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1.
J Mol Model ; 28(1): 16, 2021 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-34961887

RESUMO

In this work, we designed three new ligands by conjugating cholesterol metabolites 3-hydroxy-5-cholestenoic acid (3-HC) and 3-oxo-4-cholestenoic acid (3-OC) and the natural tri-terpenoid betulinic acid with the tumor-targeting peptide YHWYGYTPQNVI. Molecular interactions with the unconjugated peptide and the conjugates were examined with three receptors that are commonly overexpressed in pancreatic adenocarcinoma cells using ligand docking and molecular dynamics. This study demonstrated the utility of the designed conjugates as a valuable scaffold for potentially targeting EGFR and LDLR receptors. Our results indicate that the conjugates showed strong binding affinities and formation of stable complexes with EGFR, while the unconjugated peptide, BT-peptide conjugate, an 3-HC-peptide conjugate showed the formation of fairly stable complexes with LDLR receptor. For EGFR, two receptor kinase domains were explored. Interactions with the N-terminal domain of CCKA-R were relatively weaker. For LDLR, binding occurred in the beta-propeller region. For the N-terminal fragment of CCKA-R, the conjugates induced significant conformational changes in the receptor. The molecular dynamic simulations for 100 ns demonstrate that BT-peptide conjugates and the unconjugated peptide had the highest binding and formed the most stable complexes with EGFR. RMSD and trajectory analyses indicate that these molecules transit to a dynamically stable configuration in most cases within 60 ns. NMA analysis indicated that amongst the conjugates that showed relatively higher interactions with the respective receptors, the highest potential for deformability was seen for the N-terminal-47 amino acid region of the CCKA-R receptor with and the lowest for the LDLR-receptor. Thus, the newly designed compounds may be evaluated in the future toward developing drug delivery materials for targeting tumor cells overexpressing LDLR or EGFR.


Assuntos
Colesterol/química , Modelos Moleculares , Triterpenos Pentacíclicos/química , Peptídeos/química , Receptor de Colecistocinina A/química , Receptores de LDL/química , Sequência de Aminoácidos , Receptores ErbB/química , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade , Ácido Betulínico
2.
Nat Chem Biol ; 17(12): 1238-1244, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34556862

RESUMO

Cholecystokinin A receptor (CCKAR) belongs to family A G-protein-coupled receptors and regulates nutrient homeostasis upon stimulation by cholecystokinin (CCK). It is an attractive drug target for gastrointestinal and metabolic diseases. One distinguishing feature of CCKAR is its ability to interact with a sulfated ligand and to couple with divergent G-protein subtypes, including Gs, Gi and Gq. However, the basis for G-protein coupling promiscuity and ligand recognition by CCKAR remains unknown. Here, we present three cryo-electron microscopy structures of sulfated CCK-8-activated CCKAR in complex with Gs, Gi and Gq heterotrimers, respectively. CCKAR presents a similar conformation in the three structures, whereas conformational differences in the 'wavy hook' of the Gα subunits and ICL3 of the receptor serve as determinants in G-protein coupling selectivity. Our findings provide a framework for understanding G-protein coupling promiscuity by CCKAR and uncover the mechanism of receptor recognition by sulfated CCK-8.


Assuntos
Colecistocinina/química , Receptor de Colecistocinina A/química , Receptores Acoplados a Proteínas G/química , Sincalida/análogos & derivados , Sequência de Aminoácidos , Benzodiazepinonas/química , Microscopia Crioeletrônica , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Sincalida/química , Triazóis/química
3.
BMC Genomics ; 16: 431, 2015 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-26044654

RESUMO

BACKGROUND: Domestic goats (Capra hircus) have been selected to play an essential role in agricultural production systems, since being domesticated from their wild progenitor, bezoar (Capra aegagrus). A detailed understanding of the genetic consequences imparted by the domestication process remains a key goal of evolutionary genomics. RESULTS: We constructed the reference genome of bezoar and sequenced representative breeds of domestic goats to search for genomic changes that likely have accompanied goat domestication and breed formation. Thirteen copy number variation genes associated with coat color were identified in domestic goats, among which ASIP gene duplication contributes to the generation of light coat-color phenotype in domestic goats. Analysis of rapidly evolving genes identified genic changes underlying behavior-related traits, immune response and production-related traits. CONCLUSION: Based on the comparison studies of copy number variation genes and rapidly evolving genes between wild and domestic goat, our findings and methodology shed light on the genetic mechanism of animal domestication and will facilitate future goat breeding.


Assuntos
Genoma , Cabras/genética , Sequência de Aminoácidos , Animais , Animais Domésticos/genética , Animais Selvagens/genética , Evolução Biológica , Cruzamento , DNA/análise , DNA/isolamento & purificação , Variações do Número de Cópias de DNA , Variação Genética , Sistema Imunitário/metabolismo , Masculino , Dados de Sequência Molecular , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/classificação , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , Sistema Nervoso/metabolismo , Filogenia , Estrutura Terciária de Proteína , Receptor de Colecistocinina A/química , Receptor de Colecistocinina A/genética , Receptor de Colecistocinina A/metabolismo , Alinhamento de Sequência
4.
J Biol Chem ; 289(26): 18314-26, 2014 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-24825903

RESUMO

Cholecystokinin (CCK) stimulates the type 1 CCK receptor (CCK1R) to elicit satiety after a meal. Agonists with this activity, although potentially useful for treatment of obesity, can also have side effects and toxicities of concern, making the development of an intrinsically inactive positive allosteric modulator quite attractive. Positive allosteric modulators also have the potential to correct the defective receptor-G protein coupling observed in the high membrane cholesterol environment described in metabolic syndrome. Current model systems to study CCK1R in such an environment are unstable and expensive to maintain. We now report that the Y140A mutation within a cholesterol-binding motif and the conserved, class A G protein-coupled receptor-specific (E/D)RY signature sequence results in ligand binding and activity characteristics similar to wild type CCK1R in a high cholesterol environment. This is true for natural CCK, as well as ligands with distinct chemistries and activity profiles. Additionally, the Y140A construct also behaved like CCK1R in high cholesterol in regard to its internalization, sensitivity to a nonhydrolyzable GTP analog, and anisotropy of a bound fluorescent CCK analog. Chimeric CCK1R/CCK2R constructs that systematically changed the residues in the allosteric ligand-binding pocket were studied in the presence of Y140A. This established increased importance of unique residues within TM3 and reduced the importance of TM2 for binding in the presence of this mutation, with the agonist trigger likely pulled away from its Leu(356) target on TM7. The distinct conformation of this intramembranous pocket within Y140A CCK1R provides an opportunity to normalize this by using a small molecule allosteric ligand, thereby providing safe and effective correction of the coupling defect in metabolic syndrome.


Assuntos
Colesterol/metabolismo , Receptor de Colecistocinina A/genética , Receptor de Colecistocinina A/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Colesterol/química , Cricetulus , Humanos , Cinética , Modelos Moleculares , Mutação de Sentido Incorreto , Ligação Proteica , Receptor de Colecistocinina A/química
5.
PLoS One ; 8(9): e75502, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24069424

RESUMO

FMRF-NH2 peptides which contain a conserved, identical C-terminal tetrapeptide but unique N terminus modulate cardiac contractility; yet, little is known about the mechanisms involved in signaling. Here, the structure-activity relationships (SARs) of the Drosophila melanogaster FMRF-NH2 peptides, PDNFMRF-NH2, SDNFMRF-NH2, DPKQDFMRF-NH2, SPKQDFMRF-NH2, and TPAEDFMRF-NH2, which bind FMRFa-R, were investigated. The hypothesis tested was the C-terminal tetrapeptide FMRF-NH2, particularly F1, makes extensive, strong ligand-receptor contacts, yet the unique N terminus influences docking and activity. To test this hypothesis, docking, binding, and bioactivity of the C-terminal tetrapeptide and analogs, and the FMRF-NH2 peptides were compared. Results for FMRF-NH2 and analogs were consistent with the hypothesis; F1 made extensive, strong ligand-receptor contacts with FMRFa-R; Y → F (YMRF-NH2) retained binding, yet A → F (AMRF-NH2) did not. These findings reflected amino acid physicochemical properties; the bulky, aromatic residues F and Y formed strong pi-stacking and hydrophobic contacts to anchor the ligand, interactions which could not be maintained in diversity or number by the small, aliphatic A. The FMRF-NH2 peptides modulated heart rate in larva, pupa, and adult distinctly, representative of the contact sites influenced by their unique N-terminal structures. Based on physicochemical properties, the peptides each docked to FMRFa-R with one best pose, except FMRF-NH2 which docked with two equally favorable poses, consistent with the N terminus influencing docking to define specific ligand-receptor contacts. Furthermore, SDNAMRF-NH2 was designed and, despite lacking the aromatic properties of one F, it binds FMRFa-R and demonstrated a unique SAR, consistent with the N terminus influencing docking and conferring binding and activity; thus, supporting our hypothesis.


Assuntos
FMRFamida/química , FMRFamida/farmacologia , Contração Miocárdica/efeitos dos fármacos , Relação Estrutura-Atividade , Sequência de Aminoácidos , Animais , Sítios de Ligação , Colecistocinina/química , Colecistocinina/metabolismo , Drosophila melanogaster , FMRFamida/metabolismo , Feminino , Frequência Cardíaca/efeitos dos fármacos , Ligantes , Masculino , Modelos Moleculares , Simulação de Acoplamento Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Conformação Proteica , Receptor de Colecistocinina A/química , Receptor de Colecistocinina A/metabolismo , Receptores de Peptídeos de Invertebrados/química , Receptores de Peptídeos de Invertebrados/metabolismo
6.
J Biol Chem ; 288(29): 21082-21095, 2013 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-23754289

RESUMO

Understanding the molecular basis of drug action can facilitate development of more potent and selective drugs. Here, we explore the molecular basis for action of a unique small molecule ligand that is a type 1 cholecystokinin (CCK) receptor agonist and type 2 CCK receptor antagonist, GI181771X. We characterize its binding utilizing structurally related radioiodinated ligands selective for CCK receptor subtypes that utilize the same allosteric ligand-binding pocket, using wild-type receptors and chimeric constructs exchanging the distinct residues lining this pocket. Intracellular calcium assays were performed to determine biological activity. Molecular models for docking small molecule agonists to the type 1 CCK receptor were developed using a ligand-guided refinement approach. The optimal model was distinct from the previous antagonist model for the same receptor and was mechanistically consistent with the current mutagenesis data. This study revealed a key role for Leu(7.39) that was predicted to interact with the isopropyl group in the N1 position of the benzodiazepine that acts as a "trigger" for biological activity. The molecular model was predictive of binding of other small molecule agonists, effectively distinguishing these from 1065 approved drug decoys with an area under curve value of 99%. The model also selectively enriched for agonist compounds, with 130 agonists identified by ROC analysis when seeded in 2175 non-agonist ligands of the type 1 CCK receptor (area under curve 78%). Benzodiazepine agonists in this series docked in consistent pose within this pocket, with a key role played by Leu(7.39), whereas the role of this residue was less clear for chemically distinct agonists.


Assuntos
Benzodiazepinas/farmacologia , Receptor de Colecistocinina A/agonistas , Sequência de Aminoácidos , Animais , Benzodiazepinas/química , Células CHO , Cricetinae , Cricetulus , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/agonistas , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Curva ROC , Receptor de Colecistocinina A/química , Receptor de Colecistocinina A/metabolismo , Receptor de Colecistocinina B/química , Receptor de Colecistocinina B/metabolismo , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência
7.
Nucleic Acids Res ; 40(Web Server issue): W370-5, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22564899

RESUMO

Transmembrane helical segments (TMs) can be classified into two groups of so-called 'simple' and 'complex' TMs. Whereas the first group represents mere hydrophobic anchors with an overrepresentation of aliphatic hydrophobic residues that are likely attributed to convergent evolution in many cases, the complex ones embody ancestral information and tend to have structural and functional roles beyond just membrane immersion. Hence, the sequence homology concept is not applicable on simple TMs. In practice, these simple TMs can attract statistically significant but evolutionarily unrelated hits during similarity searches (whether through BLAST- or HMM-based approaches). This is especially problematic for membrane proteins that contain both globular segments and TMs. As such, we have developed the transmembrane helix: simple or complex (TMSOC) webserver for the identification of simple and complex TMs. By masking simple TM segments in seed sequences prior to sequence similarity searches, the false-discovery rate decreases without sacrificing sensitivity. Therefore, TMSOC is a novel and necessary sequence analytic tool for both the experimentalists and the computational biology community working on membrane proteins. It is freely accessible at http://tmsoc.bii.a-star.edu.sg or available for download.


Assuntos
Proteínas de Membrana/química , Software , Algoritmos , Interações Hidrofóbicas e Hidrofílicas , Internet , Estrutura Secundária de Proteína , Receptor de Colecistocinina A/química , Rodopsina/química , Análise de Sequência de Proteína , Interface Usuário-Computador
8.
Am J Physiol Cell Physiol ; 302(3): C615-27, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22049215

RESUMO

Receptor ligands, identified as antagonists, based on the absence of stimulation of signaling, can rarely stimulate receptor internalization. d-Tyr-Gly-[(Nle(28,31),d-Trp(30))CCK-26-32]-2-phenylethyl ester (d-Trp-OPE) is such a ligand that binds to the cholecystokinin (CCK) receptor and stimulates internalization. Here, the molecular basis of this trafficking event is explored, with the assumption that ligand binding initiates conformational change, exposing an epitope to direct endocytosis. Ligand-stimulated internalization was studied morphologically using fluorescent CCK and d-Trp-OPE. d-Trp-OPE occupation of Chinese hamster ovary cell receptors stimulated internalization into the same region as CCK. Arrestin-biased action was ruled out using morphological translocation of fluorescent arrestin 2 and arrestin 3, moving to the membrane in response to CCK, but not d-Trp-OPE. Possible roles of the carboxyl terminus were studied using truncated receptor constructs, eliminating the proline-rich distal tail, the serine/threonine-rich midregion, and the remainder to the vicinal cysteines. None of these constructs disrupted d-Trp-OPE-stimulated internalization. Possible contributions of transmembrane segments were studied using competitive inhibition with peptides that also had no effect. Intracellular regions were studied with a similar strategy using coexpressing cell lines. Peptides corresponding to ends of each loop region were studied, with only the peptide at the carboxyl end of the third loop inhibiting d-Trp-OPE-stimulated internalization but having no effect on CCK-stimulated internalization. The region contributing to this effect was refined to peptide 309-323, located below the recognized G protein-association motif. While a receptor in which this segment was deleted did internalize in response to d-Trp-OPE, it exhibited abnormal ligand binding and did not signal in response to CCK, suggesting an abnormal conformation and possible mechanism of internalization distinct from that being studied. This interpretation was further supported by the inability of peptide 309-323 to inhibit its d-Trp-OPE-stimulated internalization. Thus the 309-323 region of the type 1 CCK receptor affects antagonist-stimulated internalization of this receptor, although its mechanism and interacting partner are not yet clear.


Assuntos
Membrana Celular/metabolismo , Colecistocinina/análogos & derivados , Fragmentos de Peptídeos/metabolismo , Receptor de Colecistocinina A/química , Receptor de Colecistocinina A/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Arrestina/metabolismo , Células CHO , Linhagem Celular , Colecistocinina/metabolismo , Cricetinae , Endocitose , Humanos , Ligantes , Conformação Proteica , Transporte Proteico , Ratos , Deleção de Sequência
9.
Biochemistry ; 47(36): 9574-81, 2008 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-18700727

RESUMO

Fluorescence resonance energy transfer (FRET) represents a powerful tool to establish relative distances between donor and acceptor fluorophores. By utilizing several donors situated in distinct positions within a docked full agonist ligand and several acceptors distributed at distinct sites within its receptor, multiple interdependent dimensions can be determined. These can provide a unique method to establish or confirm three-dimensional structure of the molecular complex. In this work, we have utilized full agonist analogues of cholecystokinin (CCK) with Aladan distributed throughout the pharmacophore in positions 24, 29, and 33, along with receptor constructs derivatized with Alexa (546) at positions 94, 102, 204, and 341 in the helical bundle and first, second, and third extracellular loops, respectively. These provided 12 FRET distances to overlay on working models of the CCK-occupied receptor. These established that the carboxyl terminus of CCK resides at the external surface of the lipid bilayer, adjacent to the receptor amino-terminal tail, rather than being inserted into the helical bundle. They also provide important experimentally derived constraints for understanding spatial relationships between the docked ligand and the flexible extracellular loop regions. Multidimensional FRET provides a new independent method to establish and refine structural insights into ligand-receptor complexes.


Assuntos
2-Naftilamina/análogos & derivados , Alanina/análogos & derivados , Colecistocinina/química , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Compostos de Quinolínio/química , Receptor de Colecistocinina A/química , 2-Naftilamina/química , Alanina/química , Animais , Células CHO , Colecistocinina/metabolismo , Cricetinae , Cricetulus , Transferência Ressonante de Energia de Fluorescência/métodos , Ligantes , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Estrutura Secundária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Receptor de Colecistocinina A/genética , Receptor de Colecistocinina A/metabolismo , Relação Estrutura-Atividade
10.
J Med Chem ; 51(13): 3742-54, 2008 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-18540665

RESUMO

JMV-180 ( 1) and CCK-8(s) are high affinity ligands at the CCK 1 receptor that have similar and different actions via this receptor. Here we calculate the tertiary structure of 1 or CCK-8(s) in the presence of dodecylphosphocholine micelles at pH 5.0 and 35 degrees C from 2D (1)H NMR data recorded at 600 MHz. The NMR derived 3D structures of 1 and CCK-8(s) share a common type I beta-turn around residues Nle3/M3 and G4 and diverge from each other structurally at the N- and C-termini. The fluorescence and circular dichroism spectral properties of these peptides are consistent with their NMR derived structures. The structures determined in the presence of DPC micelles are compared to available models of 1 or CCK-8(s) bound to the CCK 1 receptor. For CCK and 1, these comparisons show that DPC micelle associated structures duplicate some important aspects of the models calculated from cross-linking derived constraints at the CCK 1 receptor.


Assuntos
Micelas , Modelos Moleculares , Receptor de Colecistocinina A/química , Receptor de Colecistocinina A/metabolismo , Sincalida/química , Dicroísmo Circular , Imageamento Tridimensional , Espectroscopia de Ressonância Magnética , Ligação Proteica , Estrutura Terciária de Proteína , Sincalida/análogos & derivados , Espectrometria de Fluorescência
11.
J Mol Model ; 14(4): 303-14, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18286312

RESUMO

Pyridopyrimidine-based analogues are among the most highly potent and selective antagonists of cholecystokinin receptor subtype-1 (CCK1R) described to date. To better understand the structural and chemical features responsible for the recognition mechanism, and to explore the binding pocket of these compounds, we performed automated molecular docking using GOLD2.2 software on some derivatives with structural diversity, and propose a putative binding conformation for each compound. The docking protocol was guided by the key role of the Asn333 residue, as revealed by site directed mutagenesis studies. The results suggest two putative binding modes located in the same pocket. Both are characterized by interaction with the main residues revealed by experiment, Asn333 and Arg336, and differ in the spatial position of the Boc-Trp moiety of these compounds. Hydrophobic contacts with residues Thr117, Phe107, Ile352 and Ile329 are also in agreement with experimental data. Despite the poor correlation obtained between the estimated binding energies and the experimental activity, the proposed models allow us to suggest a plausible explanation of the observed binding data in accordance with chemical characteristics of the compounds, and also to explain the observed diastereoselectivity of this family of antagonists towards CCK1R. The most reasonable selected binding conformations could be the starting point for future studies. Figure Superimposition of the two putative binding conformations revealed by molecular docking for pyridopyrimidine-based CCK1 antagonists.


Assuntos
Desenho de Fármacos , Modelos Moleculares , Pirimidinas/química , Receptor de Colecistocinina A/química , Sítios de Ligação , Simulação por Computador , Entropia , Ligantes , Conformação Molecular , Conformação Proteica , Pirimidinas/farmacologia , Receptor de Colecistocinina A/agonistas , Software , Estereoisomerismo , Relação Estrutura-Atividade
12.
J Biol Chem ; 281(37): 27072-80, 2006 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16857665

RESUMO

Ligand probes with fluorescent indicators positioned throughout the pharmacophoric domain can provide important insights into the molecular basis of receptor binding and activation as reflected in the microenvironment of each indicator while docked at a receptor. We developed three cholecystokinin-like probes with Aladan situated at the N terminus, in the mid-region, and at the C terminus (positions 24, 29, and 33, respectively). These were studied in solution and docked at type A and B cholecystokinin receptors. This study demonstrated clear differences in mechanisms of cholecystokinin binding and activation of these structurally related receptors with distinct agonist structure-activity relationships. The fluorescence characteristics of Aladan are highly sensitive to the polarity of its microenvironment. The mid-region probe was least accessible to the aqueous milieu as determined by fluorescence emission spectra and iodide quenching, which was not altered by changes in conformation from active to inactive. Accessibility of the N- and C-terminal probes was affected by receptor conformation. The position 24 probe was more easily quenched in the active than in the G protein-uncoupled conformation for both receptors. However, the position 33 probe docked at the type A cholecystokinin receptor was more easily quenched in the active conformation, whereas the same probe docked at the type B cholecystokinin receptor was more easily quenched in the inactive conformation. Fluorescence anisotropy and red edge excitation shift determinations confirmed these observations and supported the proposed movements. Although both type A and B cholecystokinin receptors bind cholecystokinin with high affinity, resulting in fully efficacious biological responses, these receptors utilize distinct molecular modes of binding.


Assuntos
Colecistocinina/química , Corantes Fluorescentes/farmacologia , Receptor de Colecistocinina A/química , Receptor de Colecistocinina B/química , Animais , Células CHO , Membrana Celular/metabolismo , Colecistocinina/metabolismo , Colecistocinina/farmacologia , Cricetinae , Cinética , Modelos Químicos , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade
13.
J Med Chem ; 49(8): 2456-62, 2006 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-16610788

RESUMO

The interaction between the 1-47 N-terminus of the CCK(1)-R and the anthranilic acid based antagonists has been investigated by fluorescence spectroscopy. These antagonists interact with W39 of the N-terminal domain of the CCK(1)-R like that of the endogenous ligand CCK-8. This specific interaction was not found in other nonpeptide ligands of the CCK(1)-R. Conformational studies, using NMR and energy minimization procedures, have allowed formulation of a new hypothesis on the CCK(1)-R binding mode of the anthranilic antagonists.


Assuntos
Receptor de Colecistocinina A/antagonistas & inibidores , Sincalida/farmacologia , ortoaminobenzoatos/química , ortoaminobenzoatos/farmacologia , Animais , Ligação Competitiva/efeitos dos fármacos , Bioensaio , Humanos , Indóis/química , Indóis/farmacologia , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Proglumida/análogos & derivados , Proglumida/química , Proglumida/farmacologia , Conformação Proteica , Ratos , Receptor de Colecistocinina A/química , Sincalida/química , Espectrometria de Fluorescência , Relação Estrutura-Atividade
14.
J Med Chem ; 49(7): 2253-61, 2006 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-16570921

RESUMO

A series of 1,3,4-benzotriazepine-based CCK(2) antagonists have been devised by consideration of the structural features that govern CCK receptor affinity and the receptor subtype selectivity of 1,4-benzodiazepine-based CCK(2) antagonists. In contrast to the latter compounds, these novel 1,3,4-benzotriazepines are achiral, yet they display similar affinity for CCK(2) receptors to the earlier molecules and are highly selective over CCK(1) receptors.


Assuntos
Benzazepinas/síntese química , Receptor de Colecistocinina A/antagonistas & inibidores , Receptor de Colecistocinina B/antagonistas & inibidores , Animais , Benzazepinas/química , Benzazepinas/farmacologia , Benzodiazepinas/química , Linhagem Celular , Cricetinae , Cricetulus , Cristalografia por Raios X , Humanos , Camundongos , Estrutura Molecular , Ensaio Radioligante , Ratos , Receptor de Colecistocinina A/química , Receptor de Colecistocinina B/química , Estereoisomerismo , Relação Estrutura-Atividade
15.
Peptides ; 27(7): 1841-5, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16472889

RESUMO

The present study was performed to determine whether the expression levels of the hypothalamic cholecystokinin (CCK) and its receptors are associated with the responsiveness to high frequency electroacupuncture (EA) analgesia in rats. EA stimulation (100 Hz, 0.5 ms pulse width, 0.2-0.3 mA) was delivered to the Zusanli (ST36) acupoint of male Sprague-Dawley rats for 20 min without anesthetics or holder restraint. The analgesic effect of EA was quantified using a tail flick latency test, and subsequently animals were allocated to responder or non-responder groups. The hypothalamus of rats in each group was dissected and RNA was purified. The mRNA expressions of CCK, and CCK-A and -B receptor were determined by real-time RT-PCR. CCK mRNA levels were not significantly different in the two groups, whereas both CCK-A and -B receptors were significantly more expressed in non-responders. These results suggest that the level of CCK receptor mRNA expression in the hypothalamus, rather than CCK mRNA, has an important relationship with the individual variations to high frequency EA analgesia in rats.


Assuntos
Eletroacupuntura , RNA Mensageiro/metabolismo , Receptor de Colecistocinina A/química , Receptor de Colecistocinina B/química , Sincalida/química , Analgesia , Animais , Hipotálamo/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sincalida/metabolismo
16.
Mol Pharmacol ; 67(6): 1892-900, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15774770

RESUMO

Understanding the structures of active and inactive agonist- and antagonist-bound receptor complexes is of great interest. In this work, we focus on position 30 of cholecystokinin (CCK) and its spatial approximation with the type A CCK receptor. For this, we developed two photoaffinity labeling probes, replacing the naturally occurring tryptophan with p-benzoyl-l-phenylalanine (Bpa) or p-nitro-phenylalanine (NO(2)-Phe). The Bpa probe was shown to represent an antagonist, whereas the NO(2)-Phe probe stimulated intracellular calcium as a fully efficacious agonist (EC(50) = 81 +/- 15 nM). Both ligands bound to the receptor specifically, although with lower affinity than CCK (K(i) values: Bpa probe, 270 +/- 72 nM; NO(2)-Phe probe, 180 +/- 40 nM). Both probes covalently labeled the receptor in an efficient manner. The Bpa antagonist labeled the receptor in two distinct regions as identified by cyanogen bromide cleavage, with labeled bands migrating at M(r) = 25,000 and 4500. The former represented the glycosylated amino-terminal fragment, with the site of attachment further localized by endoproteinase Lys-C cleavage to the region between Asn(10) and Lys(37). The latter was shown to represent the first extracellular loop using further cleavage and sequencing of the wild-type and a mutant receptor. Following the same approach, the NO(2)-Phe agonist probe was shown to also label the first extracellular loop region. Radiochemical sequencing identified that the Bpa antagonist probe labeled receptor residue Lys(105), whereas the NO(2)-Phe agonist probe labeled residue Leu(99). These data extend our understanding of the molecular basis of binding and the conformational states of this important receptor.


Assuntos
Receptor de Colecistocinina A/química , Receptor de Colecistocinina A/genética , Tetragastrina/química , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Relação Dose-Resposta a Droga , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Receptor de Colecistocinina A/metabolismo , Tetragastrina/metabolismo
17.
J Med Chem ; 48(1): 180-91, 2005 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-15634012

RESUMO

The Cholecystokinin-1 receptor (CCK1R) mediates actions of CCK in areas of the central nervous system and of the gut. It is a potential target to treat a number of diseases. As for all G-protein-coupled receptors, docking of ligands into modeled CCK1R binding site should greatly help to understand intrinsic mechanisms of activation. Here, we describe the procedure we used to progressively build a structural model for the CCK1R, to integrated, and on the basis of site-directed mutagenesis data on its binding site. Reliability of the CCK1R model was confirmed by interaction networks that involved conserved and functionally crucial motifs in G-protein-coupled receptors, such as Glu/Asp-Arg-Tyr and Asn-Pro-Xaa-Xaa-Tyr motifs. In addition, the 3-D structure of CCK1R-bound CCK resembled that determined by NMR in a lipid environment. The derived computational model was also used for revealing binding modes of several nonpeptide ligands and for rationalizing ligand structure-activity relationships known from experiments. Our findings indeed support that our "validated CCK1R model" could be used to study the intrinsic mechanism of CCK1R activation and design new ligands.


Assuntos
Modelos Moleculares , Receptor de Colecistocinina A/química , Receptor de Colecistocinina A/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Benzodiazepinonas/química , Benzodiazepinonas/metabolismo , Sítios de Ligação , Células COS , Chlorocebus aethiops , Colecistocinina/química , Colecistocinina/metabolismo , Devazepida/química , Devazepida/metabolismo , Humanos , Ácidos Indolacéticos/química , Ácidos Indolacéticos/metabolismo , Indóis/química , Indóis/metabolismo , Ligantes , Metabolismo dos Lipídeos , Lipídeos/química , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Compostos de Fenilureia/química , Compostos de Fenilureia/metabolismo , Conformação Proteica , Receptor de Colecistocinina A/agonistas , Receptor de Colecistocinina A/genética , Rodopsina/química , Rodopsina/metabolismo , Homologia de Sequência de Aminoácidos , Tiazóis/química , Tiazóis/metabolismo
18.
J Biol Chem ; 280(11): 10664-74, 2005 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-15632187

RESUMO

The cholecystokinin-1 receptor (CCK1R) is a G protein-coupled receptor (GPCR) that regulates important physiological functions. As for other GPCRs, the molecular basis of full and partial agonism is still far from clearly understood. In the present report, using both laboratory experiments and molecular modeling approaches, we have investigated the partial agonism mechanism of JMV 180, on the human CCK1R. We first showed that efficacy of the CCK1R to activate phospholipase C is dependent on the correct orientation of the C-terminal end of peptidic ligands toward residue Phe(330) of helix VI. We have previously reported that a single mutation of Met(121) (helix III) markedly reduced the receptor-mediated inositol phosphate production upon stimulation by CCK. Computational simulations predicted that residue 121 affected orientation of the C-terminal end of CCK, thus suggesting that the molecular complex with a reduced inositol phosphate production observed with the mutated CCK1R resembles that resulting from binding of JMV 180 to the WT-CCK1R. Pharmacological, biochemical, and functional characterizations of the two receptor.ligand complexes with decreased abilities to signal were carried out in different cell types. We found that they presented the same features, such as total dependence of inositol phosphate production to Galpha(q) expression, single affinity of binding sites, insensitivity of binding to non-hydrolyzable GTP, absence of GTPgamma[S(35)] binding following agonist stimulation, similarity of dose-response curves for amylase secretion, and incapacity to induce acute pancreatitis in pancreatic acini. We concluded that helices VI and III of the CCK1R are functionally linked through the CCK1R agonist binding site and that positioning of the C-terminal ends of peptidic agonists toward Phe(330) of helix VI is responsible for extent of phospholipase C activation through Galpha(q) coupling. Given the potential therapeutic interest of partial agonists such as JMV 180, our structural data will serve for target structure-based design of new CCK1R ligands.


Assuntos
Receptor de Colecistocinina A/agonistas , Receptor de Colecistocinina A/química , Adenoviridae/genética , Amilases/química , Animais , Arginina/química , Sítios de Ligação , Células COS , Membrana Celular/metabolismo , Ativação Enzimática , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Humanos , Concentração Inibidora 50 , Fosfatos de Inositol/química , Cinética , Ligantes , Lisina/química , Substâncias Macromoleculares/química , Masculino , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Pâncreas/citologia , Peptídeos/química , Fosfatos/química , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Software , Fosfolipases Tipo C/metabolismo
19.
Biochem Biophys Res Commun ; 314(3): 861-9, 2004 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-14741716

RESUMO

Key amino acids of the cholecystokinin (CCK) peptide for receptor binding are sulfated Y27, W30, D32, and F33-NH(2). Three-dimensional modeling showed that the CCK-A receptor (CCK-AR) antagonist devazepide penetrated into the transmembrane (TM) domains, whereas CCK was placed on the surface of the CCK-AR. Four types of rat CCK-AR cDNAs were transfected into CHO-K1 and COS-7 cells: normal CCK-AR cDNA transfected cells (wild type, WT); K120 substituted with V; K130V; and R352V. Binding of [3H]CCK-8 was observed in WT and K130V, but not in K120V and R352V. CCK caused Ca(2+) spiking in WT and K130V, whereas K120V and R352V had no effect. Three chimeras including the CCK-AR/3ibeta2 adrenergic receptor (beta2AR), 3Nibeta2AR, and 3Cibeta2AR were constructed. Two groups of point mutations in the CCK-AR3i were also made: Y252V, S274V, S281V, and S289V (non-phospho-acceptor Y or S); S260V, S264V, S271V, and S275V (phospho-acceptor S). WT and CCK-AR/3Cibeta2AR increased [Ca(2+)](i) in response to CCK; 3Nibeta2AR was vice versa. CCK failed to increase [IP(3)] in phospho-acceptor S to V without affecting binding. Non-phospho-acceptor S or Y to V showed normal response. Thus, Lys120 outside the TM2 and Arg352 outside the TM6 of the CCK-AR are amino acids interacting with Tyr[SO(3)H]27 and Asp32 of the CCK peptide for binding. Phospho-acceptor Ser groups in the CCK-AR 3Ni are amino acids for initiating cell signaling.


Assuntos
Receptor de Colecistocinina A/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células CHO , Células COS , Cálcio/química , Cálcio/metabolismo , Cricetinae , Devazepida/metabolismo , Inositol 1,4,5-Trifosfato/análise , Inositol 1,4,5-Trifosfato/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ensaio Radioligante , Ratos , Receptor de Colecistocinina A/química , Receptor de Colecistocinina A/genética , Transdução de Sinais , Sincalida/genética , Sincalida/metabolismo , Sincalida/farmacologia , Relação Estrutura-Atividade , Transfecção
20.
Chembiochem ; 4(11): 1176-87, 2003 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-14613109

RESUMO

A cyclic CCK8 analogue, cyclo(29,34)[Dpr(29),Lys(34)]-CCK8 (Dpr=L-2,3-diaminopropionic acid), has been designed on the basis of the NMR structure of the bimolecular complex between the N-terminal fragment of the CCK(A) receptor and its natural ligand CCK8. The conformational features of cyclo(29,34)[Dpr(29),Lys(34)]-CCK8 have been determined by NMR spectroscopy in aqueous solution and in water containing DPC-d(38) micelles (DPC=dodecylphosphocholine). The structure of the cyclic peptide in aqueous solution is found to be in a relaxed conformation, with the backbone and Dpr29 side chain atoms making a planar ring and the N-terminal tripeptide extending approximately along the plane of this ring. In DPC/water, the cyclic peptide adopts a "boat-shaped" conformation, which is more compact than that found in aqueous solution. The cyclic constraint between the Dpr29 side chain and the CCK8 carboxyl terminus (Lys34) introduces a restriction in the backbone conformational freedom. However, the interaction of cyclo(29,34)[Dpr(29),Lys(34)]-CCK8 with the micelles still plays an important role in the stabilisation of the bioactive conformation. A careful comparison of the NMR structure of the cyclic peptide in a DPC micelle aqueous solution with the structure of the rationally designed model underlines that the turn-like conformation in the Trp30-Met31 region is preserved, such that the Trp30 and Met31 side chains can adopt the proper spatial orientation to interact with the CCK(A) receptor. The binding properties of cyclo(29,34)[Dpr(29),Lys(34)]-CCK8 to the N-terminal receptor fragment have been investigated by fluorescence spectroscopy in a micellar environment. Estimates of the apparent dissociation constant, K(d), were in the range of 70-150 nM, with a mean value of 120+/-27 nM. Preliminary nuclear medicine studies on cell lines transfected with the CCK(A) receptor indicate that the sulfated-Tyr derivative of cyclo(29,34)[Dpr(29),Lys(34)]-CCK8 displaces the natural ligand with an IC(50) value of 15 microM.


Assuntos
Peptídeos Cíclicos/síntese química , Receptor de Colecistocinina A/química , Receptor de Colecistocinina A/metabolismo , Células Cultivadas , Desenho de Fármacos , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Ligação Proteica , Conformação Proteica
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