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1.
Eur J Med Chem ; 203: 112621, 2020 Oct 01.
Article in English | MEDLINE | ID: mdl-32707527

ABSTRACT

S100P, a calcium-binding protein, is known to advance tumor progression and metastasis in pancreatic and several other cancers. Herein is described the in silico identification of a putative binding pocket of S100P to identify, synthesize and evaluate novel small molecules with the potential to selectively bind S100P and inhibit its activation of cell survival and metastatic pathways. The virtual screening of a drug-like database against the S100P model led to the identification of over 100 clusters of diverse scaffolds. A representative test set identified a number of structurally unrelated hits that inhibit S100P-RAGE interaction, measured by ELISA, and reduce in vitro cell invasion selectively in S100P-expressing pancreatic cancer cells at 10 µM. This study establishes a proof of concept in the potential for rational design of small molecule S100P inhibitors for drug candidate development.


Subject(s)
Antineoplastic Agents/pharmacology , Drug Design , Pancreatic Neoplasms/pathology , S100 Proteins/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Antineoplastic Agents/chemistry , Cell Line, Tumor , Dose-Response Relationship, Drug , Gene Expression Regulation, Neoplastic/drug effects , Humans , Neoplasm Invasiveness , Small Molecule Libraries/chemistry
2.
Structure ; 24(8): 1380-1386, 2016 08 02.
Article in English | MEDLINE | ID: mdl-27452402

ABSTRACT

Cysteine string protein (CSP) is a member of the DnaJ/Hsp40 chaperone family that localizes to neuronal synaptic vesicles. Impaired CSP function leads to neurodegeneration in humans and model organisms as a result of misfolding of client proteins involved in neurotransmission. Mammalian CSP is phosphorylated in vivo on Ser10, and this modulates its protein interactions and effects on neurotransmitter release. However, there are no data on the structural consequences of CSP phosphorylation to explain these functional effects. We show that Ser10 phosphorylation causes an order-to-disorder transition that disrupts CSP's extreme N-terminal α helix. This triggers the concomitant formation of a hairpin loop stabilized by ionic interactions between phosphoSer10 and the highly conserved J-domain residue, Lys58. These phosphorylation-induced effects result in significant changes to CSP conformation and surface charge distribution. The phospho-switch revealed here provides structural insight into how Ser10 phosphorylation modulates CSP function and also has potential implications for other DnaJ phosphoproteins.


Subject(s)
Escherichia coli Proteins/chemistry , HSC70 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/chemistry , Lysine/chemistry , Membrane Proteins/chemistry , Serine/chemistry , Amino Acid Motifs , Binding Sites , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression , HSC70 Heat-Shock Proteins/genetics , HSC70 Heat-Shock Proteins/metabolism , HSP40 Heat-Shock Proteins/genetics , HSP40 Heat-Shock Proteins/metabolism , Humans , Kinetics , Lysine/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Phosphorylation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serine/metabolism , Structural Homology, Protein , Thermodynamics
3.
Life Sci ; 146: 66-72, 2016 Feb 01.
Article in English | MEDLINE | ID: mdl-26792060

ABSTRACT

UNLABELLED: Long term use of NSAIDs is linked to side effects such as gastric bleeding and myocardial infarction. AIMS: Use of in silico methods and pharmacology to investigate the potential for NSAIDs diclofenac, celecoxib and naproxen to bind to nuclear receptors. MATERIALS AND METHODS: In silico screening predicted that both diclofenac and celecoxib has the potential to bind to a number of different nuclear receptors; docking analysis confirmed a theoretical ability for diclofenac and celecoxib but not naproxen to bind to TRß. KEY FINDINGS: Results from TRß luciferase reporter assays confirmed that both diclofenac and celecoxib display TRß antagonistic properties; celecoxib, IC50 3.6 × 10(-6)M, and diclofenac IC50 5.3 × 10(-6)M, comparable to the TRß antagonist MLS (IC50 3.1 × 10(-6)M). In contrast naproxen, a cardio-sparing NSAID, lacked TRß antagonist effects. In order to determine the effects of NSAIDs in whole organ in vitro, we used isometric wire myography to measure the changes to Triiodothyronine (T3) induced vasodilation of rat mesenteric arteries. Incubation of arteries in the presence of the TRß antagonist MLS000389544 (10(-5)M), as well as diclofenac (10(-5)M) and celecoxib (10(-5)M) but not naproxen significantly inhibited T3 induced vasodilation compared to controls. SIGNIFICANCE: These results highlight the benefits of computational chemistry methods used to retrospectively analyse well known drugs for side effects. Using in silico and in vitro methods we have shown that both celecoxib and diclofenac but not naproxen exhibit off-target TRß antagonist behaviour, which may be linked to their detrimental side effects.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Celecoxib/pharmacology , Cyclooxygenase 2 Inhibitors/pharmacology , Diclofenac/pharmacology , Thyroid Hormone Receptors beta/antagonists & inhibitors , Animals , Computer Simulation , Humans , Male , Mesenteric Arteries/drug effects , Models, Molecular , Naproxen/pharmacology , Rats , Rats, Wistar , Triiodothyronine/metabolism , Vasodilation/drug effects
4.
Prostaglandins Other Lipid Mediat ; 122: 18-27, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26686607

ABSTRACT

Prostacyclin (PGI2) is a key mediator involved in cardiovascular homeostasis, acting predominantly on two receptor types; cell surface IP receptor and cytosolic peroxisome proliferator activated receptor (PPAR) ß/δ. Having a very short half-life, direct methods to determine its long term effects on cells is difficult, and little is known of its interactions with nuclear receptors. Here we used computational chemistry methods to investigate the potential for PGI2, beraprost (IP receptor agonist), and GW0742 (PPARß/δ agonist), to bind to nuclear receptors, confirmed with pharmacological methods. In silico screening predicted that PGI2, beraprost, and GW0742 have the potential to bind to different nuclear receptors, in particular thyroid hormone ß receptor (TRß) and thyroid hormone α receptor (TRα). Docking analysis predicts a binding profile to residues thought to have allosteric control on the TR ligand binding site. Luciferase reporter assays confirmed that beraprost and GW0742 display TRß and TRα antagonistic properties; beraprost IC50 6.3 × 10(-5)mol/L and GW0742 IC50 4.9 × 10(-6) mol/L. Changes to triiodothyronine (T3) induced vasodilation of rat mesenteric arteries measured on the wire myograph were measured in the presence of the TR antagonist MLS000389544 (10(-5) mol/L), beraprost (10(-5) mol/L) and GW0742 (10(-5) mol/L); all significantly inhibited T3 induced vasodilation compared to controls. We have shown that both beraprost and GW0742 exhibit TRß and TRα antagonist behaviour, and suggests that PGI2 has the ability to affect the long term function of cells through binding to and inactivating thyroid hormone receptors.


Subject(s)
Computer Simulation , Epoprostenol/pharmacology , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Thyroid Hormone/antagonists & inhibitors , Animals , Binding, Competitive/drug effects , Dose-Response Relationship, Drug , Epoprostenol/analogs & derivatives , Epoprostenol/chemistry , Epoprostenol/metabolism , Humans , Ligands , Male , Mesenteric Arteries/drug effects , Mesenteric Arteries/physiology , Models, Molecular , Myography/methods , Protein Domains , Rats, Wistar , Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Thyroid Hormone/chemistry , Receptors, Thyroid Hormone/metabolism , Thiazoles/chemistry , Thiazoles/metabolism , Thiazoles/pharmacology , Thyroid Hormone Receptors alpha/antagonists & inhibitors , Thyroid Hormone Receptors alpha/chemistry , Thyroid Hormone Receptors alpha/metabolism , Thyroid Hormone Receptors beta/antagonists & inhibitors , Thyroid Hormone Receptors beta/chemistry , Thyroid Hormone Receptors beta/metabolism , Triiodothyronine/metabolism , Triiodothyronine/pharmacology , Vasodilation/drug effects
5.
J Mol Biol ; 425(5): 929-43, 2013 Mar 11.
Article in English | MEDLINE | ID: mdl-23274113

ABSTRACT

Anterior gradient 2 (AGR2) is a normal endoplasmic reticulum protein that has two important abnormal functions, amphibian limb regeneration and human cancer metastasis promotion. These normal intracellular and abnormal extracellular roles can be attributed to the multidomain structure of AGR2. The NMR structure shows that AGR2 consists of an unstructured N-terminal region followed by a thioredoxin fold. The protein exists in monomer-dimer equilibrium with a K(d) of 8.83µM, and intermolecular salt bridges involving E60 and K64 within the folded domain serve to stabilize the dimer. The unstructured region is primarily responsible for the ability of AGR2 to promote cell adhesion, while dimerization is less important for this activity. The structural data of AGR2 show a separation between potential catalytic redox activity and adhesion function within the context of metastasis and development.


Subject(s)
Cell Adhesion/physiology , Proteins/chemistry , Thioredoxins/chemistry , Amino Acid Sequence , Chromatography, Gel , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Mucoproteins , Mutagenesis, Site-Directed , Mutation/genetics , Oncogene Proteins , Protein Multimerization , Protein Structure, Tertiary , Proteins/genetics , Proteins/metabolism , Sequence Homology, Amino Acid
6.
J Biol Chem ; 287(45): 38231-43, 2012 Nov 02.
Article in English | MEDLINE | ID: mdl-22989873

ABSTRACT

Calcium-binding protein 7 (CaBP7) is a member of the calmodulin (CaM) superfamily that harbors two high affinity EF-hand motifs and a C-terminal transmembrane domain. CaBP7 has been previously shown to interact with and modulate phosphatidylinositol 4-kinase III-ß (PI4KIIIß) activity in in vitro assays and affects vesicle transport in neurons when overexpressed. Here we show that the N-terminal domain (NTD) of CaBP7 is sufficient to mediate the interaction of CaBP7 with PI4KIIIß. CaBP7 NTD encompasses the two high affinity Ca(2+) binding sites, and structural characterization through multiangle light scattering, circular dichroism, and NMR reveals unique properties for this domain. CaBP7 NTD binds specifically to Ca(2+) but not Mg(2+) and undergoes significant conformational changes in both secondary and tertiary structure upon Ca(2+) binding. The Ca(2+)-bound form of CaBP7 NTD is monomeric and exhibits an open conformation similar to that of CaM. Ca(2+)-bound CaBP7 NTD has a solvent-exposed hydrophobic surface that is more expansive than observed in CaM or CaBP1. Within this hydrophobic pocket, there is a significant reduction in the number of methionine residues that are conserved in CaM and CaBP1 and shown to be important for target recognition. In CaBP7 NTD, these residues are replaced with isoleucine and leucine residues with branched side chains that are intrinsically more rigid than the flexible methionine side chain. We propose that these differences in surface hydrophobicity, charge, and methionine content may be important in determining highly specific interactions of CaBP7 with target proteins, such as PI4KIIIß.


Subject(s)
Calcium-Binding Proteins/metabolism , Calcium/metabolism , Golgi Apparatus/metabolism , Magnetic Resonance Spectroscopy/methods , Amino Acid Sequence , Animals , Binding Sites/genetics , Blotting, Western , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , Cattle , Circular Dichroism , Humans , Hydrophobic and Hydrophilic Interactions , Minor Histocompatibility Antigens , Models, Molecular , Molecular Sequence Data , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Transport , Sequence Homology, Amino Acid , Solutions , Surface Properties
7.
PLoS One ; 6(11): e27779, 2011.
Article in English | MEDLINE | ID: mdl-22114693

ABSTRACT

NCS-1 is a member of the neuronal calcium sensor (NCS) family of EF-hand Ca(2+) binding proteins which has been implicated in several physiological functions including regulation of neurotransmitter release, membrane traffic, voltage gated Ca(2+) channels, neuronal development, synaptic plasticity, and learning. NCS-1 binds to the dopamine D2 receptor, potentially affecting its internalisation and controlling dopamine D2 receptor surface expression. The D2 receptor binds NCS-1 via a short 16-residue cytoplasmic C-terminal tail. We have used NMR and fluorescence spectroscopy to characterise the interactions between the NCS-1/Ca(2+) and D2 peptide. The data show that NCS-1 binds D2 peptide with a K(d) of ∼14.3 µM and stoichiometry of peptide binding to NCS-1 of 2:1. NMR chemical shift mapping confirms that D2 peptide binds to the large, solvent-exposed hydrophobic groove, on one face of the NCS-1 molecule, with residues affected by the presence of the peptide spanning both the N and C-terminal portions of the protein. The NMR and mutagenesis data further show that movement of the C-terminal helix 11 of NCS-1 to fully expose the hydrophobic groove is important for D2 peptide binding. Molecular docking using restraints derived from the NMR chemical shift data, together with the experimentally-derived stoichiometry, produced a model of the complex between NCS-1 and the dopamine receptor, in which two molecules of the receptor are able to simultaneously bind to the NCS-1 monomer.


Subject(s)
Calcium Signaling/physiology , Neuronal Calcium-Sensor Proteins/metabolism , Neuropeptides/metabolism , Peptide Fragments/metabolism , Receptors, Dopamine D2/metabolism , Amino Acid Sequence , Animals , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Neuronal Calcium-Sensor Proteins/genetics , Neuropeptides/genetics , Protein Binding , Protein Multimerization , Protein Structure, Tertiary , Rats , Receptors, Dopamine D2/genetics
8.
J Mol Biol ; 379(5): 981-90, 2008 Jun 20.
Article in English | MEDLINE | ID: mdl-18495154

ABSTRACT

Mutations in the protein dysferlin, a member of the ferlin family, lead to limb girdle muscular dystrophy type 2B and Myoshi myopathy. The ferlins are large proteins characterised by multiple C2 domains and a single C-terminal membrane-spanning helix. However, there is sequence conservation in some of the ferlin family in regions outside the C2 domains. In one annotation of the domain structure of these proteins, an unusual internal duplication event has been noted where a putative domain is inserted in between the N- and C-terminal parts of a homologous domain. This domain is known as the DysF domain. Here, we present the solution structure of the inner DysF domain of the dysferlin paralogue myoferlin, which has a unique fold held together by stacking of arginine and tryptophans, mutations that lead to clinical disease in dysferlin.


Subject(s)
Membrane Proteins/chemistry , Muscle Proteins/chemistry , Muscular Dystrophies, Limb-Girdle/etiology , Amino Acid Sequence , Amino Acid Substitution , Calcium-Binding Proteins , Dysferlin , Humans , Membrane Proteins/genetics , Models, Molecular , Molecular Sequence Data , Muscle Proteins/genetics , Muscular Dystrophies, Limb-Girdle/classification , Muscular Dystrophies, Limb-Girdle/genetics , Muscular Dystrophies, Limb-Girdle/metabolism , Nuclear Magnetic Resonance, Biomolecular , Point Mutation , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Static Electricity , Thermodynamics
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