothelial cells. Following transfection with siRNA against hFABP5, human brain endothelial cell (hCMEC/D3) uptake of lipophilic ligands with varying affinity to FABP5 was assessed with intracellular concentrations quantified by liquid scintillation counting, HPLC, or LCMS/MS. The in situ BBB transport of [H]-diazepam was also assessed in wild type and FABP5-deficient mice. hFABP5 siRNA reduced FABP5 expression in hCMEC/D3 cells by 39.9 ± 3.8% (mRNA) and 38.8 ± 6.6% (protein; mean ± SEM), leading to a reduction in uptake of [C]-lauric acid, [H]-oleic acid, and [C]-stearic acid by 37.5 ± 8.8%, 41.7 ± 11.6%, and 50.7 ± 13.6%, respectively, over 1 min. No significant changes in [C]-diazepam, pioglitazone, and troglitazone uptake were detected following FABP5 knockdown in hCMEC/D3 cells. Similarly, no difference in BBB transport of [H]-diazepam was observed between wild type and FABP5-deficient mice. Therefore, although FABP5 facilitates brain endothelial cell uptake of fatty acids, it has limited effects on brain endothelial cell uptake and BBB transport of drugs with lower affinity for FABP5.
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http://dx.doi.org/10.1016/j.xphs.2017.11.024DOI Listing
April 2018

Inhibition of Diverse DsbA Enzymes in Multi-DsbA Encoding Pathogens.

Antioxid Redox Signal 2018 09 1;29(7):653-666. Epub 2018 Feb 1.

2 Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University , Bundoora, Australia .

Aims: DsbA catalyzes disulfide bond formation in secreted and outer membrane proteins in bacteria. In pathogens, DsbA is a major facilitator of virulence constituting a target for antivirulence antimicrobial development. However, many pathogens encode multiple and diverse DsbA enzymes for virulence factor folding during infection. The aim of this study was to determine whether our recently identified inhibitors of Escherichia coli K-12 DsbA can inhibit the diverse DsbA enzymes found in two important human pathogens and attenuate their virulence.

Results: DsbA inhibitors from two chemical classes (phenylthiophene and phenoxyphenyl derivatives) inhibited the virulence of uropathogenic E. coli and Salmonella enterica serovar Typhimurium, encoding two and three diverse DsbA homologues, respectively. Inhibitors blocked the virulence of dsbA null mutants complemented with structurally diverse DsbL and SrgA, suggesting that they were not selective for prototypical DsbA. Structural characterization of DsbA-inhibitor complexes showed that compounds from each class bind in a similar region of the hydrophobic groove adjacent to the Cys30-Pro31-His32-Cys33 (CPHC) active site. Modeling of DsbL- and SrgA-inhibitor interactions showed that these accessory enzymes could accommodate the inhibitors in their different hydrophobic grooves, supporting our in vivo findings. Further, we identified highly conserved residues surrounding the active site for 20 diverse bacterial DsbA enzymes, which could be exploited in developing inhibitors with a broad spectrum of activity. Innovation and Conclusion: We have developed tools to analyze the specificity of DsbA inhibitors in bacterial pathogens encoding multiple DsbA enzymes. This work demonstrates that DsbA inhibitors can be developed to target diverse homologues found in bacteria. Antioxid. Redox Signal. 29, 653-666.
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http://dx.doi.org/10.1089/ars.2017.7104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6067686PMC
September 2018

Reduced blood-brain barrier expression of fatty acid-binding protein 5 is associated with increased vulnerability of APP/PS1 mice to cognitive deficits from low omega-3 fatty acid diets.

J Neurochem 2018 01;144(1):81-92

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Vic., Australia.

Lower levels of the cognitively beneficial docosahexaenoic acid (DHA) are often observed in Alzheimer's disease (AD) brains. Brain DHA levels are regulated by the blood-brain barrier (BBB) transport of plasma-derived DHA, a process facilitated by fatty acid-binding protein 5 (FABP5). This study reports a 42.1 ± 12.6% decrease in the BBB transport of C-DHA in 8-month-old AD transgenic mice (APPswe,PSEN1∆E9) relative to wild-type mice, associated with a 34.5 ± 6.7% reduction in FABP5 expression in isolated brain capillaries of AD mice. Furthermore, short-term spatial and recognition memory deficits were observed in AD mice on a 6-month n-3 fatty acid-depleted diet, but not in AD mice on control diet. This intervention led to a dramatic reduction (41.5 ± 11.9%) of brain DHA levels in AD mice. This study demonstrates FABP5 deficiency and impaired DHA transport at the BBB are associated with increased vulnerability to cognitive deficits in mice fed an n-3 fatty acid-depleted diet, in line with our previous studies demonstrating a crucial role of FABP5 in BBB transport of DHA and cognitive function.
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http://dx.doi.org/10.1111/jnc.14249DOI Listing
January 2018

The first total synthesis and solution structure of a polypeptin, PE2, a cyclic lipopeptide with broad spectrum antibiotic activity.

Org Biomol Chem 2017 Aug;15(34):7173-7180

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

The first total synthesis of a polypeptin, PE2, as well as its solution structure is reported. Synthesis in optically pure form confirms the proposed stereochemistry of the polypeptins at the 3-position on the 3-hydroxy depsipeptide moiety. We have also determined the NMR structure of PE2 in aqueous solution, showing it to form a stable ring conformation. The synthetic peptide shows anti-bacterial activity consistent with reports for naturally derived counterparts.
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http://dx.doi.org/10.1039/c7ob01493gDOI Listing
August 2017

H, N, C and C assignments of the two periplasmic domains of Neisseria meningitidis DsbD.

Biomol NMR Assign 2017 Oct 6;11(2):181-186. Epub 2017 Jun 6.

La Trobe Institute for Molecular Science, La Trobe University, Bundoora, VIC, Australia.

DsbD is a disulfide bond reductase present in the inner membrane of many Gamma-Proteobacteria. In the human pathogen Neisseria meningitidis, DsbD is required for viability and represents a potential target for the development of antibiotics. Here we report the chemical shift assignments (H, N, C and C) for the reduced and oxidized forms of the two periplasmic domains of N. meningitidis DsbD, n-NmDsbD and c-NmDsbD. The backbone amide resonances in all four forms were completely assigned, and the secondary structures for the core regions of the proteins were calculated using C shifts. The reduced and oxidized forms of each domain have similar secondary shifts suggesting they retain the same fold. We anticipate that these data will provide an important basis for studying the interaction between n-NmDsbD and c-NmDsbD, which is required for electron transfer across the bacterial cytoplasmic membrane.
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http://dx.doi.org/10.1007/s12104-017-9743-xDOI Listing
October 2017

Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.

Sci Transl Med 2017 05;9(392)

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

Typically considered to be cell surface sensors of extracellular signals, heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) control many pathophysiological processes and are the target of 30% of therapeutic drugs. Activated receptors redistribute to endosomes, but researchers have yet to explore whether endosomal receptors generate signals that control complex processes in vivo and are viable therapeutic targets. We report that the substance P (SP) neurokinin 1 receptor (NKR) signals from endosomes to induce sustained excitation of spinal neurons and pain transmission and that specific antagonism of the NKR in endosomes with membrane-anchored drug conjugates provides more effective and sustained pain relief than conventional plasma membrane-targeted antagonists. Pharmacological and genetic disruption of clathrin, dynamin, and β-arrestin blocked SP-induced NKR endocytosis and prevented SP-stimulated activation of cytosolic protein kinase C and nuclear extracellular signal-regulated kinase, as well as transcription. Endocytosis inhibitors prevented sustained SP-induced excitation of neurons in spinal cord slices in vitro and attenuated nociception in vivo. When conjugated to cholestanol to promote endosomal targeting, NKR antagonists selectively inhibited endosomal signaling and sustained neuronal excitation. Cholestanol conjugation amplified and prolonged the antinociceptive actions of NKR antagonists. These results reveal a critical role for endosomal signaling of the NKR in the complex pathophysiology of pain and demonstrate the use of endosomally targeted GPCR antagonists.
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http://dx.doi.org/10.1126/scitranslmed.aal3447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034632PMC
May 2017

Fragment library screening identifies hits that bind to the non-catalytic surface of Pseudomonas aeruginosa DsbA1.

PLoS One 2017 27;12(3):e0173436. Epub 2017 Mar 27.

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

At a time when the antibiotic drug discovery pipeline has stalled, antibiotic resistance is accelerating with catastrophic implications for our ability to treat bacterial infections. Globally we face the prospect of a future when common infections can once again kill. Anti-virulence approaches that target the capacity of the bacterium to cause disease rather than the growth or survival of the bacterium itself offer a tantalizing prospect of novel antimicrobials. They may also reduce the propensity to induce resistance by removing the strong selection pressure imparted by bactericidal or bacteriostatic agents. In the human pathogen Pseudomonas aeruginosa, disulfide bond protein A (PaDsbA1) plays a central role in the oxidative folding of virulence factors and is therefore an attractive target for the development of new anti-virulence antimicrobials. Using a fragment-based approach we have identified small molecules that bind to PaDsbA1. The fragment hits show selective binding to PaDsbA1 over the DsbA protein from Escherichia coli, suggesting that developing species-specific narrow-spectrum inhibitors of DsbA enzymes may be feasible. Structures of a co-complex of PaDsbA1 with the highest affinity fragment identified in the screen reveal that the fragment binds on the non-catalytic surface of the protein at a domain interface. This biophysical and structural data represent a starting point in the development of higher affinity compounds, which will be assessed for their potential as selective PaDsbA1 inhibitors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0173436PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5367682PMC
August 2017

Fatty Acid-Binding Protein 5 at the Blood-Brain Barrier Regulates Endogenous Brain Docosahexaenoic Acid Levels and Cognitive Function.

J Neurosci 2016 11;36(46):11755-11767

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

Fatty acid-binding protein 5 (FABP5) at the blood-brain barrier contributes to the brain uptake of docosahexaenoic acid (DHA), a blood-derived polyunsaturated fatty acid essential for maintenance of cognitive function. Given the importance of DHA in cognition, the aim of this study was to investigate whether deletion of FABP5 results in cognitive dysfunction and whether this is associated with reduced brain endothelial cell uptake of exogenous DHA and subsequent attenuation in the brain levels of endogenous DHA. Cognitive function was assessed in male and female FABP5 and FABP5 mice using a battery of memory paradigms. FABP5 mice exhibited impaired working memory and short-term memory, and these cognitive deficits were associated with a 14.7 ± 5.7% reduction in endogenous brain DHA levels. The role of FABP5 in the blood-brain barrier transport of DHA was assessed by measuring C-DHA uptake into brain endothelial cells and capillaries isolated from FABP5 and FABP5 mice. In line with a crucial role of FABP5 in the brain uptake of DHA, C-DHA uptake into brain endothelial cells and brain capillaries of FABP5 mice was reduced by 48.4 ± 14.5% and 14.0 ± 4.2%, respectively, relative to those of FABP5 mice. These results strongly support the hypothesis that FABP5 is essential for maintaining brain endothelial cell uptake of DHA, and that cognitive deficits observed in FABP5 mice are associated with reduced CNS access of DHA.

Significance Statement: Genetic deletion of fatty acid-binding protein 5 (FABP5) in mice reduces uptake of exogenous docosahexaenoic acid (DHA) into brain endothelial cells and brain capillaries and reduces brain parenchymal levels of endogenous DHA. Therefore, FABP5 in the brain endothelial cell is a crucial contributor to the brain levels of DHA. Critically, lowered brain DHA levels in FABP5 mice occurred in tandem with cognitive deficits in a battery of memory paradigms. This study provides evidence of a critical role for FABP5 in the maintenance of cognitive function via regulating the brain uptake of DHA, and suggests that upregulation of FABP5 in neurodegenerative diseases, where brain DHA levels are possibly diminished (e.g., Alzheimer's disease), may provide a novel therapeutic approach for restoring cognitive function.
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http://dx.doi.org/10.1523/JNEUROSCI.1583-16.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6705637PMC
November 2016

Determination of ligand binding modes in weak protein-ligand complexes using sparse NMR data.

J Biomol NMR 2016 11 24;66(3):195-208. Epub 2016 Oct 24.

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

We describe a general approach to determine the binding pose of small molecules in weakly bound protein-ligand complexes by deriving distance constraints between the ligand and methyl groups from all methyl-containing residues of the protein. We demonstrate that using a single sample, which can be prepared without the use of expensive precursors, it is possible to generate high-resolution data rapidly and obtain the resonance assignments of Ile, Leu, Val, Ala and Thr methyl groups using triple resonance scalar correlation data. The same sample may be used to obtain Met CH assignments using NOESY-based methods, although the superior sensitivity of NOESY using [U-C,N]-labeled protein makes the use of this second sample more efficient. We describe a structural model for a weakly binding ligand bound to its target protein, DsbA, derived from intermolecular methyl-to-ligand nuclear Overhauser enhancements, and demonstrate that the ability to assign all methyl resonances in the spectrum is essential to derive an accurate model of the structure. Once the methyl assignments have been obtained, this approach provides a rapid means to generate structural models for weakly bound protein-ligand complexes. Such weak complexes are often found at the beginning of programs of fragment based drug design and can be challenging to characterize using X-ray crystallography.
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http://dx.doi.org/10.1007/s10858-016-0067-4DOI Listing
November 2016

The ways and means of fragment-based drug design.

Pharmacol Ther 2016 11 22;167:28-37. Epub 2016 Jul 22.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Australia. Electronic address:

Fragment-based drug design (FBDD) has emerged as a mainstream approach for the rapid and efficient identification of building blocks that can be used to develop high-affinity ligands against protein targets. One of the strengths of FBDD is the relative ease and low cost of the primary screen to identify fragments that bind. However, the fragments that emerge from primary screens often have low affinities, with K values in the high μM to mM range, and a significant challenge for FBDD is to develop the initial fragments into more potent ligands. Successful fragment elaboration often requires co-structures of the fragments bound to their target proteins, as well as a range of biophysical and biochemical assays to track potency and efficacy. These challenges have led to the development of specific chemical strategies for the elaboration of weakly-binding fragments into more potent "hits" and lead compounds. In this article we review different approaches that have been employed to meet these challenges and describe some of the strategies that have resulted in several fragment-derived compounds entering clinical trials.
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http://dx.doi.org/10.1016/j.pharmthera.2016.07.003DOI Listing
November 2016

Targeting Bacterial Dsb Proteins for the Development of Anti-Virulence Agents.

Molecules 2016 Jul 16;21(7). Epub 2016 Jul 16.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Kingsbury Drive, Bundoora, Vic 3083, Australia.

Recent years have witnessed a dramatic increase in bacterial antimicrobial resistance and a decline in the development of novel antibiotics. New therapeutic strategies are urgently needed to combat the growing threat posed by multidrug resistant bacterial infections. The Dsb disulfide bond forming pathways are potential targets for the development of antimicrobial agents because they play a central role in bacterial pathogenesis. In particular, the DsbA/DsbB system catalyses disulfide bond formation in a wide array of virulence factors, which are essential for many pathogens to establish infections and cause disease. These redox enzymes are well placed as antimicrobial targets because they are taxonomically widespread, share low sequence identity with human proteins, and many years of basic research have provided a deep molecular understanding of these systems in bacteria. In this review, we discuss disulfide bond catalytic pathways in bacteria and their significance in pathogenesis. We also review the use of different approaches to develop inhibitors against Dsb proteins as potential anti-virulence agents, including fragment-based drug discovery, high-throughput screening and other structure-based drug discovery methods.
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http://dx.doi.org/10.3390/molecules21070811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6273893PMC
July 2016

Structure-Activity Studies of β-Hairpin Peptide Inhibitors of the Plasmodium falciparum AMA1-RON2 Interaction.

J Mol Biol 2016 10 14;428(20):3986-3998. Epub 2016 Jul 14.

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

The interaction between apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2) plays a key role in the invasion of red blood cells by Plasmodium parasites. Disruption of this critical protein-protein interaction represents a promising avenue for antimalarial drug discovery. In this work, we exploited a 13-residue β-hairpin based on the C-terminal loop of RON2 to probe a conserved binding site on Plasmodium falciparum AMA1. A series of mutations was synthetically engineered into β-hairpin peptides to establish structure-activity relationships. The best mutations improved the binding affinity of the β-hairpin peptide by ~7-fold for 3D7 AMA1 and ~14-fold for FVO AMA1. We determined the crystal structures of several β-hairpin peptides in complex with AMA1 in order to define the structural features and specific interactions that contribute to improved binding affinity. The same mutations in the longer RON2sp2 peptide (residues 2027-2055 of RON2) increased the binding affinity by >30-fold for 3D7 and FVO AMA1, producing K values of 2.1nM and 0.4nM, respectively. To our knowledge, this is the most potent strain-transcending peptide reported to date and represents a valuable tool to characterize the AMA1-RON2 interaction.
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http://dx.doi.org/10.1016/j.jmb.2016.07.001DOI Listing
October 2016

Design, Synthesis, and Characterization of Cyclic Peptidomimetics of the Inducible Nitric Oxide Synthase Binding Epitope That Disrupt the Protein-Protein Interaction Involving SPRY Domain-Containing Suppressor of Cytokine Signaling Box Protein (SPSB) 2 and Inducible Nitric Oxide Synthase.

J Med Chem 2016 06 8;59(12):5799-809. Epub 2016 Jun 8.

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

SPRY domain-containing suppressor of cytokine signaling box protein (SPSB) 2-deficient macrophages have been found to exhibit prolonged expression of inducible nitric oxide synthase (iNOS) and enhanced killing of persistent pathogens, suggesting that inhibitors of the SPSB2-iNOS interaction have potential as novel anti-infectives. In this study, we describe the design, synthesis, and characterization of cyclic peptidomimetic inhibitors of the SPSB2-iNOS interaction constrained by organic linkers to improve stability and druggability. SPR, ITC, and (19)F NMR analyses revealed that the most potent cyclic peptidomimetic bound to the iNOS binding site of SPSB2 with low nanomolar affinity (KD 29 nM), a 10-fold improvement over that of the linear peptide DINNN (KD 318 nM), and showed strong inhibition of SPSB2-iNOS interaction in macrophage cell lysates. This study exemplifies a novel approach to cyclize a Type II β-turn linear peptide and provides a foundation for future development of this group of inhibitors as new anti-infectives.
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http://dx.doi.org/10.1021/acs.jmedchem.6b00386DOI Listing
June 2016

Binding of CFA/I Pili of Enterotoxigenic Escherichia coli to Asialo-GM1 Is Mediated by the Minor Pilin CfaE.

Infect Immun 2016 05 22;84(5):1642-1649. Epub 2016 Apr 22.

Menzies Health Institute Queensland, School of Medical Sciences, Griffith University, Southport, QLD, Australia

CFA/I pili are representatives of a large family of related pili that mediate the adherence of enterotoxigenic Escherichia coli to intestinal epithelial cells. They are assembled via the alternate chaperone-usher pathway and consist of two subunits, CfaB, which makes up the pilus shaft and a single pilus tip-associated subunit, CfaE. The current model of pilus-mediated adherence proposes that CFA/I has two distinct binding activities; the CfaE subunit is responsible for binding to receptors of unknown structure on erythrocyte and intestinal epithelial cell surfaces, while CfaB binds to various glycosphingolipids, including asialo-GM1. In this report, we present two independent lines of evidence that, contrary to the existing model, CfaB does not bind to asialo-GM1 independently of CfaE. Neither purified CfaB subunits nor CfaB assembled into pili bind to asialo-GM1. Instead, we demonstrate that binding activity toward asialo-GM1 resides in CfaE and this is essential for pilus binding to Caco-2 intestinal epithelial cells. We conclude that the binding activities of CFA/I pili for asialo-GM1, erythrocytes, and intestinal cells are inseparable, require the same amino acid residues in CfaE, and therefore depend on the same or very similar binding mechanisms.
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http://dx.doi.org/10.1128/IAI.01562-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862694PMC
May 2016

Redox-stable cyclic peptide inhibitors of the SPSB2-iNOS interaction.

FEBS Lett 2016 Mar 6;590(6):696-704. Epub 2016 Mar 6.

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

SPSB2 mediates the proteasomal degradation of iNOS. Inhibitors of SPSB2-iNOS interaction are expected to prolong iNOS lifetime and thereby enhance killing of persistent pathogens. Here, we describe the synthesis and characterization of two redox-stable cyclized peptides containing the DINNN motif required for SPSB2 binding. Both analogues bind with low nanomolar affinity to the iNOS binding site on SPSB, as determined by SPR and (19)F NMR, and efficiently displace full-length iNOS from binding to SPSB2 in macrophage cell lysates. These peptides provide a foundation for future development of redox-stable, potent ligands for SPSB proteins as a potential novel class of anti-infectives.
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http://dx.doi.org/10.1002/1873-3468.12115DOI Listing
March 2016

Solution NMR characterization of apical membrane antigen 1 and small molecule interactions as a basis for designing new antimalarials.

J Mol Recognit 2016 06 24;29(6):281-91. Epub 2016 Jan 24.

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

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) plays an important role in the invasion by merozoites of human red blood cells during a malaria infection. A key region of PfAMA1 is a conserved hydrophobic cleft formed by 12 hydrophobic residues. As anti-apical membrane antigen 1 antibodies and other inhibitory molecules that target this hydrophobic cleft are able to block the invasion process, PfAMA1 is an attractive target for the development of strain-transcending antimalarial agents. As solution nuclear magnetic resonance spectroscopy is a valuable technique for the rapid characterization of protein-ligand interactions, we have determined the sequence-specific backbone assignments for PfAMA1 from two P. falciparum strains, FVO and 3D7. Both selective labelling and unlabelling strategies were used to complement triple-resonance experiments in order to facilitate the assignment process. We have then used these assignments for mapping the binding sites for small molecules, including benzimidazoles, pyrazoles and 2-aminothiazoles, which were selected on the basis of their affinities measured from surface plasmon resonance binding experiments. Among the compounds tested, benzimidazoles showed binding to a similar region on both FVO and 3D7 PfAMA1, suggesting that these compounds are promising scaffolds for the development of novel PfAMA1 inhibitors. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/jmr.2529DOI Listing
June 2016

Sent packing: protein engineering generates a new crystal form of Pseudomonas aeruginosa DsbA1 with increased catalytic surface accessibility.

Acta Crystallogr D Biol Crystallogr 2015 Dec 26;71(Pt 12):2386-95. Epub 2015 Nov 26.

Institute for Molecular Bioscience, Division of Chemistry and Structural Biology, University of Queensland, 306 Carmody Road, Brisbane, Queensland 4072, Australia.

Pseudomonas aeruginosa is an opportunistic human pathogen for which new antimicrobial drug options are urgently sought. P. aeruginosa disulfide-bond protein A1 (PaDsbA1) plays a pivotal role in catalyzing the oxidative folding of multiple virulence proteins and as such holds great promise as a drug target. As part of a fragment-based lead discovery approach to PaDsbA1 inhibitor development, the identification of a crystal form of PaDsbA1 that was more suitable for fragment-soaking experiments was sought. A previously identified crystallization condition for this protein was unsuitable, as in this crystal form of PaDsbA1 the active-site surface loops are engaged in the crystal packing, occluding access to the target site. A single residue involved in crystal-packing interactions was substituted with an amino acid commonly found at this position in closely related enzymes, and this variant was successfully used to generate a new crystal form of PaDsbA1 in which the active-site surface is more accessible for soaking experiments. The PaDsbA1 variant displays identical redox character and in vitro activity to wild-type PaDsbA1 and is structurally highly similar. Two crystal structures of the PaDsbA1 variant were determined in complex with small molecules bound to the protein active site. These small molecules (MES, glycerol and ethylene glycol) were derived from the crystallization or cryoprotectant solutions and provide a proof of principle that the reported crystal form will be amenable to co-crystallization and soaking with small molecules designed to target the protein active-site surface.
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http://dx.doi.org/10.1107/S1399004715018519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4667283PMC
December 2015

Fatty Acid-Binding Protein 5 Facilitates the Blood-Brain Barrier Transport of Docosahexaenoic Acid.

Mol Pharm 2015 Dec 30;12(12):4375-85. Epub 2015 Oct 30.

Department of Organ Anatomy, Yamaguchi University Graduate School of Medicine , Minami-kogushi 1-1-1, Ube 755-8505, Japan.

The brain has a limited ability to synthesize the essential polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) from its omega-3 fatty acid precursors. Therefore, to maintain brain concentrations of this PUFA at physiological levels, plasma-derived DHA must be transported across the blood-brain barrier (BBB). While DHA is able to partition into the luminal membrane of brain endothelial cells, its low aqueous solubility likely limits its cytosolic transfer to the abluminal membrane, necessitating the requirement of an intracellular carrier protein to facilitate trafficking of this PUFA across the BBB. As the intracellular carrier protein fatty acid-binding protein 5 (FABP5) is expressed at the human BBB, the current study assessed the putative role of FABP5 in the brain endothelial cell uptake and BBB transport of DHA in vitro and in vivo, respectively. hFAPB5 was recombinantly expressed and purified from Escherichia coli C41(DE3) cells and the binding affinity of DHA to hFABP5 assessed using isothermal titration calorimetry. The impact of FABP5 siRNA on uptake of (14)C-DHA into immortalized human brain microvascular endothelial (hCMEC/D3) cells was assessed. An in situ transcardiac perfusion method was optimized in C57BL/6 mice and subsequently used to compare the BBB influx rate (Kin) of (14)C-DHA between FABP5-deficient (FABP5(-/-)) and wild-type (FABP5(+/+)) C57BL/6 mice. DHA bound to hFABP5 with an equilibrium dissociation constant of 155 ± 8 nM (mean ± SEM). FABP5 siRNA transfection decreased hCMEC/D3 mRNA and protein expression of FABP5 by 53.2 ± 5.5% and 44.8 ± 13.7%, respectively, which was associated with a 14.1 ± 2.7% reduction in (14)C-DHA cellular uptake. By using optimized conditions for the in situ transcardiac perfusion (a 1 min preperfusion (10 mL/min) followed by perfusion of (14)C-DHA (1 min)), the Kin of (14)C-DHA was 0.04 ± 0.01 mL/g/s. Relative to FABP5(+/+) mice, the Kin of (14)C-DHA decreased 36.7 ± 12.4% in FABP5(-/-) mice. This study demonstrates that FABP5 binds to DHA and is involved in the brain endothelial cell uptake and subsequent BBB transport of DHA, confirming the importance of this cytoplasmic carrier protein in the CNS exposure of this PUFA essential for neuronal function.
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http://dx.doi.org/10.1021/acs.molpharmaceut.5b00580DOI Listing
December 2015