The assessment of target protein molecular structure provides a distinct advantage in the rational drug design process. The increasing number of available G protein-coupled receptor crystal structures has enabled utilization of a varied number of computational approaches for understanding the ligand-receptor interactions, ligand selectivity and even receptor response upon ligand binding. The following dissertation examines the results from three different projects with varied objectives – i) structural modeling of human C-C chemokine receptor type 5 (CCR5) and assessment of the ligand binding pocket of the receptor, ii) assessment of the selectivity profile of naltrexone derivatives on the three opioid receptors (μ-opioid, κ-opioid, δ-opioid) with an aim towards designing selective μ-opioid receptor antagonists, and iii) structural modeling of the ‘active’ state conformation of the κ-opioid receptor in response to agonist binding and determination of a plausible molecular mechanism involved in activation ‘switch’ of the κ-opioid receptor. In absence of a crystal-based molecular structure of CCR5, a homology model of the receptor was built and the ligand binding pocket was validated. On the basis of evaluation of the ligand-receptor interactions on the validated binding pocket, structural and chemical modifications to anibamine, a natural plant product, were proposed to enhance its receptor binding. The selectivity of naltrexone (a universal antagonist) was assessed with respect to the three opioid receptors by employing ligand docking studies and the ‘message-address’ concept. Multiple address sites were identified on the opioid receptors and structural modifications were proposed for the naltrexone derivatives for their enhanced selectivity. In the third project, structural modeling of the active state conformation of the κ-opioid receptor covalently bound to a salvinorin A derivative (agonist) was attempted via molecular dynamics simulations. Although the obtained molecular model lacked the signature ‘agonist-like’ conformations, the result provides a template for such studies in the future.

Submitted by admin tede (tede@pucgoias.edu.br) on 2016-08-18T13:46:45Z No. of bitstreams: 1 MARCELO WATANABE DE MATOS.pdf: 908435 bytes, checksum: fc89cc18e73bc7e996153afa23e2e3c0 (MD5) / Made available in DSpace on 2016-08-18T13:46:45Z (GMT). No. of bitstreams: 1 MARCELO WATANABE DE MATOS.pdf: 908435 bytes, checksum: fc89cc18e73bc7e996153afa23e2e3c0 (MD5) Previous issue date: 2012-08-01 / Fibromyalgia can be defined as a syndrome of chronic and diffuse musculoskeletal pain, with non-inflammatory characteristics. With the etiological processes still unclear, this disease has assumed an increasingly important role worldwide, with a prevalence of 2% estimated in the world population. Currently, the most discussed pathological mechanism for the disease is a change in pain perception mediated by excitatory and inhibitory neurotransmitters in the central nervous system. In this context are very relevant targets the endogenous opioids and their correlated receptors. The OPRM1 gene encoding the major opioid receptor called mu-opioid receptor, presents a single nucleotide polymorphism (SNP) at position 118 (A118G) that significantly affects the response to endogenous and synthetic opioids. Objectives: The objectives of this study were to compare the frequency of A118G polymorphism in the OPRM1 gene in two groups of women, including 50 women affected by fibromyalgia and 50 unaffected, as well as to investigate the possible associations between the A118G polymorphism with clinical and functional symptoms of the disease. Methodology: Patient´s clinical and functional pain symptoms were assessed by using the Fibromyalgia Impact Questionnaire (FIQ). The A118G polymorphism of the OPRM1 gene was analyzed by polymerase chain reaction followed by analysis of restriction fragment length polymorphism DNA was extracted from peripheral blood samples obtained from the two groups of women. Descriptive and comparative statistical analysis were performed and the results obtained. Results: The results of clinical and functional pain symptoms from fibromyalgia patients, obtained by the FIQ, confirmed the clinical severity of the patients selected in this study. The allele frequencies obtained for the two groups were: A (86,0%) and G (14,0%) for patients with fibromyalgia and A (87,8%) and G (12,2%) for controls. The AA genotype frequencies were found (74,0%) and AG (24,0%) and GG (2,0%) for patients with fibromyalgia and AA (77.6%) and AG (20.4%) and GG (2,0%) for the controls. Conclusions: No statistically significant difference was detected between the group of patients with fibromyalgia and control patients. The A118G polymorphism of the OPRM1 gene was not associated with clinical and functional pain symptoms of the patients analyzed in this study. / A fibromialgia pode ser definida como uma síndrome de dor músculoesquelética difusa e crônica, de caráter não inflamatório. Com processos etiológicos ainda não elucidados, esta patologia tem assumido um papel cada vez mais importante no cenário mundial, com uma prevalência estimada de 2% da população. Atualmente, o possível mecanismo patológico mais discutido é o de uma alteração na percepção da dor, mediada por neurotransmissores excitatórios e inibitórios no Sistema Nervoso Central. Neste contexto, inserem-se os opióides endógenos e seus receptores correlatos. O gene OPRM1 codifica o principal receptor opióide, denominado receptor mu-opióide, cujo polimorfismo de nucleotídeo único (SNP) na posição 118 (A118G) afeta significativamente a resposta aos opióides endógenos e sintéticos. Objetivos: Os objetivos deste estudo foram comparar a frequência do polimorfismo A118G no gene OPRM1 em dois grupos de mulheres, incluindo 50 afetadas pela fibromialgia e 49 não afetadas, bem como investigar as possíveis associações entre o polimorfismo A118G e os sintomas clínicos e funcionais da doença. Metodologia: Os sintomas clínicos e funcionais do quadro álgico das pacientes foram avaliados por meio do Questionário de Impacto da Fibromialgia (do Inglês: Fibromyalgia Impact Questionnaire – FIQ). O polimorfismo A118G do gene OPRM1 foi analisado por meio da reação em cadeia de polimerase seguida de análise de polimorfismos de comprimento de fragmentos de restrição, a partir de DNA extraído de amostras de sangue periférico obtidas dos dois grupos de mulheres. Análise estatística descritiva e comparativa foi realizada a partir dos resultados obtidos. Resultados: A análise dos resultados dos sintomas clínicos e funcionais do quadro álgico das pacientes com fibromialgia, obtidos por meio do FIQ, comprovaram a gravidade do quadro clínico das pacientes selecionadas neste estudo. As frequências alélicas obtidas para os dois grupos foram: A (86,0%) e G (14,0%) para as pacientes com fibromialgia e A (87,8%) e G (12,2%) para os controles. As frequências genotípicas encontradas foram AA (74,0%); AG (24,0%) e GG (2,0%) para pacientes com fibromialgia e AA (77,6%); AG (20,4%) e GG (2,0%) para os controles. Conclusões: Nenhuma diferença estatisticamente significativa foi detectada entre o grupo de pacientes com fibromialgia e as pacientes do grupo controle. O polimorfismo A118G do gene OPRM1 não esteve associado com os sintomas clínicos e funcionais do quadro álgico das pacientes analizadas neste estudo.

Neuronal-glial interactions play an important role in the development of neuropathic (NP) pain states. Earlier studies in our laboratory suggest a role for activated glia in morphine-induced delta opioid receptor (DOR) trafficking by altering DOR functional competence. Thus, chronic treatment with the glial inhibitor, propentofylline (PF) blocks the anti-allodynic and anti-hyperalgesic effects of the DOR agonist deltorphin II. The present study aimed to determine whether NP pain-induced changes in DOR function and trafficking are dependent on glial activation. The first global aim of this study was to determine the molecular and behavioural effects of glial activation by two glial inhibitors, PF and PJ34 in a model of neuropathic pain. Glial activation was assessed via changes in specific proteins using fluorescent immunohistochemistry (IHC). Neuropathy-induced c-Fos activation was assessed by IHC and pain hypersensitivity was assessed, including mechanical allodynia and spontaneous pain. The second global aim determined the role of activated glia in changes in neuropathy-induced increases in DOR function and DOR subcellular localization using immunogold IHC and transmission electron microscopy (EM). Chronic PJ34 attenuated chronic constriction injury (CCI)-induced mircoglial, but not astrocyte activation. Chronic administration of either PF or PJ34 attenuated the CCI-induced increase in c-Fos immunoreactive expression. However, neither drug attenuated CCI-induced mechanical allodynia or spontaneous pain. Both chronic PF and PJ34 administration in NP animals attenuated the anti-allodynic effects of the DOR-selective agonist deltorphin II, suggesting glial inhibition blocks DOR function. However, chronic PF, but not PJ34, blocked the anti-allodynic effects of another DOR agonist, SNC80. These data suggest that SNC80 might be targeting a different DOR molecular species that is not affected by factors released from microglia. Finally, EM experiments revealed that chronic PF treatment prevented the CCI-induced increase in DOR trafficking providing a positive correlation between behaviour and receptor localization. This study suggests that activated glia contribute to changes in DOR function and trafficking in NP pain states. It also suggests that there is a dissociation between glial inhibition and pain hypersensitivity. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2010-08-31 14:45:47.888

No description available.

For decades the opioid receptors have been an attractive therapeutic target for the treatment of pain. Since the first discovery of enkephalin, approximately a dozen endogenous opioid peptides have been known to produce opioid activity and analgesia, but their therapeutics have been limited mainly due to low blood brain barrier penetration and poor resistance to proteolytic degradation. One versatile approach to overcome these drawbacks is the cyclization of linear peptides to cyclic peptides with constrained topographical structure. Compared to their linear parents, cyclic analogs exhibit better metabolic stability, lower offtarget toxicity, and improved bioavailability. Extensive structure-activity relationship studies have uncovered promising compounds for the treatment of pain as well as further elucidate structural elements required for selective opioid receptor activity. The benefits that come with employing cyclization can be further enhanced through the generation of polycyclic derivatives. Opioid ligands generally have a short peptide chain and thus the realm of polycyclic peptides has yet to be explored. In this review, a brief history of designing ligands for the opioid receptors, including classic linear and cyclic ligands, is discussed along with recent approaches and successes of cyclic peptide ligands for the receptors. Various scaffolds and approaches to improve bioavailability are elaborated and concluded with a discourse towards polycyclic peptides.

The midbrain periaqueductal gray matter (PAG) is an important brain region for the coordination of μ-opioid-induced pharmacological actions. The present study was designed to determine whether newly isolated μ-opioid peptide endomorphins can activate G proteins through μ-opioid receptors in the PAG by monitoring the binding to membranes of the non-hydrolyzable analog of GTP, guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS). An autoradiographic [35S]GTPγS binding study showed that both endomorphin-1 and -2 produced similar anatomical distributions of activated G proteins in the mouse midbrain region. In the mouse PAG, endomorphin-1 and -2 at concentrations from 0.001 to 10 μM increased [35S]GTPγS binding in a concentration-dependent manner and reached a maximal stimulation of 74.6 ± 3.8 and 72.3 ± 4.0%, respectively, at 10 μM. In contrast, the synthetic selective μ-opioid receptor agonist [D-Ala2,NHPhe4,Gly-ol]enkephalin (DAMGO) had a much greater efficacy and produced a 112.6 ± 5.1% increase of the maximal stimulation. The receptor specificity of endomorphin-stimulated [35S]GTPγS binding was verified by coincubating membranes with endomorphins in the presence of specific μ-, δ- or κ-opioid receptor antagonists. Coincubation with selective μ-opioid receptor antagonists β- funaltrexamine or D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH2 (CTOP) blocked both endomorphin-1 and-2-stimulated [35S]GTPγS binding. In contrast, neither δ- nor κ-opioid receptor antagonist had any effect on the [35S]GTPγS binding stimulated by either endomorphin-1 or -2. These findings indicate that both endomorphin-1 and -2 increase [35S]GTPγS binding by selectively stimulating μ-opioid receptors with intrinsic activity less than that of DAMGO and suggest that these new endogenous ligands might be partial agonists for μ-opioid receptors in the mouse PAG.

No description available.

An investigation of the interaction of stable opioid/ligands and unstable opioid peptides with opioid receptors in guinea pig brain, guinea pig myenteric plexus and mouse vas deferens has been carried out. The initial aim of the study was to further characterise K opioid receptors, using binding assays and isolated tissue bioassays. The second aim was to determine the true affinity and potency of small dynorphin peptides for the K opioid receptor and to determine if metabolism of the peptides to non K opioid receptor-preferring products contributes to their observed in vitro pharmacology.

The opioid receptors and their endogenous ligands have long been implicated in a variety of traits including addiction, impulsive behaviors and substance dependence. Using phenotypic measurements collected from the IASPSAD, data from a latent class analysis and data from a SNP array and additional genotyping assays, association and regression tests were performed to determine the effects of common SNPs encoded in the genes of the opioid receptors and ligands on various traits relating to alcohol dependence. Although only one SNP can be reported as significant for substance dependence within alcoholics, there were a few results approaching significance that may offer some insight into variation within alcoholism.

Effect of lipids on binding characteristics of opioid receptors in membranes prepared from rat brain were studied. Lipid concentrations causing changes in specific binding of [3H]Endomorphin-1 (ProE1), an opioid agonist highly specific to mu-type opioid, [3H]Ile5,6deltorphin II (DIDI), an agonist ligand highly specific to delta type receptor and [3H]Naloxone (Nlx), a universal opioid receptor antagonist were determined. Inhibition of [3H]ProE1, [3H]DIDI and [3H]Nlx specific binding was also examined by homologous displacement experiments in the presence and absence of lipids. In order to understand whether the changes occurring in the specific binding is due to changes in equilibrium dissociation constant (KD) or maximum number of binding sites (Bmax), the equilibrium binding experiments were performed. Arachidonic acid (AA) inhibited binding of both agonist and antagonist ligand in a dose dependent manner with IC50 values of 0.15, 0.1, and 0.6 mM for [3H]ProE1, [3H]DIDI and [3H]Nlx, respectively. Kd values were not affected while Bmax values decreased 38 % and 76 % for mu, and delta receptor subtypes, respectively. For [3H]Nlx, Bmax values decreased 20 and 56 % in the absence and presence of 100 mM NaCl, respectively. Cholesteryl hemisuccinate (CHS) enhances (100 % of control) ligand binding at mu-sites however no effect was encountered at delta sites. Furthermore, CHS also enhances (50 % of control) the binding of antagonist ligand in the absence of NaCl. Bmax values were increased by 70 % for mu sites and 40% for antagonist ligand binding site. Under similar conditions Kd values were not affected. Phosphatidic acid (PA) and phosphatidylcholine (PC) exhibited negligible effect on ligand binding. PA decreased specific binding of ProE1 and DIDI by 16 and 10 %, respectively. Specific binding of antagonist ligand Nlx decreased 11 % in the presence of NaCl whereas in the absence of NaCl specific binding is very close to control. In the presence of PC specific binding of both agonist and antagonist ligands were around control values. In this study modulatory effect of lysophospholipids, lysophosphatidic acid and lysophosphatidylcholine on opioid binding sites were evaluated for the first time. Both lysophospholipids exhibited similar effects: decreasing specific binding in receptor subtype independent manner between 0.1 to 1 mM range. Kd values were not significantly affected, while remarkable decrease (45-75 %) in Bmax values were observed.