Synthesis and PET Imaging Biodistribution Studies of Radiolabeled Iododiflunisal, a Transthyretin Tetramer Stabilizer, Candidate Drug for Alzheimer's Disease.

The small-molecule iododiflunisal (IDIF) is a transthyretin (TTR) tetramer stabilizer and acts as a chaperone of the TTR-Amyloid beta interaction. Oral administration of IDIF improves Alzheimer's Disease (AD)-like pathology in mice, although the mechanism of action and pharmacokinetics remain unknown. Radiolabeling IDIF with positron or gamma emitters may aid in the in vivo evaluation of IDIF using non-invasive nuclear imaging techniques. In this work, we report an isotopic exchange reaction to obtain IDIF radiolabeled with 18F. [19F/18F]exchange reaction over IDIF in dimethyl sulfoxide at 160 °C resulted in the formation of [18F]IDIF in 7 ± 3% radiochemical yield in a 20 min reaction time, with a final radiochemical purity of >99%. Biodistribution studies after intravenous administration of [18F]IDIF in wild-type mice using positron emission tomography (PET) imaging showed capacity to cross the blood-brain barrier (ca. 1% of injected dose per gram of tissue in the brain at t > 10 min post administration), rapid accumulation in the liver, long circulation time, and progressive elimination via urine. Our results open opportunities for future studies in larger animal species or human subjects.

Binding of common organic UV-filters to the thyroid hormone transport protein transthyretin using in vitro and in silico studies: Potential implications in health.

Several anthropogenic contaminants have been identified as competing with the thyroid hormone thyroxine (T4) for binding to transport proteins as transthyretin (TTR). This binding can potentially create toxicity mechanisms posing a threat to human health. Many organic UV filters (UVFs) and paraben preservatives (PBs), widely used in personal care products, are chemicals of emerging concern due to their adverse effects as potential thyroid-disrupting compounds. Recently, organic UVFs have been found in paired maternal and fetal samples and PBs have been detected in placenta, which opens the possibility of the involvement of TTR in the transfer of these chemicals across physiological barriers. We aimed to investigate a discrete set of organic UVFs and PBs to identify novel TTR binders. The binding affinities of target UVFs towards TTR were evaluated using in vitro T4 competitive binding assays. The ligand-TTR affinities were determined by isothermal titration calorimetry (ITC) and compared with known TTR ligands. In parallel, computational studies were used to predict the 3-D structures of the binding modes of these chemicals to TTR. Some organic UVFs, compounds 2,2',4,4'-tetrahydroxybenzophenone (BP2, Kd = 0.43 µM); 2,4-dihydroxybenzophenone (BP1, Kd = 0.60 µM); 4,4'-dihydroxybenzophenone (4DHB, Kd = 0.83 µM), and 4-hydroxybenzophenone (4HB, Kd = 0.93 µM), were found to display a high affinity to TTR, being BP2 the strongest TTR binder (ΔH = -14.93 Kcal/mol). Finally, a correlation was found between the experimental ITC data and the TTR-ligand docking scores obtained by computational studies. The approach integrating in vitro assays and in silico methods constituted a useful tool to find TTR binders among common organic UVFs. Further studies on the involvement of the transporter protein TTR in assisting the transplacental transfer of these chemicals across physiological barriers and the long-term consequences of prenatal exposure to them should be pursued.

Longitudinal evaluation of neuroinflammation and oxidative stress in a mouse model of Alzheimer disease using positron emission tomography.

BACKGROUND: Validation of new biomarkers of Alzheimer disease (AD) is crucial for the successful development and implementation of treatment strategies. Additional to traditional AT(N) biomarkers, neuroinflammation biomarkers, such as translocator protein (TSPO) and cystine/glutamine antiporter system (xc-), could be considered when assessing AD progression. Herein, we report the longitudinal investigation of [18F]DPA-714 and [18F]FSPG for their ability to detect TSPO and xc- biomarkers, respectively, in the 5xFAD mouse model for AD. METHODS: Expression of TSPO and xc- system was assessed longitudinally (2-12 months of age) on 5xFAD mice and their respective controls by positron emission tomography (PET) imaging using radioligands [18F]DPA-714 and [18F]FSPG. In parallel, in the same mice, amyloid-ß plaque deposition was assessed with the amyloid PET radiotracer [18F]florbetaben. In vivo findings were correlated to ex vivo immunofluorescence staining of TSPO and xc- in microglia/macrophages and astrocytes on brain slices. Physiological changes of the brain tissue were assessed by magnetic resonance imaging (MRI) in 12-month-old mice. RESULTS: PET studies showed a significant increase in the uptake of [18F]DPA-714 and [18F]FSPG in the cortex, hippocampus, and thalamus in 5xFAD but not in WT mice over time. The results correlate with Aß plaque deposition. Ex vivo staining confirmed higher TSPO overexpression in both, microglia/macrophages and astrocytes, and overexpression of xc- in non-glial cells of 5xFAD mice. Additionally, the results show that Aß plaques were surrounded by microglia/macrophages overexpressing TSPO. MRI studies showed significant tissue shrinkage and microstructural alterations in 5xFAD mice compared to controls. CONCLUSIONS: TSPO and xc- overexpression can be assessed by [18F]DPA-714 and [18F]FSPG, respectively, and correlate with the level of Aß plaque deposition obtained with a PET amyloid tracer. These results position the two tracers as promising imaging tools for the evaluation of disease progression. Longitudinal in vivo study in the 5xFAD mouse model shows that TSPO and oxidative stress assessment through [18F]DPA-714 and [18F]FSPG-PET imaging, respectively, could serve as a potential tool for the evaluation of Alzheimer disease progression.

Targeting transthyretin in Alzheimer's disease: Drug discovery of small-molecule chaperones as disease-modifying drug candidates for Alzheimer's disease.

Transthyretin (TTR) has a well-established role in neuroprotection in Alzheimer's Disease (AD). We have setup a drug discovery program of small-molecule compounds that act as chaperones enhancing TTR/Amyloid-beta peptide (Aß) interactions. A combination of computational drug repurposing approaches and in vitro biological assays have resulted in a set of molecules which were then screened with our in-house validated high-throughput screening ternary test. A prioritized list of chaperones was obtained and corroborated with ITC studies. Small-molecule chaperones have been discovered, among them our lead compound Iododiflunisal (IDIF), a molecule in the discovery phase; one investigational drug (luteolin); and 3 marketed drugs (sulindac, olsalazine and flufenamic), which could be directly repurposed or repositioned for clinical use. Not all TTR tetramer stabilizers behave as chaperones in vitro. These chemically diverse chaperones will be used for validating TTR as a target in vivo, and to select one repurposed drug as a candidate to enter clinical trials as AD disease-modifying drug.

Neuroprotection in early stages of Alzheimer's disease is promoted by transthyretin angiogenic properties.

BACKGROUND: While still controversial, it has been demonstrated that vascular defects can precede the onset of other AD hallmarks features, making it an important therapeutic target. Given that the protein transthyretin (TTR) has been established as neuroprotective in AD, here we investigated the influence of TTR in the vasculature. METHODS: We evaluated the thickness of the basement membrane and the length of brain microvessels, by immunohistochemistry, in AßPPswe/PS1A246E (AD) transgenic mice and non-transgenic mice (NT) bearing one (TTR+/-) or two (TTR+/+) copies of the TTR gene. The angiogenic potential of TTR was evaluated in vitro using the tube formation assay, and in vivo using the chick chorioallantoic membrane (CAM) assay. RESULTS: AD transgenic mice with TTR genetic reduction, AD/TTR+/-, exhibited a thicker BM in brain microvessels and decreased vessel length than animals with normal TTR levels, AD/TTR+/+. Further in vivo investigation, using the CAM assay, revealed that TTR is a pro-angiogenic molecule, and the neovessels formed are functional. Also, TTR increased the expression of key angiogenic molecules such as proteins interleukins 6 and 8, angiopoietin 2, and vascular endothelial growth factor, by endothelial cells, in vitro, under tube formation conditions. We showed that while TTR reduction also leads to a thicker BM in NT mice, this effect is more pronounced in AD mice than in NT animals, strengthening the idea that TTR is a neuroprotective protein. We also studied the effect of TTR tetrameric stabilization on BM thickness, showing that AD mice treated with the TTR tetrameric stabilizer iododiflunisal (IDIF) displayed a significant reduction of BM thickness and increased vessel length, when compared to non-treated littermates. CONCLUSION: Our in vivo results demonstrate the involvement of TTR in angiogenesis, particularly as a modulator of vascular alterations occurring in AD. Since TTR is decreased early in AD, its tetrameric stabilization can represent a therapeutic avenue for the early treatment of AD through the maintenance of the vascular structure.

Preparative Scale Production of Recombinant Human Transthyretin for Biophysical Studies of Protein-Ligand and Protein-Protein Interactions.

Human transthyretin (hTTR), a serum protein with a main role in transporting thyroid hormones and retinol through binding to the retinol-binding protein, is an amyloidogenic protein involved in familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy, and central nervous system selective amyloidosis. hTTR also has a neuroprotective role in Alzheimer disease, being the major Aß binding protein in human cerebrospinal fluid (CSF) that prevents amyloid-ß (Aß) aggregation with consequent abrogation of toxicity. Here we report an optimized preparative expression and purification protocol of hTTR (wt and amyloidogenic mutants) for in vitro screening assays of TTR ligands acting as amyloidogenesis inhibitors or acting as molecular chaperones to enhance the TTR:Aß interaction. Preparative yields were up to 660 mg of homogenous protein per L of culture in fed-batch bioreactor. The recombinant wt protein is mainly unmodified at Cys10, the single cysteine in the protein sequence, whereas the highly amyloidogenic Y78F variant renders mainly the S-glutathionated form, which has essentially the same amyloidogenic behavior than the reduced protein with free Cys10. The TTR production protocol has shown inter-batch reproducibility of expression and protein quality for in vitro screening assays.

Repurposing Benzbromarone for Familial Amyloid Polyneuropathy: A New Transthyretin Tetramer Stabilizer.

Transthyretin (TTR) is a homotetrameric protein involved in human amyloidosis, including familial amyloid polyneuropathy (FAP). Discovering small-molecule stabilizers of the TTR tetramer is a therapeutic strategy for these diseases. Tafamidis, the only approved drug for FAP treatment, is not effective for all patients. Herein, we discovered that benzbromarone (BBM), a uricosuric drug, is an effective TTR stabilizer and inhibitor against TTR amyloid fibril formation. BBM rendered TTR more resistant to urea denaturation, similarly to iododiflunisal (IDIF), a very potent TTR stabilizer. BBM competes with thyroxine for binding in the TTR central channel, with an IC50 similar to IDIF and tafamidis. Results obtained by isothermal titration calorimetry (ITC) demonstrated that BBM binds TTR with an affinity similar to IDIF, tolcapone and tafamidis, confirming BBM as a potent binder of TTR. The crystal structure of the BBM-TTR complex shows two molecules binding deeply in the thyroxine binding channel, forming strong intermonomer hydrogen bonds and increasing the stability of the TTR tetramer. Finally, kinetic analysis of the ability of BBM to inhibit TTR fibrillogenesis at acidic pH and comparison with other stabilizers revealed that benzbromarone is a potent inhibitor of TTR amyloidogenesis, adding a new interesting scaffold for drug design of TTR stabilizers.

Optimization of kinetic stabilizers of tetrameric transthyretin: A prospective ligand efficiency-guided approach.

In the past few years, attempts have been made to use decision criteria beyond Lipinski's guidelines (Rule of five) to guide drug discovery projects more effectively. Several variables and formulations have been proposed and investigated within the framework of multiparameter optimization methods to guide drug discovery. In this context, the combination of Ligand Efficiency Indices (LEI) has been predominantly used to map and monitor the drug discovery process in a retrospective fashion. Here we provide an example of the use of a novel application of the LEI methodology for prospective lead optimization by using the transthyretin (TTR) fibrillogenesis inhibitor iododiflunisal (IDIF) as example. Using this approach, a number of compounds with theoretical efficiencies higher than the reference compound IDIF were identified. From this group, ten compounds were selected, synthesized and biologically tested. Half of the compounds (5, 6, 7, 8 and 10) showed potencies in terms of IC50 inhibition of TTR aggregation equal or higher than the lead compound. These optimized compounds mapped within the region of more efficient candidates in the corresponding experimental nBEI-NSEI plot, matching their position in the theoretical optimization plane that was used for the prediction. Due to their upstream (North-Eastern) position in the progression lines of NPOL = 3 or 4 of the nBEI-NSEI plot, three of them (5, 6 and 8) are more interesting candidates than iododiflunisal because they have been optimized in the three crucial LEI variables of potency, size and polarity at the same time. This is the first example of the effectiveness of using the combined LEIs within the decision process to validate the application of the LEI formulation for the prospective optimization of lead compounds.

Oral Treatment with Iododiflunisal Delays Hippocampal Amyloid-ß Formation in a Transgenic Mouse Model of Alzheimer's Disease: A Longitudinal in vivo Molecular Imaging Study1.

BACKGROUND: Transthyretin (TTR) is a tetrameric, amyloid-ß (Aß)-binding protein, which reduces Aß toxicity. The TTR/Aß interaction can be enhanced by a series of small molecules that stabilize its tetrameric form. Hence, TTR stabilizers might act as disease-modifying drugs in Alzheimer's disease. OBJECTIVE: We monitored the therapeutic efficacy of two TTR stabilizers, iododiflunisal (IDIF), which acts as small-molecule chaperone of the TTR/Aß interaction, and tolcapone, which does not behave as a small-molecule chaperone, in an animal model of Alzheimer's disease using positron emission tomography (PET). METHODS: Female mice (AßPPswe/PS1A246E/TTR+/-) were divided into 3 groups (n = 7 per group): IDIF-treated, tolcapone-treated, and non-treated. The oral treatment (100 mg/Kg/day) was started at 5 months of age. Treatment efficacy assessment was based on changes in longitudinal deposition of Aß in the hippocampus (HIP) and the cortex (CTX) and determined using PET-[18F]florbetaben. Immunohistochemical analysis was performed at age = 14 months. RESULTS: Standard uptake values relative to the cerebellum (SUVr) of [18F]florbetaben in CTX and HIP of non-treated animals progressively increased from age = 5 to 11 months and stabilized afterwards. In contrast, [18F]florbetaben uptake in HIP of IDIF-treated animals remained constant between ages = 5 and 11 months and significantly increased at 14 months. In the tolcapone-treated group, SUVr progressively increased with time, but at lower rate than in the non-treated group. No significant treatment effect was observed in CTX. Results from immunohistochemistry matched the in vivo data at age = 14 months. CONCLUSION: Our work provides encouraging preliminary results on the ability of small-molecule chaperones to ameliorate Aß deposition in certain brain regions.

An Assay for Screening Potential Drug Candidates for Alzheimer's Disease That Act as Chaperones of the Transthyretin and Amyloid-ß Peptides Interaction.

The protein transthyretin (TTR) modulates amyloid-ß (Aß) peptides deposition and processing and this physiological effect is further enhanced by treatment with iododiflunisal (IDIF), a small-molecule compound (SMC) with TTR tetramer stabilization properties, which behaves as chaperone of the complex. This knowledge has prompted us to design and optimize a rapid and simple high-throughput assay that relies on the ability of test compounds to form ternary soluble complexes TTR/Aß/SMC that prevent Aß aggregation. The method uses the shorter Aß(12-28) sequence which is cheaper and simpler to use while retaining the aggregation properties of their parents Aß(1-40) and Aß(1-42). The test is carried out in 96-plate wells that are UV monitored for turbidity during 6 h. Given its reproducibility, we propose that this test can be a powerful tool for efficient screening of SMCs that act as chaperones of the TTR/Aß interaction that may led to potential AD therapies.

Calorimetric Studies of Binary and Ternary Molecular Interactions between Transthyretin, Aß Peptides, and Small-Molecule Chaperones toward an Alternative Strategy for Alzheimer's Disease Drug Discovery.

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).

Undiscovered Roles for Transthyretin: From a Transporter Protein to a New Therapeutic Target for Alzheimer's Disease.

Transthyretin (TTR), an homotetrameric protein mainly synthesized by the liver and the choroid plexus, and secreted into the blood and the cerebrospinal fluid, respectively, has been specially acknowledged for its functions as a transporter protein of thyroxine and retinol (the latter through binding to the retinol-binding protein), in these fluids. Still, this protein has managed to stay in the spotlight as it has been assigned new and varied functions. In this review, we cover knowledge on novel TTR functions and the cellular pathways involved, spanning from neuroprotection to vascular events, while emphasizing its involvement in Alzheimer's disease (AD). We describe details of TTR as an amyloid binding protein and discuss its interaction with the amyloid Aß peptides, and the proposed mechanisms underlying TTR neuroprotection in AD. We also present the importance of translating advances in the knowledge of the TTR neuroprotective role into drug discovery strategies focused on TTR as a new target in AD therapeutics.

Does Antigen Glycosylation Impact the HIV-Specific T Cell Immunity?

It is largely unknown how post-translational protein modifications, including glycosylation, impacts recognition of self and non-self T cell epitopes presented by HLA molecules. Data in the literature indicate that O- and N-linked glycosylation can survive epitope processing and influence antigen presentation and T cell recognition. In this perspective, we hypothesize that glycosylation of viral proteins and processed epitopes contribute to the T cell response to HIV. Although there is some evidence for T cell responses to glycosylated epitopes (glyco-epitopes) during viral infections in the literature, this aspect has been largely neglected for HIV. To explore the role of glyco-epitope specific T cell responses in HIV infection we conducted in silico and ex vivo immune studies in individuals with chronic HIV infection. We found that in silico viral protein segments with potentially glycosylable epitopes were less frequently targeted by T cells. Ex vivo synthetically added glycosylation moieties generally masked T cell recognition of HIV derived peptides. Nonetheless, in some cases, addition of simple glycosylation moieties produced neo-epitopes that were recognized by T cells from HIV infected individuals. Herein, we discuss the potential importance of these observations and compare limitations of the employed technology with new methodologies that may have the potential to provide a more accurate assessment of glyco-epitope specific T cell immunity. Overall, this perspective is aimed to support future research on T cells recognizing glycosylated epitopes in order to expand our understanding on how glycosylation of viral proteins could alter host T cell immunity against viral infections.

Radiochemical examination of transthyretin (TTR) brain penetration assisted by iododiflunisal, a TTR tetramer stabilizer and a new candidate drug for AD.

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.

Insights on the Interaction between Transthyretin and Aß in Solution. A Saturation Transfer Difference (STD) NMR Analysis of the Role of Iododiflunisal.

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.

Role of the sugar moiety on the opioid receptor binding and conformation of a series of enkephalin neoglycopeptides.

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.

Benzyl-2-Acetamido-2-Deoxy-α-d-Galactopyranoside Increases Human Immunodeficiency Virus Replication and Viral Outgrowth Efficacy In Vitro.

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.

Influence of polar side chains modifications on the dual enkephalinase inhibitory activity and conformation of human opiorphin, a pain perception related peptide.

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.

Modulation of the Interaction between a Peptide Ligand and a G Protein-Coupled Receptor by Halogen Atoms.

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.

Optimized Proteomic Mass Spectrometry Characterization of Recombinant Human µ-Opioid Receptor Functionally Expressed in Pichia pastoris Cell Lines.

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.