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1.
J Med Chem ; 63(6): 3205-3214, 2020 03 26.
Article in English | MEDLINE | ID: mdl-32124607

ABSTRACT

Transthyretin (TTR) modulates the deposition, processing, and toxicity of Abeta (Aß) peptides. We have shown that this effect is enhanced in mice by treatment with small molecules such as iododiflunisal (IDIF, 4), a good TTR stabilizer. Here, we describe the thermodynamics of the formation of binary and ternary complexes among TTR, Aß(1-42) peptide, and TTR stabilizers using isothermal titration calorimetry (ITC). A TTR/Aß(1-42) (1:1) complex with a dissociation constant of Kd = 0.94 µM is formed; with IDIF (4), this constant improves up to Kd = 0.32 µM, indicating the presence of a ternary complex TTR/IDIF/Aß(1-42). However, with the drugs diflunisal (1) or Tafamidis (2), an analogous chaperoning effect could not be observed. Similar phenomena could be recorded with the shorter peptide Aß(12-28) (7). We propose the design of a simple assay system for the search of other chaperones that behave like IDIF and may become potential candidate drugs for Alzheimer's disease (AD).


Subject(s)
Amyloid beta-Peptides/metabolism , Benzoxazoles/metabolism , Diflunisal/analogs & derivatives , Diflunisal/metabolism , Peptide Fragments/metabolism , Prealbumin/metabolism , Protein Multimerization/drug effects , Biological Assay/methods , Calorimetry/methods , Humans , Thermodynamics
2.
Sci Rep ; 9(1): 13672, 2019 09 20.
Article in English | MEDLINE | ID: mdl-31541162

ABSTRACT

It is well settled that the amyloidogenic properties of the plasma protein transporter transthyretin (TTR) can be modulated by compounds that stabilize its native tetrameric conformation. TTR is also present in cerebrospinal fluid where it can bind to Aß-peptides and prevent Aß aggregation. We have previously shown that treatment of Alzheimer's Disease (AD) model mice with iododiflunisal (IDIF), a TTR tetramer stabilizing compound, prevents AD pathologies. This evidence positioned IDIF as a new lead drug for AD. In dissecting the mechanism of action of IDIF, we disclose here different labeling strategies for the preparation of 131I-labeled IDIF and 131I- and 124I-labeled TTR, which have been further used for the preparation of IDIF-TTR complexes labeled either on the compound or the protein. The biodistribution of all labeled species after intravenous administration has been investigated in mice using ex vivo and in vivo techniques. Our results confirm the capacity of TTR to cross the blood brain barrier (BBB) and suggest that the formation of TTR-IDIF complexes enhances BBB permeability of both IDIF and TTR. The increased TTR and IDIF brain concentrations may result in higher Aß-peptide sequestration capacity with the subsequent inhibition of AD symptoms as we have previously observed in mice.


Subject(s)
Brain/diagnostic imaging , Diflunisal/analogs & derivatives , Iodine Radioisotopes/chemistry , Prealbumin/chemistry , Prealbumin/pharmacokinetics , Administration, Intravenous , Amyloid beta-Peptides/metabolism , Animals , Autoradiography , Blood-Brain Barrier/chemistry , Brain/metabolism , Diflunisal/administration & dosage , Diflunisal/chemistry , Diflunisal/pharmacokinetics , Mice , Positron-Emission Tomography , Prealbumin/administration & dosage , Tissue Distribution
3.
J Med Chem ; 60(13): 5749-5758, 2017 07 13.
Article in English | MEDLINE | ID: mdl-28587455

ABSTRACT

Several strategies against Alzheimer disease (AD) are directed to target Aß-peptides. The ability of transthyretin (TTR) to bind Aß-peptides and the positive effect exerted by some TTR stabilizers for modulating the TTR-Aß interaction have been previously studied. Herein, key structural features of the interaction between TTR and the Aß(12-28) peptide (3), the essential recognition element of Aß, have been unravelled by STD-NMR spectroscopy methods in solution. Molecular aspects related to the role of the TTR stabilizer iododiflunisal (IDIF, 5) on the TTR-Aß complex have been also examined. The NMR results, assisted by molecular modeling protocols, have provided a structural model for the TTR-Aß interaction, as well as for the ternary complex formed in the presence of IDIF. This basic structural information could be relevant for providing light on the mechanisms involved in the ameliorating effects of AD symptoms observed in AD/TTR± animal models after IDIF treatment and eventually for designing new molecules toward AD therapeutic drugs.


Subject(s)
Amyloid beta-Peptides/metabolism , Diflunisal/analogs & derivatives , Prealbumin/metabolism , Protein Interaction Maps/drug effects , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Amyloid beta-Peptides/chemistry , Crystallography, X-Ray , Diflunisal/chemistry , Diflunisal/pharmacology , Humans , Magnetic Resonance Spectroscopy , Molecular Docking Simulation , Prealbumin/chemistry
4.
Bioorg Med Chem ; 25(7): 2260-2265, 2017 04 01.
Article in English | MEDLINE | ID: mdl-28284867

ABSTRACT

Glycosylation by simple sugars is a drug discovery alternative that has been explored with varying success for enhancing the potency and bioavailability of opioid peptides. Long ago we described two O-glycosides having either ß-Glucose and ß-Galactose of (d-Met2, Pro5)-enkephalinamide showing one of the highest antinociceptive activities known. Here, we report the resynthesis of these two analogs and the preparation of three novel neoglycopeptide derivatives (α-Mannose, ß-Lactose and ß-Cellobiose). Binding studies to cloned zebrafish opioid receptors showed very small differences of affinity between the parent compound and the five glycopeptides thus suggesting that the nature of the carbohydrate moiety plays a minor role in determining the binding mode. Indeed, NMR conformational studies, combined with molecular mechanics calculations, indicated that all glycopeptides present the same major conformation either in solution or membrane-like environment. The evidences provided here highlight the relevance for in vivo activity of the conjugating bond between the peptide and sugar moieties in opioid glycopeptides.


Subject(s)
Carbohydrates/chemistry , Enkephalins/chemistry , Glycopeptides/metabolism , Receptors, Opioid/metabolism , Animals , Glycopeptides/chemistry , Glycosylation , Magnetic Resonance Spectroscopy , Male , Mice , Mice, Inbred C57BL , Protein Conformation , Structure-Activity Relationship
5.
Front Immunol ; 8: 2010, 2017.
Article in English | MEDLINE | ID: mdl-29472913

ABSTRACT

Glycosylation of host and viral proteins is an important posttranslational modification needed to ensure correct function of glycoproteins. For this reason, we asked whether inhibition of O-glycosylation during human immunodeficiency virus (HIV) in vitro replication could affect HIV infectivity and replication rates. We used benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside (BAGN), a compound that has been widely used to inhibit O-glycosylation in several cell lines. Pretreatment and culture of PHA-blast target cells with BAGN increased the percentage of HIV-infected cells (7.6-fold, p = 0.0115), the per-cell amount of HIV p24 protein (1.3-fold, p = 0.2475), and the viral particles in culture supernatants (7.1-fold, p = 0.0029) compared to BAGN-free cultures. Initiating infection with virus previously grown in the presence of BAGN further increased percentage of infected cells (30-fold, p < 0.0001), intracellular p24 (1.5-fold, p = 0.0433), and secreted viral particles (74-fold, p < 0.0001). BAGN-treated target cells showed less CD25 and CCR5 expression, but increased HLA-DR surface expression, which positively correlated with the number of infected cells. Importantly, BAGN improved viral outgrowth kinetics in 66% of the samples tested, including samples from HIV controllers and subjects in whom no virus could be expanded in the absence of BAGN. Sequencing of the isolated virus indicated no skewing of viral quasi-species populations when compared to BAGN-free culture conditions. BAGN also increased virus production in the ACH2 latency model when used together with latency-reversing agents. Taken together, our results identify BAGN treatment as a simple strategy to improve viral outgrowth in vitro and may provide novel insights into host restriction mechanisms and O-glycosylation-related therapeutic targets for HIV control strategies.

6.
Bioorg Med Chem Lett ; 25(22): 5190-3, 2015 Nov 15.
Article in English | MEDLINE | ID: mdl-26463133

ABSTRACT

The dual inhibitory action of the pain related peptide opiorphin (H-Gln-Arg-Phe-Ser-Arg-OH) against neutral endopeptidase (NEP) and aminopeptidase N (AP-N) was further investigated by a SAR study involving minor modifications on the polar side chains of Arg residues and glycosylation with monosaccharides at Ser. None of them exerted dual or individual inhibitory potency superior than opiorphin. However, the correlations deduced offer further proof for the key role of these residues upon the binding and bioactive conformational stabilization of opiorphin. NMR conformational studies on the glycopeptides suggest that they are still very flexible compounds that may attain their respective bioactive conformations.


Subject(s)
CD13 Antigens/antagonists & inhibitors , Neprilysin/antagonists & inhibitors , Oligopeptides/chemistry , Salivary Proteins and Peptides/chemistry , Acetylgalactosamine/chemistry , Acetylglucosamine/chemistry , Amino Acid Substitution , Arginine/chemistry , Glycopeptides/chemistry , Humans , Models, Molecular , Protein Structure, Tertiary , Serine/chemistry , Structure-Activity Relationship
7.
ACS Med Chem Lett ; 6(8): 872-6, 2015 Aug 13.
Article in English | MEDLINE | ID: mdl-26288687

ABSTRACT

Systematic halogenation of two native opioid peptides has shown that halogen atoms can modulate peptide-receptor interactions in different manners. First, halogens may produce a steric hindrance that reduces the binding of the peptide to the receptor. Second, chlorine, bromine, or iodine may improve peptide binding if their positive σ-hole forms a halogen bond interaction with negatively charged atoms of the protein. Lastly, the negative electrostatic potential of fluorine can interact with positively charged atoms of the protein to improve peptide binding.

8.
J Proteome Res ; 14(8): 3162-73, 2015 Aug 07.
Article in English | MEDLINE | ID: mdl-26090583

ABSTRACT

Human µ-opioid receptor (hMOR) is a class-A G-protein-coupled receptor (GPCR), a prime therapeutic target for the management of moderate and severe pain. A chimeric form of the receptor has been cocrystallized with an opioid antagonist and resolved by X-ray diffraction; however, further direct structural analysis is still required to identify the active form of the receptor to facilitate the rational design of hMOR-selective agonist and antagonists with therapeutic potential. Toward this goal and in spite of the intrinsic difficulties posed by the highly hydrophobic transmembrane motives of hMOR, we have comprehensively characterized by mass spectrometry (MS) analysis the primary sequence of the functional hMOR. Recombinant hMOR was overexpressed as a C-terminal c-myc and 6-his tagged protein using an optimized expression procedure in Pichia pastoris cells. After membrane solubilization and metal-affinity chromatography purification, a procedure was devised to enhance the concentration of the receptor. Subsequent combinations of in-solution and in-gel digestions using either trypsin, chymotrypsin, or proteinase K, followed by matrix-assisted laser desorption ionization time-of-flight MS or nanoliquid chromatography coupled with tandem MS analyses afforded an overall sequence coverage of up to >80%, a level of description first attained for an opioid receptor and one of the six such high-coverage MS-based analyses of any GPCR.


Subject(s)
Chromatography, Liquid/methods , Receptors, Opioid, mu/chemistry , Recombinant Fusion Proteins/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Tandem Mass Spectrometry/methods , Amino Acid Sequence , Blotting, Western , Chymotrypsin/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Peptides/metabolism , Pichia/genetics , Protein Structure, Secondary , Proteomics/instrumentation , Proteomics/methods , Receptors, Opioid, mu/genetics , Receptors, Opioid, mu/metabolism , Recombinant Fusion Proteins/metabolism , Trypsin/metabolism
9.
ACS Comb Sci ; 17(1): 32-8, 2015 Jan 12.
Article in English | MEDLINE | ID: mdl-25394203

ABSTRACT

Two series of iododiflunisal and diflunisal analogues have been obtained by using a two step sequential reaction solution-phase parallel synthesis. The synthesis combined an aqueous Suzuki-Miyaura cross-coupling and a mild electrophilic aromatic iodination step using a new polymer-supported iodonium version of Barluenga's reagent. From a selected set of 77 noniodinated and 77 iodinated diflunisal analogues, a subset of good transthyretin amyloid inhibitors has been obtained with improved turbidimetry inhibition constants, high binding affinity to transthyretin, and good selectivity for TTR compared to other thyroxine binding proteins.


Subject(s)
Amyloid Neuropathies, Familial/prevention & control , Combinatorial Chemistry Techniques , Diflunisal/analogs & derivatives , Diflunisal/therapeutic use , Humans , Structure-Activity Relationship
10.
J Alzheimers Dis ; 39(2): 357-70, 2014.
Article in English | MEDLINE | ID: mdl-24169237

ABSTRACT

Alzheimer's disease (AD) is the most common form of dementia and now represents 50-70% of total dementia cases. Over the last two decades, transthyretin (TTR) has been associated with AD and, very recently, a novel concept of TTR stability has been established in vitro as a key factor in TTR/amyloid-ß (Aß) interaction. Small compounds, TTR stabilizers (usually non-steroid anti-inflammatory drugs), bind to the thyroxine (T4) central binding channel, increasing TTR tetrameric stability and TTR/Aß interaction. In this work, we evaluated in vivo the effects of one of the TTR stabilizers identified as improving TTR/Aß interaction, iododiflunisal (IDIF), in Aß deposition and other AD features, using AßPPswe/PS1A246E transgenic mice, either carrying two or just one copy of the TTR gene (AD/TTR+/+ or AD/TTR+/-, respectively), available and characterized in our laboratory. The results showed that IDIF administered orally bound TTR in plasma and stabilized the protein, as assessed by T4 displacement assays, and was able to enter the brain as revealed by mass spectrometry analysis of cerebrospinal fluid. TTR levels, both in plasma and cerebrospinal fluid, were not altered. In AD/TTR+/- mice, IDIF administration resulted not only in decreased brain Aß levels and deposition but also in improved cognitive function associated with the AD-like neuropathology in this mouse model, although no improvements were detectable in the AD/TTR+/+ animals. Further, in AD/TTR+/- mice, Aß levels were reduced in plasma suggesting TTR promoted Aß clearance from the brain and from the periphery. Taken together, these results strengthen the importance of TTR stability in the design of therapeutic drugs, highlighting the capacity of IDIF to be used in AD treatment to prevent and to slow the progression of the disease.


Subject(s)
Amyloid beta-Peptides/metabolism , Cognition Disorders/drug therapy , Diflunisal/analogs & derivatives , Nootropic Agents/pharmacology , Prealbumin/metabolism , Alzheimer Disease , Amyloid beta-Protein Precursor/genetics , Animals , Brain/drug effects , Brain/metabolism , Brain/pathology , Cognition Disorders/metabolism , Cognition Disorders/pathology , Diflunisal/analysis , Diflunisal/chemical synthesis , Diflunisal/pharmacology , Humans , Maze Learning/drug effects , Mice , Mice, Transgenic , Nootropic Agents/analysis , Nootropic Agents/chemical synthesis , Peptide Fragments/metabolism , Plaque, Amyloid/drug therapy , Plaque, Amyloid/metabolism , Plaque, Amyloid/pathology , Prealbumin/genetics , Presenilin-1/genetics
11.
J Med Chem ; 56(22): 9110-21, 2013 Nov 27.
Article in English | MEDLINE | ID: mdl-24147937

ABSTRACT

The amyloidogenic protein transthyretin (TTR) is thought to aggregate into amyloid fibrils by tetramer dissociation which can be inhibited by a number of small molecule compounds. Our analysis of a series of crystallographic protein-inhibitor complexes has shown no clear correlation between the observed molecular interactions and the in vitro activity of the inhibitors. From this analysis, it emerged that halogen bonding (XB) could be mediating some key interactions. Analysis of the halogenated derivatives of two well-known TTR inhibitors has shown that while flufenamic acid affinity for TTR was unchanged by halogenation, diflunisal gradually improves binding up to 1 order of magnitude after iodination through interactions that can be interpreted as a suboptimal XB (carbonyl Thr106: I...O distance 3.96-4.05 Å; C-I...O angle 152-156°) or as rather optimized van der Waals contacts or as a mixture of both. These results illustrate the potential of halogenation strategies in designing and optimizing TTR fibrillogenesis inhibitors.


Subject(s)
Diflunisal/chemistry , Diflunisal/pharmacology , Flufenamic Acid/chemistry , Flufenamic Acid/pharmacology , Halogenation , Prealbumin/chemistry , Protein Multimerization/drug effects , Diflunisal/metabolism , Flufenamic Acid/metabolism , Humans , Kinetics , Ligands , Models, Molecular , Prealbumin/metabolism , Protein Structure, Secondary , Structure-Activity Relationship
12.
J Med Chem ; 55(3): 1181-8, 2012 Feb 09.
Article in English | MEDLINE | ID: mdl-22224710

ABSTRACT

Toward developing new potential analgesics, this first structure-activity relationship study of opiorphin (H-Gln-Arg-Phe-Ser-Arg-OH), a human peptide inhibiting enkephalin degradation, was performed. A systematic Ala scanning proved that Phe(3) is a key residue for neprilysin and aminopeptidase N (AP-N) ectoenkephalinase inhibition. A series of Phe(3)-halogenated analogues revealed that halogen bonding based optimization strategies are not applicable to this residue. Additional substituted Phe(3) derivatives showed that replacing l-Phe(3) for d-Phe(3) increased the AP-N inhibition potency by 1 order of magnitude. NMR studies and molecular mechanics calculations indicated that the improved potency may be due to CH-π stacking interactions between the aromatic ring of d-Phe(3) and the Hγ protons of Arg(2). This structural motif is not possible for the native opiorphin and may be useful for the design of further potent and metabolically stable analogues.


Subject(s)
Analgesics/chemical synthesis , CD13 Antigens/antagonists & inhibitors , Neprilysin/antagonists & inhibitors , Oligopeptides/chemical synthesis , Protease Inhibitors/chemical synthesis , Salivary Proteins and Peptides/chemical synthesis , Analgesics/chemistry , CD13 Antigens/chemistry , Enzyme Assays , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Neprilysin/chemistry , Oligopeptides/chemistry , Protease Inhibitors/chemistry , Quantum Theory , Salivary Proteins and Peptides/chemistry , Solid-Phase Synthesis Techniques , Solutions , Structure-Activity Relationship
13.
ACS Med Chem Lett ; 3(1): 20-4, 2012 Jan 12.
Article in English | MEDLINE | ID: mdl-24900367

ABSTRACT

The conformational profiles for the endogenous peptide Opiorphin and a set of seven analogues exhibiting different inhibitory activities toward human aminopeptidase N (hAPN) and human neprilysin (hNEP) were independently computed to deduce a bioactive conformation that Opiorphin may adopt when binding these two enzymes. The conformational space was thoroughly sampled using an iterative simulated annealing protocol, and a library of low-energy conformers was generated for each peptide. Bioactive Opiorphin conformations fitting our experimental structure-activity relationship data were identified for hAPN and hNEP using computational pairwise comparisons between each of the unique low-energy conformations of Opiorphin and its analogues. The obtained results provide a structural explanation for the dual hAPN and hNEP inhibitory activity of Opiorphin and show that the inborn flexibility of Opiorphin is essential for its analgesic activity.

14.
Org Biomol Chem ; 9(17): 6133-42, 2011 Sep 07.
Article in English | MEDLINE | ID: mdl-21773621

ABSTRACT

To examine if the biological activity of the N/OFQ peptide, which is the native ligand of the pain-related and viable drug target NOP receptor, could be modulated by glycosylation and if such effects could be conformationally related, we have synthesized three N/OFQ glycopeptide analogues, namely: [Thr(5)-O-α-D-GalNAc-N/OFQ] (glycopeptide 1), [Ser(10)-O-α-D-GalNAc]-N/OFQ (glycopeptide 2) and [Ser(10)-O-ß-D-GlcNAc]-N/OFQ] (glycopeptide 3). They were tested for biological activity in competition binding assays using the zebrafish animal model in which glycopeptide 2 exhibited a slightly improved binding affinity, whereas glycopeptide 1 showed a remarkably reduced binding affinity compared to the parent compound and glycopeptide 3. The structural analysis of these glycopeptides and the parent N/OFQ peptide by NMR and circular dichroism indicated that their aqueous solutions are mainly populated by random coil conformers. However, in membrane mimic environments a certain proportion of the molecules of all these peptides exist as α-helix structures. Interestingly, under these experimental conditions, glycopeptide 1 (glycosylated at Thr-5) exhibited a population of folded hairpin-like geometries. From these facts it is tempting to speculate that nociceptin analogues showing linear helical structures are more complementary and thus interact more efficiently with the native NOP receptor than folded structures, since glycopeptide 1 showed a significantly reduced binding affinity for the NOP receptor.


Subject(s)
Glycopeptides/chemistry , Glycopeptides/pharmacology , Opioid Peptides/chemistry , Opioid Peptides/pharmacology , Receptors, G-Protein-Coupled/metabolism , Amino Acid Sequence , Animals , Binding, Competitive , Cell Line , Glycopeptides/chemical synthesis , Humans , Models, Molecular , Molecular Sequence Data , Opioid Peptides/chemical synthesis , Protein Binding , Receptors, Opioid/agonists , Zebrafish , Nociceptin
15.
Bioorg Med Chem Lett ; 19(17): 5270-3, 2009 Sep 01.
Article in English | MEDLINE | ID: mdl-19651509

ABSTRACT

The isatin core structure was found to be a novel chemical scaffold in transthyretin (TTR) fibrillogenesis inhibitor design. Among the series of isatin analogues prepared and tested, the nitro compound 1,3-dihydro-3-[(4-nitrophenyl)imino]-2H-indol-2-one (2r) is as potent as triiodophenol, which is one of the most active known TTR inhibitors. The E/Z stereochemistry of these molecules in solution, elucidated by (1)H NMR, does not influence their biological activity. The compounds do not bind to the native tetrameric TTR suggesting that their inhibitory action is independent of the protein binding and stabilization.


Subject(s)
Isatin/analogs & derivatives , Prealbumin/antagonists & inhibitors , Drug Design , Isatin/chemistry , Isatin/pharmacology , Prealbumin/metabolism , Protein Binding , Stereoisomerism , Structure-Activity Relationship
16.
J Med Chem ; 52(9): 2656-66, 2009 May 14.
Article in English | MEDLINE | ID: mdl-19351163

ABSTRACT

A synthetic mannoside derivative, namely, 6-morphinyl-alpha-D-mannopyranoside, shows a naloxone-reversible antinociception that is 100-fold more potent and twice as long lasting compared to morphine when administered intraperitoneally to rats in paw pressure and tail flick tests. The compound does not produce tolerance and binds to rat mu opioid receptors with twice the affinity of morphine. NMR studies suggest that differences of activity between the derivative and its parent compound M6G might be related to their differing molecular dynamic behavior.


Subject(s)
Analgesics/chemistry , Analgesics/pharmacology , Mannosides/chemistry , Molecular Conformation , Morphine Derivatives/chemistry , Morphine Derivatives/pharmacology , Morphine/chemistry , Morphine/pharmacology , Analgesics/chemical synthesis , Analgesics/metabolism , Animals , Cell Line , Drug Tolerance , Male , Mannosides/chemical synthesis , Mannosides/metabolism , Mannosides/pharmacology , Models, Molecular , Morphine/chemical synthesis , Morphine/metabolism , Morphine Derivatives/chemical synthesis , Morphine Derivatives/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Opioid, mu/metabolism , Time Factors
17.
PLoS One ; 4(1): e4124, 2009.
Article in English | MEDLINE | ID: mdl-19125186

ABSTRACT

The thyroid hormone and retinol transporter protein known as transthyretin (TTR) is in the origin of one of the 20 or so known amyloid diseases. TTR self assembles as a homotetramer leaving a central hydrophobic channel with two symmetrical binding sites. The aggregation pathway of TTR into amiloid fibrils is not yet well characterized but in vitro binding of thyroid hormones and other small organic molecules to TTR binding channel results in tetramer stabilization which prevents amyloid formation in an extent which is proportional to the binding constant. Up to now, TTR aggregation inhibitors have been designed looking at various structural features of this binding channel others than its ability to host iodine atoms. In the present work, greatly improved inhibitors have been designed and tested by taking into account that thyroid hormones are unique in human biochemistry owing to the presence of multiple iodine atoms in their molecules which are probed to interact with specific halogen binding domains sitting at the TTR binding channel. The new TTR fibrillogenesis inhibitors are based on the diflunisal core structure because diflunisal is a registered salicylate drug with NSAID activity now undergoing clinical trials for TTR amyloid diseases. Biochemical and biophysical evidence confirms that iodine atoms can be an important design feature in the search for candidate drugs for TTR related amyloidosis.


Subject(s)
Amyloid , Anti-Inflammatory Agents, Non-Steroidal , Diflunisal , Iodine/chemistry , Prealbumin , Protein Multimerization/drug effects , Amyloid/chemistry , Amyloid/metabolism , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Binding Sites , Diflunisal/chemistry , Diflunisal/pharmacology , Drug Design , Humans , Models, Molecular , Molecular Structure , Prealbumin/chemistry , Prealbumin/metabolism , Protein Structure, Quaternary , Thyroid Hormones/chemistry , Thyroid Hormones/metabolism
18.
Org Lett ; 10(15): 3243-5, 2008 Aug 07.
Article in English | MEDLINE | ID: mdl-18598051

ABSTRACT

An efficient arylation in water of tyrosine and phenylalanine side chains from unprotected iodopeptides is accomplished by using Suzuki-Miyaura cross-coupling processes. The method is compatible with the hydrophilic and thermolabile nature of biologically active peptides. Also of interest, the arylated tyrosine peptides can be accessed in one-pot mode starting from native peptides.


Subject(s)
Iodoproteins/chemistry , Peptides/chemical synthesis , Phenylalanine/chemistry , Tyrosine/chemistry , Enkephalin, Leucine/chemistry , Onium Compounds/chemistry , Peptides/chemistry , Pyridines/chemistry
19.
Biochim Biophys Acta ; 1784(3): 512-7, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18155178

ABSTRACT

Transthyretin (TTR) is a plasma homotetrameric protein associated with senile systemic amyloidosis and familial amyloidotic polyneuropathy. In theses cases, TTR dissociation and misfolding induces the formation of amyloidogenic intermediates that assemble into toxic oligomeric species and lead to the formation of fibrils present in amyloid deposits. The four TTR monomers associate around a central hydrophobic channel where two thyroxine molecules can bind simultaneously. In each thyroxine binding site there are three pairs of symmetry related halogen binding pockets which can accommodate the four iodine substituents of thyroxine. A number of structurally diverse small molecules that bind to the TTR channel increasing the protein stability and thereafter inhibiting amyloid fibrillogenesis have been tested. In order to take advantage of the high propensity to interactions between iodine substituents and the TTR channel we have identified two iodinated derivatives of salicylic acid, 5-iodosalicylic acid and 3,5-diiodosalicylic acid, available commercially. We report in this paper the relative binding affinities of salicylic acid and the two iodinated derivatives and the crystal structure of TTR complexed with 3,5-diiodosalicylic acid, to elucidate the higher binding affinity of this compound towards TTR.


Subject(s)
Iodobenzoates/chemistry , Prealbumin/chemistry , Salicylates/chemistry , Salicylic Acid/chemistry , Crystallography, X-Ray , Halogenation , Humans , Iodine/chemistry , Protein Conformation
20.
Biochem J ; 408(1): 131-8, 2007 Nov 15.
Article in English | MEDLINE | ID: mdl-17683281

ABSTRACT

Destabilization of the tetrameric fold of TTR (transthyretin) is important for aggregation of the protein which culminates in amyloid fibril formation. Many TTR mutations interfere with tetramer stability, increasing the amyloidogenic potential of the protein. The vast majority of proposed TTR fibrillogenesis inhibitors are based on in vitro assays with isolated protein, limiting their future use in clinical assays. In the present study we investigated TTR fibrillogenesis inhibitors using a cellular system that produces TTR intermediates/aggregates in the medium. Plasmids carrying wild-type TTR, V30M or L55P cDNA were transfected into a rat Schwannoma cell line and TTR aggregates were investigated in the medium using a dot-blot filter assay followed by immunodetection. Results showed that, in 24 h, TTR L55P forms aggregates in the medium, whereas, up to 72 h, wild-type TTR and V30M do not. A series of 12 different compounds, described in the literature as in vitro TTR fibrillogenesis inhibitors, were tested for their ability to inhibit L55P aggregate formation; in this system, 2-[(3,5-dichlorophenyl) amino] benzoic acid, benzoxazole, 4-(3,5-difluorophenyl) benzoic acid and tri-iodophenol were the most effective inhibitors, as compared with the reference iododiflunisal, previously shown by ex vivo and in vitro procedures to stabilize TTR and inhibit fibrillogenesis. Among these drugs, 2-[(3,5-dichlorophenyl) amino] benzoic acid and tri-iodophenol stabilized TTR from heterozygotic carriers of V30M in the same ex vivo conditions as those used previously for iododiflunisal. The novel cellular-based test herein proposed for TTR fibrillogenesis inhibitor screens avoids not only lengthy and cumbersome large-scale protein isolation steps but also artefacts associated with most current in vitro first-line screening methods, such as those associated with acidic conditions and the absence of serum proteins.


Subject(s)
Drug Design , Prealbumin/metabolism , Animals , Cell Line, Tumor , Gene Expression , Humans , Mass Spectrometry , Microscopy, Electron, Transmission , Mutation/genetics , Prealbumin/genetics , Prealbumin/ultrastructure , Protein Binding/drug effects , Protein Folding , Rats
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