Publications by authors named "Biswaranjan Mohanty"

46 Publications

Effect of sorbitan monopalmitate on the polymorphic transitions and physicochemical properties of mango butter.

Food Chem 2021 Jun 6;347:128987. Epub 2021 Jan 6.

Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, India. Electronic address:

The present study reports the effect of sorbitan monopalmitate (SM) as a crystallization modifier on the physicochemical properties of mango butter (MB). The concentration of SM was varied in the range of 1 and 5 wt%. The addition of SM promoted the aggregation of globular MB crystals. The FTIR patterns did not show any significant changes when SM was added. XRD and DSC analyses confirmed the crystallization of MB crystals in stable β' and β (V) polymorphic states. However, SM also introduced imperfections in the crystal lattices of MB. Among all formulations, M2 (SM; 1% w/w) possessed a mechanically stable network structure. The crystallization rate of MB was tailored by SM in a concentration-dependent manner. The solid content was highest in M4 (SM; 5% w/w) at 10 °C and 30 °C among all the oleogels. In gist, SM in manageable quantities can be utilized for preparing custom-tailored MB-based products.
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http://dx.doi.org/10.1016/j.foodchem.2020.128987DOI Listing
June 2021

Graphene Oxide Increases Corneal Permeation of Ciprofloxacin Hydrochloride from Oleogels: A Study with Cocoa Butter-Based Oleogels.

Gels 2020 Nov 23;6(4). Epub 2020 Nov 23.

Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela 769008, India.

In this work, oleogels of cocoa butter (CB), rice bran oil (RBO), and graphene oxide (GO) were prepared. The prepared oleogels were subjected to various characterization techniques such as bright-field microscopy, X-ray diffraction (XRD), crystallization kinetics, differential scanning calorimetry (DSC), and mechanical studies. The influence of increasing GO content on the in vitro drug release and ex vivo corneal permeation of the model drug (ciprofloxacin HCl-CPH) from the oleogels was also investigated. Bright-field micrographs showed that increment in GO content reduced the size of the globular particles of CB. XRD analysis revealed that CB was crystallized in its β' and β polymorphic forms in the oleogels, which was in agreement with thermal studies. The mechanical characterization demonstrated that the presence of GO improved the elastic nature and stress-bearing properties of the oleogels. Moreover, GO altered the crystallization kinetics of CB in the oleogels in a composition-dependent manner. The in vitro release of CPH from the oleogels occurred through either Fickian diffusion or fat network relaxation or a combination thereof. Furthermore, the inclusion of GO enhanced the ex vivo permeation of CPH molecules across the caprine cornea. Hence, we concluded that the prepared oleogels could be explored as potential delivery systems for ophthalmic applications.
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http://dx.doi.org/10.3390/gels6040043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7709633PMC
November 2020

Synthesis and characterization of novel tamarind gum and rice bran oil-based emulgels for the ocular delivery of antibiotics.

Int J Biol Macromol 2020 Dec 5;164:1608-1620. Epub 2020 Aug 5.

Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, India. Electronic address:

In this study, we developed tamarind gum (TG) and rice bran oil (RBO)-based emulgels. The control formulation (TR0), did not contain RBO. The emulgels were named as TR1, TR2, TR3, and TR4, which contained 5% (w/w), 10% (w/w), 15% (w/w), and 20% (w/w/) of RBO, respectively. The microscopic studies showed that the emulgels were biphasic in nature. FTIR spectroscopy revealed the reduction in the hydrogen bonding with an increase in the RBO content. Impedance profiles suggested that the resistive component of the emulgels was increased as the RBO content was increased. The thermal analysis suggested that the addition of RBO reduced the water holding capacity of the emulgels. Stress relaxation studies revealed that the fluidic component was considerably higher in TG/RBO-based emulgels as compared to TR0. In vitro release study of the model drug (ciprofloxacin HCl; a hydrochloride salt of ciprofloxacin) suggested a significantly lower release from the emulgel matrices (TR1-TR4) in comparison to TR0. However, ex vivo corneal permeation of the drug increased with an increase in the RBO content. Since the emulgels were able to improve the corneal permeation of the model drug, the emulgels can be explored to deliver drugs to the internal structures of the eye.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.07.231DOI Listing
December 2020

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

Silanization improves biocompatibility of graphene oxide.

Mater Sci Eng C Mater Biol Appl 2020 May 7;110:110647. Epub 2020 Jan 7.

Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India. Electronic address:

Evaluation of the biological properties of silanized graphene oxide is important in the context of biomedical applications of the material. In this study, we have evaluated the toxicity, immunogenicity and other biological properties like osteogenicity of silanized graphene oxide (SiGO). Graphene oxide (GO) was silanized using a common silanizing agent namely (3-aminopropyl) triethoxysilane (APTES). Silanization was confirmed through infrared spectroscopy and elemental mapping. Post-silanization, we did not observe any significant changes in the morphology of GO. Silanization leads to an increase in the interlayer distance and disorder in the lattice. Study of in vitro toxicity of SiGO on three different cell lines namely primary human dermal fibroblast, murine embryonic fibroblast and human osteosarcoma cell lines revealed that toxicity of SiGO was significantly less than GO. We further showed that in vitro immune activation of macrophage was less in the case of SiGO in comparison to GO. Profiling of osteogenic differentiation of human mesenchymal stem cell revealed that SiGO is less osteogenic than GO. Study of acute toxicity in the murine model indicated that GO was hepatotoxic at experimental concentration whereas SiGO did not show any significant toxicity. This study implied that SiGO is a better biocompatible material than GO.
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http://dx.doi.org/10.1016/j.msec.2020.110647DOI Listing
May 2020

The uncharacterized bacterial protein YejG has the same architecture as domain III of elongation factor G.

Proteins 2019 08 22;87(8):699-705. Epub 2019 Apr 22.

School of Biological Sciences, Victoria University, Wellington, New Zealand.

InterPro family IPR020489 comprises ~1000 uncharacterized bacterial proteins. Previously we showed that overexpressing the Escherichia coli representative of this family, EcYejG, conferred low-level resistance to aminoglycoside antibiotics. In an attempt to shed light on the biochemical function of EcYejG, we have solved its structure using multinuclear solution NMR spectroscopy. The structure most closely resembles that of domain III from elongation factor G (EF-G). EF-G catalyzes ribosomal translocation and mutations in EF-G have also been associated with aminoglycoside resistance. While we were unable to demonstrate a direct interaction between EcYejG and the ribosome, the protein might play a role in translation.
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http://dx.doi.org/10.1002/prot.25687DOI Listing
August 2019

Classification of the human phox homology (PX) domains based on their phosphoinositide binding specificities.

Nat Commun 2019 04 4;10(1):1528. Epub 2019 Apr 4.

Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD, 4072, Australia.

Phox homology (PX) domains are membrane interacting domains that bind to phosphatidylinositol phospholipids or phosphoinositides, markers of organelle identity in the endocytic system. Although many PX domains bind the canonical endosome-enriched lipid PtdIns3P, others interact with alternative phosphoinositides, and a precise understanding of how these specificities arise has remained elusive. Here we systematically screen all human PX domains for their phospholipid preferences using liposome binding assays, biolayer interferometry and isothermal titration calorimetry. These analyses define four distinct classes of human PX domains that either bind specifically to PtdIns3P, non-specifically to various di- and tri-phosphorylated phosphoinositides, bind both PtdIns3P and other phosphoinositides, or associate with none of the lipids tested. A comprehensive evaluation of PX domain structures reveals two distinct binding sites that explain these specificities, providing a basis for defining and predicting the functional membrane interactions of the entire PX domain protein family.
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http://dx.doi.org/10.1038/s41467-019-09355-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6449406PMC
April 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

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

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

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

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 Chromatin Remodelling Protein CHD1 Contains a Previously Unrecognised C-Terminal Helical Domain.

J Mol Biol 2016 10 31;428(21):4298-4314. Epub 2016 Aug 31.

Department of Genome Sciences, The John Curtin School of Medical Research, Building 131, Garran Road, The Australian National University, Canberra, Australian Capital Territory, 2601, Australia. Electronic address:

The packaging of eukaryotic DNA into nucleosomes, and the organisation of these nucleosomes into chromatin, plays a critical role in regulating all DNA-associated processes. Chromodomain helicase DNA-binding protein 1 (CHD1) is an ATP-dependent chromatin remodelling protein that is conserved throughout eukaryotes and has an ability to assemble and organise nucleosomes both in vitro and in vivo. This activity is involved in the regulation of transcription and is implicated in mammalian development and stem cell biology. CHD1 is classically depicted as possessing a pair of tandem chromodomains that directly precede a core catalytic helicase-like domain that is then followed by a SANT-SLIDE DNA-binding domain. Here, we have identified an additional conserved domain C-terminal to the SANT-SLIDE domain and determined its structure by multidimensional heteronuclear NMR spectroscopy. We have termed this domain the CHD1 helical C-terminal (CHCT) domain as it is comprised of five α-helices arranged in a variant helical bundle topology. CHCT has a conserved, positively charged surface and is able to bind DNA and nucleosomes. In addition, we have identified another group of proteins, the as yet uncharacterised C17orf64 proteins, as also containing a conserved CHCT domain. Our data provide new structural insights into the CHD1 enzyme family.
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http://dx.doi.org/10.1016/j.jmb.2016.08.028DOI Listing
October 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

NMR in structural genomics to increase structural coverage of the protein universe: Delivered by Prof. Kurt Wüthrich on 7 July 2013 at the 38th FEBS Congress in St. Petersburg, Russia.

FEBS J 2016 11 9;283(21):3870-3881. Epub 2016 Jun 9.

Joint Center for Structural Genomics, The Scripps Research Institute, La Jolla, CA, USA.

For more than a decade, the Joint Center for Structural Genomics (JCSG; www.jcsg.org) worked toward increased three-dimensional structure coverage of the protein universe. This coordinated quest was one of the main goals of the four high-throughput (HT) structure determination centers of the Protein Structure Initiative (PSI; www.nigms.nih.gov/Research/specificareas/PSI). To achieve the goals of the PSI, the JCSG made use of the complementarity of structure determination by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy to increase and diversify the range of targets entering the HT structure determination pipeline. The overall strategy, for both techniques, was to determine atomic resolution structures for representatives of large protein families, as defined by the Pfam database, which had no structural coverage and could make significant contributions to biological and biomedical research. Furthermore, the experimental structures could be leveraged by homology modeling to further expand the structural coverage of the protein universe and increase biological insights. Here, we describe what could be achieved by this structural genomics approach, using as an illustration the contributions from 20 NMR structure determinations out of a total of 98 JCSG NMR structures, which were selected because they are the first three-dimensional structure representations of the respective Pfam protein families. The information from this small sample is representative for the overall results from crystal and NMR structure determination in the JCSG. There are five new folds, which were classified as domains of unknown functions (DUF), three of the proteins could be functionally annotated based on three-dimensional structure similarity with previously characterized proteins, and 12 proteins showed only limited similarity with previous deposits in the Protein Data Bank (PDB) and were classified as DUFs.
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http://dx.doi.org/10.1111/febs.13751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5099109PMC
November 2016

NMR reveals structural rearrangements associated to substrate insertion in nucleotide-adding enzymes.

Protein Sci 2016 Apr 20;25(4):917-25. Epub 2016 Jan 20.

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, 92037.

The protein NP_344798.1 from Streptococcus pneumoniae TIGR4 exhibits a head and base-interacting neck domain architecture, as observed in class II nucleotide-adding enzymes. Although it has less than 20% overall sequence identity with any member of this enzyme family, the residues involved in substrate-recognition and catalysis are highly conserved in NP_344798.1. NMR studies showed binding affinity of NP_344798.1 for nucleotides and revealed μs to ms time scale rate processes involving residues constituting the active site. The results thus obtained indicate that large-amplitude rearrangements of regular secondary structures facilitate the penetration of the substrate into the occluded nucleotide-binding site of NP_344798.1 and, by inference based on sequence and structural homology, probably a wide range of other nucleotide-adding enzymes.
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http://dx.doi.org/10.1002/pro.2872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4941227PMC
April 2016

1H, 13C and 15N resonance assignments of a C-terminal domain of human CHD1.

Biomol NMR Assign 2016 Apr 19;10(1):31-4. Epub 2015 Aug 19.

Department of Genome Sciences, The John Curtin School of Medical Research, The Australian National University, Building 131, Garran Road, Canberra, ACT, 2601, Australia.

Chromatin remodelling proteins are an essential family of eukaryotic proteins. They harness the energy from ATP hydrolysis and apply it to alter chromatin structure in order to regulate all aspects of genome biology. Chromodomain helicase DNA-binding protein 1 (CHD1) is one such remodelling protein that has specialised nucleosome organising abilities and is conserved across eukaryotes. CHD1 possesses a pair of tandem chromodomains that directly precede the core catalytic Snf2 helicase-like domain, and a C-terminal SANT-SLIDE DNA-binding domain. We have identified an additional conserved domain in the C-terminal region of CHD1. Here, we report the backbone and side chain resonance assignments for this domain from human CHD1 at pH 6.5 and 25 °C (BMRB No. 25638).
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http://dx.doi.org/10.1007/s12104-015-9631-1DOI Listing
April 2016

Structure and Functional Characterization of the Conserved JAK Interaction Region in the Intrinsically Disordered N-Terminus of SOCS5.

Biochemistry 2015 Aug 27;54(30):4672-82. Epub 2015 Jul 27.

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

SOCS5 can negatively regulate both JAK/STAT and EGF-receptor pathways and has therefore been implicated in regulating both the immune response and tumorigenesis. Understanding the molecular basis for SOCS5 activity may reveal novel ways to target key components of these signaling pathways. The N-terminal region of SOCS5 coordinates critical protein interactions involved in inhibition of JAK/STAT signaling, and a conserved region within the N-terminus of SOCS5 mediates direct binding to the JAK kinase domain. Here we have characterized the solution conformation of this conserved JAK interaction region (JIR) within the largely disordered N-terminus of SOCS5. Using nuclear magnetic resonance (NMR) chemical shift analysis, relaxation measurements, and NOE analysis, we demonstrate the presence of preformed structural elements in the JIR of mouse SOCS5 (mSOCS5175-244), consisting of an α-helix encompassing residues 224-233, preceded by a turn and an extended structure. We have identified a phosphorylation site (Ser211) within the JIR of mSOCS5 and have investigated the role of phosphorylation in modulating JAK binding using site-directed mutagenesis.
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http://dx.doi.org/10.1021/acs.biochem.5b00619DOI Listing
August 2015

Structure of amylase-binding protein A of Streptococcus gordonii: a potential receptor for human salivary α-amylase enzyme.

Protein Sci 2015 Jun 2;24(6):1013-8. Epub 2015 Apr 2.

Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, 30 Flemington Road, Parkville, Victoria, 3010, Australia.

Amylase-binding protein A (AbpA) of a number of oral streptococci is essential for the colonization of the dental pellicle. We have determined the solution structure of residues 24-195 of AbpA of Streptococcus gordonii and show a well-defined core of five helices in the region of 45-115 and 135-145. (13) Cα/β chemical shift and heteronuclear (15) N-{(1) H} NOE data are consistent with this fold and that the remainder of the protein is unstructured. The structure will inform future molecular experiments in defining the mechanism of human salivary α-amylase binding and biofilm formation by streptococci.
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http://dx.doi.org/10.1002/pro.2671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4456114PMC
June 2015

Small molecule inhibitors of disulfide bond formation by the bacterial DsbA-DsbB dual enzyme system.

ACS Chem Biol 2015 Apr 27;10(4):957-64. Epub 2015 Jan 27.

†Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.

The DsbA:DsbB redox machinery catalyzes disulfide bond formation in secreted proteins and is required for bacterial virulence factor assembly. Both enzymes have been identified as targets for antivirulence drugs. Here, we report synthetic analogues of ubiquinone (dimedone derivatives) that inhibit disulfide bond formation (IC50∼1 μM) catalyzed by E. coli DsbA:DsbB. The mechanism involves covalent modification of a single free cysteine leaving other cysteines unmodified. A vinylogous anhydride in each inhibitor is cleaved by the thiol, which becomes covalently modified to a thioester by a propionyl substituent. Cysteines and lysines on DsbA and DsbB and a nonredox enzyme were modified in a manner that implies some specificity. Moreover, human thioredoxin was not inhibited under the same conditions that inhibited EcDsbA. This proof of concept work uses small molecules that target specific cysteines to validate the DsbA and DsbB dual enzyme system as a viable and potentially druggable antivirulence target.
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http://dx.doi.org/10.1021/cb500988rDOI Listing
April 2015

Promiscuous 2-aminothiazoles (PrATs): a frequent hitting scaffold.

J Med Chem 2015 Feb 16;58(3):1205-14. Epub 2015 Jan 16.

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

We have identified a class of molecules, known as 2-aminothiazoles (2-ATs), as frequent-hitting fragments in biophysical binding assays. This was exemplified by 4-phenylthiazol-2-amine being identified as a hit in 14/14 screens against a diverse range of protein targets, suggesting that this scaffold is a poor starting point for fragment-based drug discovery. This prompted us to analyze this scaffold in the context of an academic fragment library used for fragment-based drug discovery (FBDD) and two larger compound libraries used for high-throughput screening (HTS). This analysis revealed that such "promiscuous 2-aminothiazoles" (PrATs) behaved as frequent hitters under both FBDD and HTS settings, although the problem was more pronounced in the fragment-based studies. As 2-ATs are present in known drugs, they cannot necessarily be deemed undesirable, but the combination of their promiscuity and difficulties associated with optimizing them into a lead compound makes them, in our opinion, poor scaffolds for fragment libraries.
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http://dx.doi.org/10.1021/jm501402xDOI Listing
February 2015

Application of fragment-based screening to the design of inhibitors of Escherichia coli DsbA.

Angew Chem Int Ed Engl 2015 Feb 30;54(7):2179-84. Epub 2014 Dec 30.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052 (Australia) http://www.pharm.monash.edu.au.

The thiol-disulfide oxidoreductase enzyme DsbA catalyzes the formation of disulfide bonds in the periplasm of Gram-negative bacteria. DsbA substrates include proteins involved in bacterial virulence. In the absence of DsbA, many of these proteins do not fold correctly, which renders the bacteria avirulent. Thus DsbA is a critical mediator of virulence and inhibitors may act as antivirulence agents. Biophysical screening has been employed to identify fragments that bind to DsbA from Escherichia coli. Elaboration of one of these fragments produced compounds that inhibit DsbA activity in vitro. In cell-based assays, the compounds inhibit bacterial motility, but have no effect on growth in liquid culture, which is consistent with selective inhibition of DsbA. Crystal structures of inhibitors bound to DsbA indicate that they bind adjacent to the active site. Together, the data suggest that DsbA may be amenable to the development of novel antibacterial compounds that act by inhibiting bacterial virulence.
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http://dx.doi.org/10.1002/anie.201410341DOI Listing
February 2015

NMR structure determination of the protein NP_344798.1 as the first representative of Pfam PF06042.

J Biomol NMR 2015 Jan 28;61(1):83-7. Epub 2014 Nov 28.

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA.

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http://dx.doi.org/10.1007/s10858-014-9878-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4304887PMC
January 2015

Molecular insights into the interaction between Plasmodium falciparum apical membrane antigen 1 and an invasion-inhibitory peptide.

PLoS One 2014 24;9(10):e109674. Epub 2014 Oct 24.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia; Australian Research Council Centre of Excellence for Coherent X-ray Science, Monash University, Parkville, Victoria, Australia.

Apical membrane antigen 1 (AMA1) of the human malaria parasite Plasmodium falciparum has been implicated in invasion of the host erythrocyte. It interacts with malarial rhoptry neck (RON) proteins in the moving junction that forms between the host cell and the invading parasite. Agents that block this interaction inhibit invasion and may serve as promising leads for anti-malarial drug development. The invasion-inhibitory peptide R1 binds to a hydrophobic cleft on AMA1, which is an attractive target site for small molecules that block parasite invasion. In this work, truncation and mutational analyses show that Phe5-Phe9, Phe12 and Arg15 in R1 are the most important residues for high affinity binding to AMA1. These residues interact with two well-defined binding hot spots on AMA1. Computational solvent mapping reveals that one of these hot spots is suitable for small molecule targeting. We also confirm that R1 in solution binds to AMA1 with 1:1 stoichiometry and adopts a secondary structure consistent with the major form of R1 observed in the crystal structure of the complex. Our results provide a basis for designing high affinity inhibitors of the AMA1-RON2 interaction.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0109674PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4208761PMC
June 2015

Characterization of two distinct modes of drug binding to human intestinal fatty acid binding protein.

ACS Chem Biol 2014 Nov 2;9(11):2526-34. Epub 2014 Sep 2.

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

The aqueous cytoplasm of cells poses a potentially significant barrier for many lipophilic drugs to reach their sites of action. Fatty acid binding proteins (FABPs) bind to poorly water-soluble fatty acids (FAs) and lipophilic compounds and facilitate their intracellular transport. Several structures of FA in complex with FABPs have been described, but data describing the binding sites of other lipophilic ligands including drugs are limited. Here the environmentally sensitive fluorophores, 1-anilinonapthalene 8-sulfonic acid (ANS), and 11-dansylamino undecanoic acid (DAUDA) were used to investigate drug binding to human intestinal FABP (hIFABP). Most drugs that bound hIFABP were able to displace both ANS and DAUDA. A notable exception was ketorolac, a non-steroidal anti-inflammatory drug that bound to hIFABP and displaced DAUDA but failed to displace ANS. Isothermal titration calorimetry revealed that for the majority of ligands including FA, ANS, and DAUDA, binding to hIFABP was exothermic. In contrast, ketorolac binding to hIFABP was endothermic and entropy-driven. The X-ray crystal structure of DAUDA-hIFABP revealed a FA-like binding mode where the carboxylate of DAUDA formed a network of hydrogen bonds with residues at the bottom of the binding cavity and the dansyl group interacted with residues in the portal region. In contrast, NMR chemical shift perturbation (CSP) data suggested that ANS bound only toward the bottom of the hIFABP cavity, whereas ketorolac occupied only the portal region. The CSP data further suggested that ANS and ketorolac were able to bind simultaneously to hIFABP, consistent with the lack of displacement of ANS observed by fluorescence and supported by a model of the ternary complex. The NMR solution structure of the ketorolac-hIFABP complex therefore describes a newly characterized, hydrophobic ligand binding site in the portal region of hIFABP.
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http://dx.doi.org/10.1021/cb5005178DOI Listing
November 2014

Kv1.3 channel-blocking immunomodulatory peptides from parasitic worms: implications for autoimmune diseases.

FASEB J 2014 Sep 2;28(9):3952-64. Epub 2014 Jun 2.

Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, California, USA;

The voltage-gated potassium (Kv) 1.3 channel is widely regarded as a therapeutic target for immunomodulation in autoimmune diseases. ShK-186, a selective inhibitor of Kv1.3 channels, ameliorates autoimmune diseases in rodent models, and human phase 1 trials of this agent in healthy volunteers have been completed. In this study, we identified and characterized a large family of Stichodactyla helianthus toxin (ShK)-related peptides in parasitic worms. Based on phylogenetic analysis, 2 worm peptides were selected for study: AcK1, a 51-residue peptide expressed in the anterior secretory glands of the dog-infecting hookworm Ancylostoma caninum and the human-infecting hookworm Ancylostoma ceylanicum, and BmK1, the C-terminal domain of a metalloprotease from the filarial worm Brugia malayi. These peptides in solution adopt helical structures closely resembling that of ShK. At doses in the nanomolar-micromolar range, they block native Kv1.3 in human T cells and cloned Kv1.3 stably expressed in L929 mouse fibroblasts. They preferentially suppress the proliferation of rat CCR7(-) effector memory T cells without affecting naive and central memory subsets and inhibit the delayed-type hypersensitivity (DTH) response caused by skin-homing effector memory T cells in rats. Further, they suppress IFNγ production by human T lymphocytes. ShK-related peptides in parasitic worms may contribute to the potential beneficial effects of probiotic parasitic worm therapy in human autoimmune diseases.
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http://dx.doi.org/10.1096/fj.14-251967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4139903PMC
September 2014

NMR structures of α-proteobacterial ATPase-regulating ζ-subunits.

J Mol Biol 2014 Jul 13;426(14):2547-53. Epub 2014 May 13.

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA; Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA; Joint Center for Structural Genomics (http://www.jcsg.org.), La Jolla, CA 92037, USA.

NMR structures of ζ-subunits, which are recently discovered α-proteobacterial F1F0-ATPase-regulatory proteins representing a Pfam protein family of 246 sequences from 219 species (PF07345), exhibit a four-helix bundle, which is different from all other known F1F0-ATPase inhibitors. Chemical shift mapping reveals a conserved ADP/ATP binding site in ζ-subunit, which mediates long-range conformational changes related to function, as revealed by the structure of the Paracoccus denitrificans ζ-subunit in complex with ADP. These structural data suggest a new mechanism of F1F0-ATPase regulation in α-proteobacteria.
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http://dx.doi.org/10.1016/j.jmb.2014.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4089900PMC
July 2014