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1.
Psychopharmacology (Berl) ; 237(3): 723-734, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31822924

RESUMO

RATIONALE: The N-phenylpropyl-N'-substituted piperazines SA-4503 (N-phenylpropyl-N'-(3,4-dimethoxyphenethyl)piperazine) and YZ-185 (N-phenylpropyl-N'-(3-methoxyphenethyl)piperazine) bind to sigma (σ) receptors and block the development of cocaine-induced conditioned place preference at concentrations that inhibit cocaine-induced hyperactivity. YZ-067 (N-phenylpropyl-N'-(4-methoxyphenethyl)piperazine) also binds to sigma receptors and attenuates cocaine-induced hyperactivity in mice. OBJECTIVES: The present study determined the effect of YZ-067 on the development and expression of cocaine (66 µmol/kg or 33 µmol/kg) conditioned place preference (CPP) and locomotor sensitization in mice. RESULTS: YZ-067 (10 or 31.6 µmol/kg) did not have intrinsic effects on place preference or place aversion. Interestingly, the 31.6 µmol/kg YZ-067 dose enhanced the development of cocaine place preference, while 10 µmol/kg YZ-067 attenuated the development of cocaine-induced locomotor sensitization. However, YZ-067 did not alter the expression of cocaine place preference nor cocaine-induced locomotor sensitization. In follow-up studies, YZ-067 did not affect performance in the zero maze or rotarod, indicating that sigma receptors probed by this ligand do not regulate anxiety-like or coordinated motor skill behaviors, respectively. CONCLUSION: Overall, these results are consistent with previous studies demonstrating a role for sigma receptors in the behavioral effects of cocaine. However, the present findings also indicate that N-phenylpropyl-N'-substituted piperazines do not strictly block cocaine's behavioral effects and that sigma receptor may differentially mediate cocaine-induced hyperactivity and place conditioning.


Assuntos
Cocaína/metabolismo , Condicionamento Psicológico/efeitos dos fármacos , Piperazinas/metabolismo , Receptores sigma/agonistas , Receptores sigma/metabolismo , Recompensa , Animais , Cocaína/farmacologia , Condicionamento Psicológico/fisiologia , Inibidores da Captação de Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Masculino , Camundongos , Piperazinas/química , Piperazinas/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia
2.
Psychopharmacology (Berl) ; 236(11): 3147-3158, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31139878

RESUMO

RATIONALE: Previous research indicates that the selective sigma-1 receptor ligand PD144418 and the selective sigma-2 ligands YUN-252 can inhibit cocaine-induced hyperactivity. The effects of these ligands on other stimulants, such as methamphetamine, have not been reported. OBJECTIVES: The present study examined the effects of PD144418 and YUN-252 pretreatment on methamphetamine-induced hyperactivity after acute treatment. METHODS: Mice (n = 8-14/group) were injected with PD144418 (3.16, 10, or 31.6 µmol/kg), YUN-252 (0.316, 3.16, 31.6 µmol/kg), or saline. After 15 min, mice injected with 2.69 µmol/kg methamphetamine or saline vehicle, where distance traveled during a 60-min period was recorded. Additionally, the effect of PD144418 on the initiation and expression of methamphetamine sensitization was determined by treating mice (n = 8-14/group) with PD144418, methamphetamine or saline repeatedly over a 5-day period, and testing said mice with a challenge dose after a 7-day withdrawal period. RESULTS: Results indicate that both PD144418 and YUN-252, in a dose-dependent manner, attenuated hyperactivity induced by an acute methamphetamine injection. Specifically, 10 µmol/kg or 31.6 µmol/kg of PD144418 and 31 µmol/kg of YUN-252 suppressed methamphetamine-induced hyperactivity. In regard to methamphetamine sensitization, while 10 µmol/kg PD144418 prevented the initiation of methamphetamine sensitization, it did not have an effect on the expression. CONCLUSIONS: Overall, the current results suggest an intriguing potential for this novel sigma receptor ligand as a treatment for the addictive properties of methamphetamine. Future analysis of this novel sigma receptor ligand in assays directly measuring reinforcement properties will be critical.


Assuntos
Estimulantes do Sistema Nervoso Central/farmacologia , Isoxazóis/metabolismo , Locomoção/efeitos dos fármacos , Metanfetamina/farmacologia , Piridinas/metabolismo , Receptores sigma/metabolismo , Animais , Estimulantes do Sistema Nervoso Central/antagonistas & inibidores , Isoxazóis/farmacologia , Ligantes , Locomoção/fisiologia , Masculino , Camundongos , Piridinas/farmacologia , Receptores sigma/antagonistas & inibidores , Reforço Psicológico , Receptor Sigma-1
3.
Nucl Med Biol ; 70: 53-66, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30933866

RESUMO

INTRODUCTION: Peripheral mu (µ) opioid receptors are implicated in pain, bowel dysfunction and the progression of certain cancers. In an effort to identify radioligands well suited for imaging these peripheral sites, we have prepared and evaluated four hydrophilic 111In labeled DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) conjugated µ tetrapeptides. METHODS: Peptides were prepared by solid-phase techniques, using orthogonal strategies to achieve branching to DOTA, and then characterized by HPLC, mass spectroscopy and amino acid analysis. Scaffolds included novel peptide H-Dmt-D-Ala-Phe-Orn-NH2 (DAPO), where Dmt = 2',6'-dimethyltyrosine, and known peptide H-Dmt-D-Arg-Phe-Lys-NH2 ([Dmt1]DALDA). Constructs had DOTA conjugation at the Orn4 or Lys4 side chains, or to the C-terminal through a hexanoic acid-lysine linker. Indium(III) complexation and 111In radiolabeling were accomplished by standard methods. Protein binding and Log D7.4 were determined. Binding and pharmacological profiles were obtained in vitro. Biodistribution and radiometabolite studies were conducted using male CD-1 mice. RESULTS: All four indium(III)-DOTA conjugates derived from DAPO and [Dmt1]DALDA showed good selectivity and subnanomolar affinity for µ opioid receptors. One radioligand, H-Dmt-D-Ala-Phe-Orn(δ-[111In]In-DOTA)-NH2, showed 25% specific binding in vivo to µ sites in mouse gut. Notably, this was the least polar of the series, and also showed low sensitivity to modulation of binding by sodium ions. All radioligands showed high kidney uptake of radiometabolites. CONCLUSIONS: Visualizing peripheral µ opioid receptors using 111In labeled DOTA-conjugated tetrapeptides appears feasible, but structural modifications to enhance specific binding and metabolic stability, as well as to reduce kidney uptake, will be required. ADVANCES IN KNOWLEDGE: This study shows in vivo labeling of peripheral µ opioid receptors by a tetrapeptide radioligand, and provides information that should prove useful in the design of peptide radioligands having optimal properties.


Assuntos
Desenho de Fármacos , Compostos Heterocíclicos com 1 Anel/química , Radioisótopos de Índio , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Receptores Opioides mu/metabolismo , Sequência de Aminoácidos , Animais , Técnicas de Química Sintética , Marcação por Isótopo , Ligantes , Masculino , Camundongos , Oligopeptídeos/síntese química , Oligopeptídeos/farmacocinética , Ligação Proteica , Especificidade por Substrato , Distribuição Tecidual
4.
Pharmacol Res ; 142: 87-100, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30721730

RESUMO

ABT-737, ABT-263 (Navitoclax) and ABT-199 (Venetoclax) are under intensive preclinical and clinical investigation as treatments for hematologic and other malignancies. These small molecules mimic pro-death B-cell lymphoma-2 (Bcl-2) Homology 3 (BH3) domain-only proteins. They also bear a structural resemblance to certain sigma (σ) receptor ligands. Moreover, the Bcl-2 and σ receptor protein families are both located primarily at the endoplasmic reticulum, mediate cell death and survival through protein-protein interactions, and physically associate. Accordingly, we examined the ability of the ABT series of BH3 mimetics to interact with σ receptors using radioligand-binding techniques. Negative allosteric modulation of [3H](+)-pentazocine, an agonist, binding to σ1 receptors in guinea pig brain membranes was observed for ABT-737, ABT-263 and ABT-199. Findings included reduction of specific binding to distinct plateaus in concentration-dependent fashion, significant slowing of radioligand dissociation kinetics, and decreases in radioligand affinity with no or modest changes in maximal receptor densities. Using a ternary complex model, dissociation constants (KX) for modulator binding to the σ1 receptor ranged from 1 to 2.5 µM, while negative cooperativity factors (α), representing the changes in affinity of ligand and modulator when bound as a ternary complex with the receptor, ranged from 0.15 to 0.42. These observations were extended and reinforced by studies using intact small cell (NCI-H69) and non-small cell (NCI-H23) lung cancer cells, and by using an antagonist σ1 receptor radioligand, E-N-1-(3'-[125I]iodoallyl)-N'-4-(3″,4″-dimethoxyphenethyl)piperazine, in mouse brain membranes. By contrast, exploratory studies indicate marked enhancement of the σ2 receptor binding of [3H]1,3-di-(o-tolyl)guanidine/(+)-pentazocine in NCI-H23 cells and guinea pig brain membranes. These findings raise intriguing questions regarding mechanism and potential functional outcomes.


Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Compostos de Bifenilo/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Nitrofenóis/farmacologia , Receptores sigma/metabolismo , Sulfonamidas/farmacologia , Regulação Alostérica , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular Tumoral , Cobaias , Humanos , Masculino , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2 , Ensaio Radioligante , Receptor Sigma-1
5.
Neurochem Int ; 123: 34-45, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30125594

RESUMO

The dopamine transporter (DAT) is a neuronal membrane protein that is responsible for reuptake of dopamine (DA) from the synapse and functions as a major determinant in control of DA neurotransmission. Cocaine and many psychostimulant drugs bind to DAT and block reuptake, inducing DA overflow that forms the neurochemical basis for euphoria and addiction. Paradoxically, however, some ligands such as benztropine (BZT) bind to DAT and inhibit reuptake but do not produce these effects, and it has been hypothesized that differential mechanisms of binding may stabilize specific transporter conformations that affect downstream neurochemical or behavioral outcomes. To investigate the binding mechanisms of BZT on DAT we used the photoaffinity BZT analog [125I]N-[n-butyl-4-(4‴-azido-3‴-iodophenyl)]-4',4″-difluoro-3α-(diphenylmethoxy)tropane ([125I]GA II 34) to identify the site of cross-linking and predict the binding pose relative to that of previously-examined cocaine photoaffinity analogs. Biochemical findings show that adduction of [125I]GA II 34 occurs at residues Asp79 or Leu80 in TM1, with molecular modeling supporting adduction to Leu80 and a pharmacophore pose in the central S1 site similar to that of cocaine and cocaine analogs. Substituted cysteine accessibility method protection analyses verified these findings, but identified some differences in structural stabilization relative to cocaine that may relate to BZT neurochemical outcomes.


Assuntos
Benzotropina/farmacologia , Sítios de Ligação/efeitos dos fármacos , Cocaína/farmacologia , Dopamina/metabolismo , Relação Estrutura-Atividade , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Humanos , Radioisótopos do Iodo/farmacologia
6.
Biochem Pharmacol ; 142: 204-215, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28734777

RESUMO

Dopamine transporter (DAT) blockers like cocaine and many other abused and therapeutic drugs bind and stabilize an inactive form of the transporter inhibiting reuptake of extracellular dopamine (DA). The resulting increases in DA lead to the ability of these drugs to induce psychomotor alterations and addiction, but paradoxical findings in animal models indicate that not all DAT antagonists induce cocaine-like behavioral outcomes. How this occurs is not known, but one possibility is that uptake inhibitors may bind at multiple locations or in different poses to stabilize distinct conformational transporter states associated with differential neurochemical endpoints. Understanding the molecular mechanisms governing the pharmacological inhibition of DAT is therefore key for understanding the requisite interactions for behavioral modulation and addiction. Previously, we leveraged complementary computational docking, mutagenesis, peptide mapping, and substituted cysteine accessibility strategies to identify the specific adduction site and binding pose for the crosslinkable, photoactive cocaine analog, RTI 82, which contains a photoactive azide attached at the 2ß position of the tropane pharmacophore. Here, we utilize similar methodology with a different cocaine analog N-[4-(4-azido-3-I-iodophenyl)-butyl]-2-carbomethoxy-3-(4-chlorophenyl)tropane, MFZ 2-24, where the photoactive azide is attached to the tropane nitrogen. In contrast to RTI 82, which crosslinked into residue Phe319 of transmembrane domain (TM) 6, our findings show that MFZ 2-24 adducts to Leu80 in TM1 with modeling and biochemical data indicating that MFZ 2-24, like RTI 82, occupies the central S1 binding pocket with the (+)-charged tropane ring nitrogen coordinating with the (-)-charged carboxyl side chain of Asp79. The superimposition of the tropane ring in the three-dimensional binding poses of these two distinct ligands provides strong experimental evidence for cocaine binding to DAT in the S1 site and the importance of the tropane moiety in competitive mechanisms of DA uptake inhibition. These findings set a structure-function baseline for comparison of typical and atypical DAT inhibitors and how their interactions with DAT could lead to the loss of cocaine-like behaviors.


Assuntos
Cocaína/análogos & derivados , Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Transtornos Relacionados ao Uso de Substâncias/metabolismo , Tropanos/metabolismo , Animais , Azidas/química , Azidas/metabolismo , Sítios de Ligação , Cocaína/química , Cocaína/metabolismo , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Radioisótopos do Iodo , Células LLC-PK1 , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mapeamento de Peptídeos , Marcadores de Fotoafinidade , Ligação Proteica , Relação Estrutura-Atividade , Transtornos Relacionados ao Uso de Substâncias/psicologia , Suínos , Tropanos/química
7.
Nucl Med Biol ; 44: 4-30, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27755986

RESUMO

INTRODUCTION: Aspects of radiopharmaceutical development are illustrated through preclinical studies of [125I]-(E)-1-(2-(2,3-dihydrobenzofuran-5-yl)ethyl)-4-(iodoallyl)piperazine ([125I]-E-IA-BF-PE-PIPZE), a radioligand for sigma-1 (σ1) receptors, coupled with examples from the recent literature. Findings are compared to those previously observed for [125I]-(E)-1-(2-(2,3-dimethoxy-5-yl)ethyl)-4-(iodoallyl)piperazine ([125I]-E-IA-DM-PE-PIPZE). METHODS: Syntheses of E-IA-BF-PE-PIPZE and [125I]-E-IA-BF-PE-PIPZE were accomplished by standard methods. In vitro receptor binding studies and autoradiography were performed, and binding potential was predicted. Measurements of lipophilicity and protein binding were obtained. In vivo studies were conducted in mice to evaluate radioligand stability, as well as specific binding to σ1 sites in brain, brain regions and peripheral organs in the presence and absence of potential blockers. RESULTS: E-IA-BF-PE-PIPZE exhibited high affinity and selectivity for σ1 receptors (Ki = 0.43 ± 0.03 nM, σ2/σ1 = 173). [125I]-E-IA-BF-PE-PIPZE was prepared in good yield and purity, with high specific activity. Radioligand binding provided dissociation (koff) and association (kon) rate constants, along with a measured Kd of 0.24 ± 0.01 nM and Bmax of 472 ± 13 fmol/mg protein. The radioligand proved suitable for quantitative autoradiography in vitro using brain sections. Moderate lipophilicity, Log D7.4 2.69 ± 0.28, was determined, and protein binding was 71 ± 0.3%. In vivo, high initial whole brain uptake, >6% injected dose/g, cleared slowly over 24 h. Specific binding represented 75% to 93% of total binding from 15 min to 24 h. Findings were confirmed and extended by regional brain biodistribution. Radiometabolites were not observed in brain (1%). CONCLUSIONS: Substitution of dihydrobenzofuranylethyl for dimethoxyphenethyl increased radioligand affinity for σ1 receptors by 16-fold. While high specific binding to σ1 receptors was observed for both radioligands in vivo, [125I]-E-IA-BF-PE-PIPZE displayed much slower clearance kinetics than [125I]-E-IA-DM-PE-PIPZE. Thus, minor structural modifications of σ1 receptor radioligands lead to major differences in binding properties in vitro and in vivo.


Assuntos
Compostos Radiofarmacêuticos/metabolismo , Receptores sigma/metabolismo , Animais , Traçadores Radioativos , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/química , Reprodutibilidade dos Testes
8.
Pharmacol Biochem Behav ; 150-151: 198-206, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27851908

RESUMO

This study examined the effect of the N-phenylpropyl-N'-substituted piperazine ligands SA4503 (3.4-dimethoxyphenethyl), YZ-067 (4-methoxyphenethyl), YZ-185 (3-methoxyphenethyl) and Nahas-3h (4-methoxybenzyl) on methamphetamine-induced hyperactivity in mice. In a previous study in rats, SA4503 increased methamphetamine-induced hyperactivity at a lower ligand dose and enhanced it at a higher dose. The other ligands have not been investigated in this assay. Presently, mice were administered sigma ligands, and specific [125I]E-IA-DM-PE-PIPZE and [125I]RTI-121 binding was measured to determine σ1 sigma receptor and dopamine transporter occupancy, respectively. Mice were also administered sigma ligands followed by methamphetamine, and locomotor activity was measured. Each of the ligands occupied σ1 sigma receptors (ED50=0.2-0.6µmol/kg) with similar potency, but none occupied the transporter (ED50>10µmol/kg). At the highest dose tested (31.6µmol/kg) all four sigma ligands significantly attenuated methamphetamine-induced hyperactivity. Interestingly, SA4503, YZ-067 and Nahas-3h, but not YZ-185, enhanced methamphetamine-induced hyperactivity at lower ligand doses (1-3.16µmol/kg). These results suggest that these ligands function as stimulant agonists at lower doses and as antagonists at higher does, with subtle changes in the substitution pattern at the 3- and 4-positions of the phenethyl group contributing to the nature of the interactions. Overall, these data indicate a complex role for σ1 sigma receptor ligands in methamphetamine's behavioral effects.


Assuntos
Hipercinese/induzido quimicamente , Metanfetamina/farmacologia , Piperazinas/farmacologia , Receptores sigma/efeitos dos fármacos , Animais , Cocaína/análogos & derivados , Cocaína/metabolismo , Masculino , Camundongos , Receptores sigma/agonistas , Receptores sigma/fisiologia , Relação Estrutura-Atividade
9.
Tetrahedron ; 72(40): 6127-6135, 2016 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-28190898

RESUMO

Radiolabeled diprenorphine (DPN) and analogs are widely used ligands for non-invasive brain imaging of opioid receptors. To develop complementary radioligands optimized for studies of the peripheral opioid receptors, we prepared a pair of hydrophilic DPN derivatives, conjugated to the macrocyclic chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), for complexation with trivalent metals. The non-radioactive indium (III) complexes, tethered to the C6-oxygen position of the DPN scaffold by 6- to 9-atom spacers, displayed high affinities for binding to µ, δ and κ opioid receptors in vitro. Use of the 9-atom linker conferred picomolar affinities equipotent to those of the parent ligand DPN. The [111In]-labeled complexes were prepared in good yield (>70%), with high radiochemical purity (~99%) and high specific radioactivity (>4000 mCi/µmol). Their log D7.4 values were -2.21 to -1.66. In comparison, DPN is lipophilic, with a log D7.4 of +2.25. Further study in vivo is warranted to assess the suitability of these [111In]-labeled DPN-DOTA conjugates for imaging trials.

10.
Synapse ; 70(3): 98-111, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26618331

RESUMO

Activation of sigma1 (σ1) receptors contributes to the behavioral and toxic effects of (-)-cocaine. We studied a key step, the ability of (-)-cocaine to occupy σ1 receptors in vivo, using CD-1(®) mice and the novel radioligand [(125) I]E-N-1-(3'-iodoallyl)-N'-4-(3",4"-dimethoxyphenethyl)-piperazine ([(125) I]E-IA-DM-PE-PIPZE). (-)-Cocaine displayed an ED50 of 68 µmol/kg for inhibition of specific radioligand binding in whole brain, with values between 73 and 80 µmol/kg for heart, lung, and spleen. For comparison, an ED50 of 26 µmol/kg for (-)-cocaine occupancy of striatal dopamine transporters (DAT) was determined by inhibition of [(125) I]3ß-(4-iodophenyl)tropan-2ß-carboxylic acid isopropyl ester ([(125) I]RTI-121) binding. A chief finding is the relatively small potency difference between (-)-cocaine occupancy of σ1 receptors and the DAT, although the DAT occupancy is likely underestimated. Interactions of (-)-cocaine with σ1 receptors were assessed further using [(125) I]E-IA-DM-PE-PIPZE for regional cerebral biodistribution studies and quantitative ex vivo autoradiography of brain sections. (-)-Cocaine binding to cerebral σ1 receptors proved directly proportional to the relative site densities known for the brain regions. Nonradioactive E-IA-DM-PE-PIPZE gave an ED50 of 0.23 µmol/kg for occupancy of cerebral σ1 receptors, and a 3.16 µmol/kg (i.p.) dose attenuated (-)-cocaine-induced locomotor hyperactivity by 30%. This effect did not reach statistical significance, but suggests that E-IA-DM-PE-PIPZE is a probable σ1 receptor antagonist. As groundwork for the in vivo studies, we used standard techniques in vitro to determine ligand affinities, site densities, and pharmacological profiles for the σ1 and σ2 receptors expressed in CD-1(®) mouse brain.


Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Cocaína/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Receptores sigma/metabolismo , Animais , Autorradiografia , Ligação Competitiva , Cocaína/farmacocinética , Inibidores da Captação de Dopamina/farmacocinética , Relação Dose-Resposta a Droga , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Receptor Sigma-1
11.
J Med Chem ; 58(14): 5609-19, 2015 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-26153715

RESUMO

Three photoaffinity ligands (PALs) for the human serotonin transporter (hSERT) were synthesized based on the selective serotonin reuptake inhibitor (SSRI), (S)-citalopram (1). The classic 4-azido-3-iodo-phenyl group was appended to either the C-1 or C-5 position of the parent molecule, with variable-length linkers, to generate ligands 15, 22, and 26. These ligands retained high to moderate affinity binding (K(i) = 24-227 nM) for hSERT, as assessed by [(3)H]5-HT transport inhibition. When tested against Ser438Thr hSERT, all three PALs showed dramatic rightward shifts in inhibitory potency, with Ki values ranging from 3.8 to 9.9 µM, consistent with the role of Ser438 as a key residue for high-affinity binding of many SSRIs, including (S)-citalopram. Photoactivation studies demonstrated irreversible adduction to hSERT by all ligands, but the reduced (S)-citalopram inhibition of labeling by [(125)I]15 compared to that by [(125)I]22 and [(125)I]26 suggests differences in binding mode(s). These radioligands will be useful for characterizing the drug-protein binding interactions for (S)-citalopram at hSERT.


Assuntos
Citalopram/metabolismo , Desenho de Fármacos , Processos Fotoquímicos , Inibidores Seletivos de Recaptação de Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Azidas/química , Citalopram/síntese química , Citalopram/química , Células HEK293 , Humanos , Ligantes , Inibidores Seletivos de Recaptação de Serotonina/síntese química , Inibidores Seletivos de Recaptação de Serotonina/química
12.
Eur J Pharmacol ; 762: 118-26, 2015 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-26004528

RESUMO

This study establishes the expression of appreciable populations of sites on mouse lung membranes that exhibit radioligand binding properties and pharmacology consistent with assignment as sigma1 and sigma2 receptors. Specific binding of the sigma1 receptor radioligand [(3)H](+)-pentazocine reached steady state within 6h at 37°C. Saturation studies revealed high affinity binding to a single class of sites (Kd 1.36±0.04nM; Bmax 967±11fmol/mg protein). Inhibition studies showed appropriate sigma1 receptor pharmacology, including higher affinity for (+)-N-allylnormetazocine with respect to the (-)-enantiomer, and positive allosteric modulation of dextromethorphan binding by phenytoin. Using [(3)H]1,3-di(2-tolyl)guanidine in the presence of (+)-pentazocine to assess sigma2 receptor binding, steady state was achieved within 2min at 25°C. Cold saturation studies revealed one high affinity, low capacity binding site (Kd 31.8±8.3nM; Bmax 921±228fmol/mg protein) that displayed sigma2 receptor pharmacology. A very low affinity, high capacity interaction also was observed that represents saturable, but not sigma receptor specific, binding. A panel of ligands showed rank order inhibition of radioligand binding appropriate for the sigma2 receptor, with ifenprodil displaying the highest apparent affinity. In vivo, dextromethorphan inhibited the specific binding of a radioiodinated sigma1 receptor ligand in lung with an ED50 of 1.2µmol/kg, a value near the recommended dosage for the drug as a cough suppressant. Overall, the present work provides a foundation for studies of drug interactions with pulmonary sigma1 and sigma2 receptors in vitro and in vivo.


Assuntos
Pulmão/metabolismo , Ensaio Radioligante , Receptores sigma/metabolismo , Animais , Membrana Celular/metabolismo , Pulmão/citologia , Masculino , Camundongos
13.
Bioorg Med Chem ; 23(1): 222-30, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25468036

RESUMO

Two series of novel ether analogs of the sigma (σ) receptor ligand 1-[2-(3,4-dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine (SA4503) have been prepared. In one series, the alkyl portion of the 4-methoxy group was replaced with allyl, propyl, bromoethyl, benzyl, phenethyl, and phenylpropyl moieties. In the second series, the 3,4-dimethoxy was replaced with cyclic methylenedioxy, ethylenedioxy and propylenedioxy groups. These ligands, along with 4-O-des-methyl SA4503, were evaluated for σ1 and σ2 receptor affinity, and compared to SA4503 and several known ether analogs. SA4503 and a subset of ether analogs were also evaluated for dopamine transporter (DAT) and serotonin transporter (SERT) affinity. The highest σ1 receptor affinities, Ki values of 1.75-4.63 nM, were observed for 4-O-des-methyl SA4503, SA4503 and the methylenedioxy analog. As steric bulk increased, σ1 receptor affinity decreased, but only to a point. Allyl, propyl and bromoethyl substitutions gave σ1 receptor Ki values in the 20-30 nM range, while bulkier analogs having phenylalkyl, and Z- and E-iodoallyl, ether substitutions showed higher σ1 affinities, with Ki values in the 13-21 nM range. Most ligands studied exhibited comparable σ1 and σ2 affinities, resulting in little to no subtype selectivity. SA4503, the fluoroethyl analog and the methylenedioxy congener showed modest six- to fourteen-fold selectivity for σ1 sites. DAT and SERT interactions proved much more sensitive than σ receptor interactions to these structural modifications. For example, the benzyl congener (σ1Ki=20.8 nM; σ2Ki=16.4 nM) showed over 100-fold higher DAT affinity (Ki=121 nM) and 6-fold higher SERT affinity (Ki=128nM) than the parent SA4503 (DAT Ki=12650 nM; SERT Ki=760 nM). Thus, ether modifications to the SA4503 scaffold can provide polyfunctional ligands having a broader spectrum of possible pharmacological actions.


Assuntos
Éter/química , Piperazinas/química , Receptores sigma/química , Éter/metabolismo , Piperazinas/metabolismo , Ligação Proteica , Receptores sigma/agonistas , Receptores sigma/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Receptor Sigma-1
14.
J Biol Chem ; 289(43): 29712-27, 2014 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-25179220

RESUMO

The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3ß-(p-chlorophenyl)tropane-2ß-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([(125)I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [(125)I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies overcomes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors.


Assuntos
Azidas/metabolismo , Cocaína/análogos & derivados , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Simulação de Acoplamento Molecular , Animais , Azidas/química , Sítios de Ligação , Cocaína/química , Cocaína/metabolismo , Brometo de Cianogênio/metabolismo , Células HeLa , Humanos , Células LLC-PK1 , Ligantes , Mesilatos/metabolismo , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ratos , Especificidade por Substrato , Suínos
15.
J Pharmacol Exp Ther ; 351(1): 153-63, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25100754

RESUMO

Psychostimulant effects of cocaine are mediated partly by agonist actions at sigma-1 (σ1) receptors. Selective σ1 receptor antagonists attenuate these effects and provide a potential avenue for pharmacotherapy. However, the selective and high affinity σ1 antagonist PD144418 (1,2,3,6-tetrahydro-5-[3-(4-methylphenyl)-5-isoxazolyl]-1-propylpyridine) has been reported not to inhibit cocaine-induced hyperactivity. To address this apparent paradox, we evaluated aspects of PD144418 binding in vitro, investigated σ1 receptor and dopamine transporter (DAT) occupancy in vivo, and re-examined effects on locomotor activity. PD144418 displayed high affinity for σ1 sites (Ki 0.46 nM) and 3596-fold selectivity over σ2 sites (Ki 1654 nM) in guinea pig brain membranes. No appreciable affinity was noted for serotonin and norepinephrine transporters (Ki >100 µM), and the DAT interaction was weak (Ki 9.0 µM). In vivo, PD144418 bound to central and peripheral σ1 sites in mouse, with an ED50 of 0.22 µmol/kg in whole brain. No DAT occupancy by PD144418 (10.0 µmol/kg) or possible metabolites were observed. At doses that did not affect basal locomotor activity, PD144418 (1, 3.16, and 10 µmol/kg) attenuated cocaine-induced hyperactivity in a dose-dependent manner in mice. There was good correlation (r(2) = 0.88) of hyperactivity reduction with increasing cerebral σ1 receptor occupancy. The behavioral ED50 of 0.79 µmol/kg corresponded to 80% occupancy. Significant σ1 receptor occupancy and the ability to mitigate cocaine's motor stimulatory effects were observed for 16 hours after a single 10.0 µmol/kg dose of PD144418.


Assuntos
Cocaína/farmacologia , Isoxazóis/farmacologia , Córtex Motor/efeitos dos fármacos , Antagonistas de Entorpecentes/farmacocinética , Piridinas/farmacologia , Receptores sigma/metabolismo , Animais , Sítios de Ligação , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Cobaias , Hipercinese/metabolismo , Isoxazóis/química , Isoxazóis/farmacocinética , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Córtex Motor/metabolismo , Antagonistas de Entorpecentes/farmacologia , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Ligação Proteica , Piridinas/química , Piridinas/farmacocinética , Receptores sigma/antagonistas & inibidores , Receptores sigma/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Receptor Sigma-1
16.
Synapse ; 68(2): 73-84, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24123353

RESUMO

Cocaine functions, in part, through agonist actions at sigma-1 (σ1 ) receptors, while roles played by sigma-2 (σ2 ) receptors are less established. Attempts to discriminate σ2 receptor-mediated effects of cocaine in locomotor hyperactivity assays have been hampered by the lack of potent and selective antagonists. Certain tetrahydroisoquinolinyl benzamides display high σ2 receptor affinity, and excellent selectivity for binding to σ2 over σ1 receptors. The behavioral properties of this structural class of σ ligands have not yet been investigated. The present study evaluated 5-bromo-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl)]-2,3-dimethoxy-benzamide, 1, a ligand shown by others to bind preferentially to σ2 over σ1 receptors, as well as dopamine D2 and D3 sites. First, we determined binding to monoamine transporters and opioid receptors, and noted 57-fold selectivity for σ2 receptors over the serotonin transporter, and >800-fold selectivity for σ2 receptors over the other sites tested. We then examined 1 in locomotor activity studies using male CD-1® mice, and saw no alteration of basal activity at doses up to 31.6 µmol/kg. Cocaine produced a fivefold increase in locomotor activity, which was attenuated by 66% upon pretreatment of mice with 1 at 31.6 µmol/kg. In vivo radioligand binding studies also were performed, and showed no occupancy of σ1 receptors or the dopamine transporter by 1, or its possible metabolites, at the 31.6 µmol/kg dose. Thus, ligand 1 profiles behaviorally as a σ2 receptor-selective antagonist that is able to counteract cocaine's motor stimulatory effects.


Assuntos
Benzamidas/farmacologia , Cocaína/farmacologia , Isoquinolinas/farmacologia , Locomoção/efeitos dos fármacos , Receptores sigma/antagonistas & inibidores , Animais , Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Ligantes , Camundongos , Ligação Proteica , Receptores sigma/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Receptor Sigma-1
17.
ISRN Pharmacol ; 2013: 546314, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24089641

RESUMO

Sigma receptor antagonists diminish the effects of cocaine in behavioral assays, including conditioned place preference. Previous locomotor activity experiments in mice determined that the sigma receptor ligand YZ-185 (N-phenylpropyl-N'-(3-methoxyphenethyl)piperazine) enhanced cocaine-induced hyperactivity at a lower (0.1 µ mol/kg) dose and dose-dependently attenuated cocaine-induced hyperactivity at higher (3.16-31.6 µ mol/kg) doses. The present study investigated the effect of YZ-185 on cocaine's conditioned-rewarding properties in mice. YZ-185 (0.1, 0.316, 3.16, and 31.6 µ mol/kg) did not have intrinsic activity to produce conditioned place preference or aversion. A higher (31.6 µ mol/kg) YZ-185 dose, but not lower (0.1-3.16 µ mol/kg) YZ-185 doses, prevented the development of place preference to cocaine (66 µ mol/kg). YZ-185 did not alter the expression of cocaine place preference. To further characterize YZ-185's behavioral profile, its effects in the elevated zero maze and rotarod procedures were also determined; YZ-185 produced no significant change from baseline in either assay, indicating that the sigma receptors probed by YZ-185 do not regulate anxiety-like or coordinated motor skill behaviors. Overall, these results suggest that YZ-185 is a sigma receptor antagonist at the 31.6 µ mol/kg dose and demonstrate that sigma receptors can mediate the development of the conditioned-rewarding properties of cocaine.

18.
Pharmacol Biochem Behav ; 110: 201-7, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23891829

RESUMO

The present study examined N-phenylpropyl-N'-substituted piperazine sigma receptor ligands on cocaine-induced changes in locomotor activity in mice. Previous reports indicate that N-phenylpropyl-N'-(4-methoxybenzyl)piperazine (Nahas-3h), N-phenylpropyl-N'-(4-methoxyphenethyl)piperazine (YZ-067), and N-phenylpropyl-N'-(3-methoxyphenethyl)piperazine (YZ-185) bind with high affinity (Ki values≈1 nM) to σ1 sigma receptors. YZ-067 and YZ-185 are known to attenuate cocaine-induced convulsions, while Nahas-3h has not been tested in behavioral studies. Nahas-3h significantly attenuated cocaine-induced hyperactivity. YZ-067 decreased the effect of cocaine in a dose-dependent manner. Interestingly, YZ-185 inhibited cocaine's effect at higher doses, but enhanced cocaine's effect at a low dose. The YZ-185 inhibition of cocaine-induced hyperactivity was not surmounted by increasing the cocaine dose. Overall, this study is consistent with previous work showing the ability of certain sigma receptor ligands to affect cocaine-induced hyperactivity. Further, subtle alterations of ligand structure and the specific dosage levels employed influence the behavioral effects observed, with a 3-methoxy substituent apparently conferring the ability of a ligand to enhance cocaine's locomotor stimulatory effects.


Assuntos
Cocaína/farmacologia , Hipercinese/induzido quimicamente , Piperazinas/farmacologia , Receptores sigma/metabolismo , Animais , Relação Dose-Resposta a Droga , Ligantes , Masculino , Camundongos
19.
ACS Med Chem Lett ; 3(5): 378-382, 2012 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-23066448

RESUMO

The development of photoaffinity ligands for determining covalent points of attachment to the dopamine transporter (DAT) has predominantly focused on tropane-based compounds bearing variable-length linkers between the photoreactive group and inhibitor pharmacophore. In order to expand the array of photoprobes useful for mapping inhibitor-binding pockets within the DAT, a compact non-tropane ligand was synthesized featuring a photoreactive azide and iodine tag directly attached to the aromatic ring of (±)-threo-methylphenidate. (±)-threo-4-Azido-3-iodomethylphenidate ((±)-6); K(i) = 4.0 ± 0.8 nM) displayed high affinity for hDAT. Moreover, a radioiodinated analog of (±)-6 demonstrated covalent ligation to the DAT in cultured cells and rat striatal membranes, thus suggesting the potential utility of this photoprobe in DAT structure-function studies.

20.
Proc Natl Acad Sci U S A ; 109(31): 12426-31, 2012 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-22802668

RESUMO

Systemic delivery of therapeutic agents to solid tumors is hindered by vascular and interstitial barriers. We hypothesized that prostate tumor specific epigallocatechin-gallate (EGCg) functionalized radioactive gold nanoparticles, when delivered intratumorally (IT), would circumvent transport barriers, resulting in targeted delivery of therapeutic payloads. The results described herein support our hypothesis. We report the development of inherently therapeutic gold nanoparticles derived from the Au-198 isotope; the range of the (198)Au ß-particle (approximately 11 mm in tissue or approximately 1100 cell diameters) is sufficiently long to provide cross-fire effects of a radiation dose delivered to cells within the prostate gland and short enough to minimize the radiation dose to critical tissues near the periphery of the capsule. The formulation of biocompatible (198)AuNPs utilizes the redox chemistry of prostate tumor specific phytochemical EGCg as it converts gold salt into gold nanoparticles and also selectively binds with excellent affinity to Laminin67R receptors, which are over expressed in prostate tumor cells. Pharmacokinetic studies in PC-3 xenograft SCID mice showed approximately 72% retention of (198)AuNP-EGCg in tumors 24 h after intratumoral administration. Therapeutic studies showed 80% reduction of tumor volumes after 28 d demonstrating significant inhibition of tumor growth compared to controls. This innovative nanotechnological approach serves as a basis for designing biocompatible target specific antineoplastic agents. This novel intratumorally injectable (198)AuNP-EGCg nanotherapeutic agent may provide significant advances in oncology for use as an effective treatment for prostate and other solid tumors.


Assuntos
Anticarcinógenos/farmacocinética , Catequina/análogos & derivados , Ouro/farmacocinética , Nanopartículas Metálicas , Neoplasias da Próstata/tratamento farmacológico , Animais , Anticarcinógenos/farmacologia , Catequina/farmacocinética , Catequina/farmacologia , Linhagem Celular Tumoral , Feminino , Ouro/farmacologia , Radioisótopos de Ouro/farmacocinética , Radioisótopos de Ouro/farmacologia , Humanos , Masculino , Camundongos , Camundongos SCID , Tamanho da Partícula , Neoplasias da Próstata/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
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