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1.
Life (Basel) ; 13(12)2023 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-38137889

RESUMO

We previously reported on a novel peptaibol, named Tripleurin XIIc (TPN), an 18-residue long sequence produced by the fungus Trichoderma pleuroti. We elucidated its 3D structure via classical and accelerated molecular dynamics simulation (aMD) methods and reported the folding dynamics of TPN in water and chloroform solvents. Peptaibols, in general, are insoluble in water, as they are amphipathic and may prefer hydrophobic environments like transmembrane regions. In this study, we attempted to use aMD simulations to model an all-atom bacterial membrane system while placing a TPN molecule in its vicinity. The results highlighted that TPN was able to introduce some disorder into the membrane and caused lipid clustering. It could also enter the transmembrane region from the water-bilayer interface. The structural dynamics of TPN in the transmembrane region revealed a single energetically stable conformation similar to the one obtained from water and chloroform solvent simulations reported by us previously. However, this linear structure was found to be at the local energy minimum (stable) in water but at a metastable intermediate state (higher energy) in chloroform. Therefore, it could be said that the water solvent can be successfully used for folding simulations of peptaibols.

2.
Sci Rep ; 13(1): 20305, 2023 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-37985681

RESUMO

Opiate alkaloids and their synthetic derivatives are still widely used in pain management, drug addiction, and abuse. To avoid serious side effects, compounds with properly designed pharmacological profiles at the opioid receptor subtypes are long needed. Here a series of 17-N-substituted derivatives of normorphine and noroxymorphone analogues with five- and six-membered ring substituents have been synthesized for structure-activity study. Some compounds showed nanomolar affinity to MOR, DOR and KOR in in vitro competition binding experiments with selective agonists [3H]DAMGO, [3H]Ile5,6-deltorphin II and [3H]HS665, respectively. Pharmacological characterization of the compounds in G-protein signaling was determined by [35S]GTPγS binding assays. The normorphine analogues showed higher affinity to KOR compared to MOR and DOR, while most of the noroxymorphone derivatives did not bind to KOR. The presence of 14-OH substituent resulted in a shift in the pharmacological profiles in the agonist > partial agonist > antagonist direction compared to the parent compounds. A molecular docking-based in silico method was also applied to estimate the pharmacological profile of the compounds. Docking energies and the patterns of the interacting receptor atoms, obtained with experimentally determined active and inactive states of MOR, were used to explain the observed pharmacological features of the compounds.


Assuntos
Receptores Opioides mu , Receptores Opioides , Receptores Opioides mu/metabolismo , Simulação de Acoplamento Molecular , Receptores Opioides/metabolismo , Ligação Competitiva , Relação Estrutura-Atividade , Receptores Opioides kappa/metabolismo
3.
BMC Chem ; 17(1): 167, 2023 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-38007463

RESUMO

Like most phosphinic acids, the potent and selective RXP03 inhibitor of different MMPs exhibited moderate absorption and low bioavailability, which impaired its use. In an unprecedented attempt, we present an interesting synthetic approach to a new class of phosphinate prodrug, glycosyl ester of RXP03, to provide a potentially improved blood-brain barrier (BBB) behavior compared to the former lead compound RXP03. To validate this speculation, a predictive study for permeability enhancer of glycosyl ester of RXP03 showed encouraging insights to improve drug delivery across biological barriers.

5.
Eur J Med Chem ; 191: 112145, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32092588

RESUMO

Morphine and its derivatives play inevitably important role in the µ-opioid receptor (MOR) targeted antinociception. A structure-activity relationship study is presented for novel and known orvinol and thevinol derivatives with varying 3-O, 6-O, 17-N and 20-alkyl substitutions starting from agonists, antagonists and partial agonists. In vitro competition binding experiments with [3H]DAMGO showed low subnanomolar affinity to MOR. Generally, 6-O-demethylation increased the affinity toward MOR and decreased the efficacy changing the pharmacological profile in some cases. In vivo tests in osteoarthritis inflammation model showed significant antiallodynic effects of thevinol derivatives while orvinol derivatives did not. The pharmacological character was modelled by computational docking to both active and inactive state models of MOR. Docking energy difference for the two states separates agonists and antagonists well while partial agonists overlapped with them. An interaction pattern of the ligands, involving the interacting receptor atoms, showed more efficient separation of the pharmacological profiles. In rats, thevinol derivatives showed antiallodynic effect in vivo. The orvinol derivatives, except for 6-O-desmethyl-dihydroetorfin (2c), did not show antiallodynic effect.


Assuntos
Analgésicos Opioides/farmacologia , Inflamação/tratamento farmacológico , Morfinanos/farmacologia , Osteoartrite/tratamento farmacológico , Receptores Opioides mu/agonistas , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/química , Animais , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Cobaias , Masculino , Simulação de Acoplamento Molecular , Estrutura Molecular , Morfinanos/administração & dosagem , Morfinanos/química , Ratos Wistar , Receptores Opioides mu/antagonistas & inibidores , Relação Estrutura-Atividade
6.
Int J Mol Sci ; 20(17)2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31480404

RESUMO

The use of enhanced sampling molecular dynamics simulations to facilitate the folding of proteins is a relatively new approach which has quickly gained momentum in recent years. Accelerated molecular dynamics (aMD) can elucidate the dynamic path from the unfolded state to the near-native state, "flattened" by introducing a non-negative boost to the potential. Alamethicin F30/3 (Alm F30/3), chosen in this study, belongs to the class of peptaibols that are 7-20 residue long, non-ribosomally synthesized, amphipathic molecules that show interesting membrane perturbing activity. The recent studies undertaken on the Alm molecules and their transmembrane channels have been reviewed. Three consecutive simulations of ~900 ns each were carried out where N-terminal folding could be observed within the first 100 ns, while C-terminal folding could only be achieved almost after 800 ns. It took ~1 µs to attain the near-native conformation with stronger potential boost which may take several µs worth of classical MD to produce the same results. The Alm F30/3 hexamer channel was also simulated in an E. coli mimicking membrane under an external electric field that correlates with previous experiments. It can be concluded that aMD simulation techniques are suited to elucidate peptaibol structures and to understand their folding dynamics.


Assuntos
Simulação de Dinâmica Molecular , Peptaibols/química , Peptaibols/metabolismo , Dobramento de Proteína , Bicamadas Lipídicas/química , Análise de Componente Principal , Eletricidade Estática , Termodinâmica , Água/química
7.
Pharmaceutics ; 11(2)2019 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-30744154

RESUMO

The absorption of drugs is limited by the epithelial barriers of the gastrointestinal tract. One of the strategies to improve drug delivery is the modulation of barrier function by the targeted opening of epithelial tight junctions. In our previous study the 18-mer amphiphilic PN159 peptide was found to be an effective tight junction modulator on intestinal epithelial and blood⁻brain barrier models. PN159, also known as KLAL or MAP, was described to interact with biological membranes as a cell-penetrating peptide. In the present work we demonstrated that the PN159 peptide as a penetration enhancer has a dual action on intestinal epithelial cells. The peptide safely and reversibly enhanced the permeability of Caco-2 monolayers by opening the intercellular junctions. The penetration of dextran molecules with different size and four efflux pump substrate drugs was increased several folds. We identified claudin-4 and -7 junctional proteins by docking studies as potential binding partners and targets of PN159 in the opening of the paracellular pathway. In addition to the tight junction modulator action, the peptide showed cell membrane permeabilizing and antimicrobial effects. This dual action is not general for cell-penetrating peptides (CPPs), since the other three CPPs tested did not show barrier opening effects.

8.
Molecules ; 24(2)2019 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-30669493

RESUMO

Peptaibols are a special class of fungal peptides with an acetylated N-terminus and a C-terminal 1,2-amino alcohol along with non-standard amino acid residues. New peptaibols named tripleurins were recently identified from a strain of the filamentous fungal species Trichoderma pleuroti, which is known to cause green mould disease on cultivated oyster mushrooms. To understand the mode of action of these peptaibols, the three-dimensional structure of tripleurin (TPN) XIIc, an 18-mer peptide, was elucidated using an enhanced sampling method, accelerated MD, in water and chloroform solvents. Non-standard residues were parameterized by the Restrained Electrostatic Potential (RESP) charge fitting method. The dihedral distribution indicated towards a right-handed helical formation for TPN XIIc in both solvents. Dihedral angle based principal component analysis revealed a propensity for a slightly bent, helical folded conformation in water solvent, while two distinct conformations were revealed in chloroform: One that folds into highly bent helical structure that resembles a beta-hairpin and another with an almost straight peptide backbone appearing as a rare energy barrier crossing event. The hinge-like movement of the terminals was also observed and is speculated to be functionally relevant. The convergence and efficient sampling is addressed using Cartesian PCA and Kullback-Leibler divergence methods.


Assuntos
Simulação de Dinâmica Molecular , Peptaibols/química , Dobramento de Proteína , Solventes/química , Trichoderma/química , Sequência de Aminoácidos , Aminoácidos/química , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Análise de Componente Principal , Estrutura Secundária de Proteína , Eletricidade Estática , Termodinâmica , Água/química
9.
Peptides ; 112: 106-113, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30513351

RESUMO

The endogenous ligand nociceptin (N/OFQ) and a positively charged synthetic peptide RYYRIK are both selective for the nociceptin opioid receptor (NOPr). Despite their structural dissimilarity, N/OFQ and RYYRIK compete for the same binding site of NOP receptor possessing full and partial agonistic character, respectively. In the view of the message-address concept, hybrid peptide constructs were probed for the NOP receptor combining different regions of N/OFQ and RYYRIK related peptide sequences. Nine novel nociceptin- or Ac-RYYRIK-NH2 peptide variants or hybrid peptides were synthesized and characterized. Peptides P2 and P8 contain fragments of native N/OFQ. The other seven analogues (P1, P3-7, P9) are composed of Ac-RYYRIK-NH2 fragments and parts of the original nociceptin sequence. The analogues were characterized in receptor binding assays and G-protein activation experiments on rat brain membranes, as well as by electrically stimulated mouse vas deferens bioassay. In receptor binding assays ligands P2, P4, P6 (Ki 0.37 nM) and P7 showed higher affinity (Ki 0.65 nM, 0.6 nM, 0.37 nM and 0.44 nM, respectively) for NOP receptor than their parent compounds N/OFQ (Ki 2.8 nM) or Ac-RYYRIK-NH2 (Ki 4.2 nM). In [35S]GTPγS binding experiments P2 and P3 behaved as full agonists. The other variants exhibited partial agonist properties characterized by submaximal stimulatory effects. In mouse vas deferens bioassay only P2 showed agonist activity. P4, P5, P6 inhibited the biological activity of N/OFQ more effectively than the NOP receptor selective antagonist JTC-801. In summary, hybrid peptides P4, P5 and P6 proved to be NOP receptor partial agonists even antagonists, while P2 peptide retained the full agonist property.


Assuntos
Peptídeos Opioides/farmacologia , Receptores Opioides/agonistas , Animais , Cobaias , Ligantes , Masculino , Ratos , Receptores Opioides/efeitos dos fármacos , Receptor de Nociceptina , Nociceptina
10.
Peptides ; 99: 205-216, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29038035

RESUMO

In an attempt to design opioid-nociceptin hybrid peptides, three novel bivalent ligands, H-YGGFGGGRYYRIK-NH2, H-YGGFRYYRIK-NH2 and Ac-RYYRIKGGGYGGFL-OH were synthesized and studied by biochemical, pharmacological, biophysical and molecular modelling tools. These chimeric molecules consist of YGGF sequence, a crucial motif in the N-terminus of natural opioid peptides, and Ac-RYYRIK-NH2, which was isolated from a combinatorial peptide library as an antagonist or partial agonist that inhibits the biological activity of the endogenously occurring heptadecapeptide nociceptin. Solution structures for the peptides were studied by analysing their circular dichroism spectra. Receptor binding affinities were measured by equilibrium competition experiments using four highly selective radioligands. G-protein activating properties of the multitarget peptides were estimated in [35S]GTPγS binding tests. The three compounds were also measured in electrically stimulated mouse vas deferens (MVD) bioassay. H-YGGFGGGRYYRIK-NH2 (BA55), carrying N-terminal opioid and C-terminal nociceptin-like sequences interconnected with GGG tripeptide spacer displayed a tendency of having either unordered or ß-sheet structures, was moderately potent in MVD and possessed a NOP/KOP receptor preference. A similar peptide without spacer H-YGGFRYYRIK-NH2 (BA62) exhibited the weakest effect in MVD, more α-helical periodicity was present in its structure and it exhibited the most efficacious agonist actions in the G-protein stimulation assays. The third hybrid peptide Ac-RYYRIKGGGYGGFL-OH (BA61) unexpectedly displayed opioid receptor affinities, because the opioid message motif is hidden within the C-terminus. The designed chimeric peptide ligands presented in this study accommodate well into a group of multitarget opioid compounds that include opioid-non-opioid peptide dimer analogues, dual non-peptide dimers and mixed peptide- non-peptide bifunctional ligands.


Assuntos
Modelos Moleculares , Peptídeos Opioides , Engenharia de Proteínas/métodos , Receptores Acoplados a Proteínas G/agonistas , Animais , Feminino , Masculino , Camundongos , Peptídeos Opioides/química , Peptídeos Opioides/genética , Peptídeos Opioides/farmacologia , Estrutura Secundária de Proteína , Ratos , Ratos Wistar , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Nociceptina
11.
Cell Stress Chaperones ; 22(5): 717-728, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28474205

RESUMO

Defects in cellular protein homeostasis are associated with many severe and prevalent pathological conditions such as neurodegenerative diseases, muscle dystrophies, and metabolic disorders. One way to counteract these defects is to improve the protein homeostasis capacity through induction of the heat shock response. Despite numerous attempts to develop strategies for chemical activation of the heat shock response by heat shock transcription factor 1 (HSF1), the underlying mechanisms of drug candidates' mode of action are poorly understood. To lower the threshold for the heat shock response activation, we used the chaperone co-inducer BGP-15 that was previously shown to have beneficial effects on several proteinopathic disease models. We found that BGP-15 treatment combined with heat stress caused a substantial increase in HSF1-dependent heat shock protein 70 (HSPA1A/B) expression already at a febrile range of temperatures. Moreover, BGP-15 alone inhibited the activity of histone deacetylases (HDACs), thereby increasing chromatin accessibility at multiple genomic loci including the stress-inducible HSPA1A. Intriguingly, treatment with well-known potent HDAC inhibitors trichostatin A and valproic acid enhanced the heat shock response and improved cytoprotection. These results present a new pharmacological strategy for restoring protein homeostasis by inhibiting HDACs, increasing chromatin accessibility, and lowering the threshold for heat shock response activation.


Assuntos
Cromatina/metabolismo , Resposta ao Choque Térmico/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Oximas/farmacologia , Piperidinas/farmacologia , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Imunoprecipitação da Cromatina , Proteínas Correpressoras , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Fatores de Transcrição de Choque Térmico/genética , Fatores de Transcrição de Choque Térmico/metabolismo , Histona Desacetilases/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Ligação Proteica , Receptor Notch4/metabolismo
12.
J Enzyme Inhib Med Chem ; 31(6): 1638-47, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27063555

RESUMO

Fentanyl is a powerful opiate analgesic typically used for the treatment of severe and chronic pain, but its prescription is strongly limited by the well-documented side-effects. Different approaches have been applied to develop strong analgesic drugs with reduced pharmacologic side-effects. One of the most promising is the design of multitarget drugs. In this paper we report the synthesis, characterization and biological evaluation of twelve new 4-anilidopiperidine (fentanyl analogues). In vivo hot-Plate test, shows a moderate antinociceptive activity for compounds OMDM585 and OMDM586, despite the weak binding affinity on both µ and δ-opioid receptors. A strong inverse agonist activity in the GTP-binding assay was revealed suggesting the involvement of alternative systems in the brain. Fatty acid amide hydrolase inhibition was evaluated, together with binding assays of cannabinoid receptors. We can conclude that compounds OMDM585 and 586 are capable to elicit antinociception due to their multitarget activity on different systems involved in pain modulation.


Assuntos
Analgésicos/farmacologia , Carbamatos/análise , Piperidinas/farmacologia , Ureia/análise , Analgésicos/química , Animais , Feminino , Cobaias , Masculino , Camundongos , Piperidinas/química , Ratos , Ratos Wistar , Análise Espectral/métodos
13.
J Pharm Sci ; 105(2): 754-765, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26869428

RESUMO

The intercellular junctions restrict the free passage of hydrophilic compounds through the paracellular clefts. Reversible opening of the tight junctions of biological barriers is investigated as one of the ways to increase drug delivery to the systemic circulation or the central nervous system. Six peptides, ADT-6, HAV-6, C-CPE, 7-mer (FDFWITP, PN-78), AT-1002, and PN-159, acting on different integral membrane and linker junctional proteins were tested on Caco-2 intestinal epithelial cell line and a coculture model of the blood-brain barrier. All peptides tested in nontoxic concentrations showed a reversible tight junctions modulating effect and were effective to open the paracellular pathway for the marker molecules fluorescein and albumin. The change in the structure of cell-cell junctions was verified by immunostaining for occludin, claudin-4,-5, ZO-1, ß-catenin, and E-cadherin. Expression levels of occludin and claudins were measured in both models. We could demonstrate a selectivity of C-CPE, ADT-6, and HAV-6 peptides for epithelial cells and 7-mer and AT-1002 peptides for brain endothelial cells. PN-159 was the most effective modulator of junctional permeability in both models possibly acting via claudin-1 and -5. Our results indicate that these peptides can be effectively and selectively used as potential pharmaceutical excipients to improve drug delivery across biological barriers.


Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Junções Íntimas/efeitos dos fármacos , Animais , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/metabolismo , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Células Endoteliais/metabolismo , Humanos , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/metabolismo , Mucosa Intestinal/metabolismo , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Wistar , Junções Íntimas/metabolismo
14.
Mol Pain ; 9: 30, 2013 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-23800232

RESUMO

This review aims to create an overview of the currently available results of site-directed mutagenesis studies on transient receptor potential vanilloid type 1 (TRPV1) receptor. Systematization of the vast number of data on the functionally important amino acid mutations of TRPV1 may provide a clearer picture of this field, and may promote a better understanding of the relationship between the structure and function of TRPV1. The review summarizes information on 112 unique mutated sites along the TRPV1, exchanged to multiple different residues in many cases. These mutations influence the effect or binding of different agonists, antagonists, and channel blockers, alter the responsiveness to heat, acid, and voltage dependence, affect the channel pore characteristics, and influence the regulation of the receptor function by phosphorylation, glycosylation, calmodulin, PIP2, ATP, and lipid binding. The main goal of this paper is to publish the above mentioned data in a form that facilitates in silico molecular modelling of the receptor by promoting easier establishment of boundary conditions. The better understanding of the structure-function relationship of TRPV1 may promote discovery of new, promising, more effective and safe drugs for treatment of neurogenic inflammation and pain-related diseases and may offer new opportunities for therapeutic interventions.


Assuntos
Mutação , Canais de Cátion TRPV/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Modelos Biológicos , Mutagênese Sítio-Dirigida , Ratos , Canais de Cátion TRPV/química , Canais de Cátion TRPV/genética
15.
Biol Trace Elem Res ; 151(3): 451-61, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23264033

RESUMO

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel involved in pain sensation and in a wide range of non-pain-related physiological and pathological conditions. The aim of the present study was to explore the effects of selected heavy metal cations on the function of TRPV1. The cations ranked in the following sequence of pore-blocking activity: Co(2+) [half-maximal inhibitory concentration (IC(50)) = 13 µM] > Cd(2+) (I (50) = 38 µM) > Ni(2+) (IC(50) = 62 µM) > Cu(2+) (IC(50) = 200 µM). Zn(2+) proved to be a weak (IC(50) = 27 µM) and only partial inhibitor of the channel function, whereas Mg(2+), Mn(2+) and La(3+) did not exhibit any substantial effect. Co(2+), the most potent channel blocker, was able not only to compete with Ca(2+) but also to pass with it through the open channel of TRPV1. In response to heat activation or vanilloid treatment, Co(2+) accumulation was verified in TRPV1-transfected cell lines and in the TRPV1+ dorsal root ganglion neurons. The inhibitory effect was also demonstrated in vivo. Co(2+) applied together with vanilloid agonists attenuated the nocifensive eye wipe response in mice. Different rat TRPV1 pore point mutants (Y627W, N628W, D646N and E651W) were created that can validate the binding site of previously used channel blockers in agonist-evoked (45)Ca(2+) influx assays in cells expressing TRPV1. The IC(50) of Co(2+) on these point mutants were determined to be reasonably comparable to those on the wild type, which suggests that divalent cations passing through the TRPV1 channel use the same negatively charged amino acids as Ca(2+).


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Metais Pesados/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Células 3T3 , Animais , Células COS , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/química , Cátions Bivalentes/química , Cátions Bivalentes/farmacologia , Linhagem Celular , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Humanos , Metais Pesados/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Ratos , Relação Estrutura-Atividade , Canais de Cátion TRPV/metabolismo
16.
Life Sci ; 91(3-4): 115-26, 2012 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-22749862

RESUMO

AIMS: A homology modeling methodology was developed and used to obtain the 3D structures for four putative catalases of Rhizopus oryzae in order to assess their functionality. MAIN METHODS: Homology models were built using different modeling strategies using non-protein compounds as steric constraints, a symmetry constraint to force identical chains and an additional loop modeling algorithm. Percent structural overlap values (SO) were calculated for each model-template pair to qualify the homology models. KEY FINDINGS: Comparing the different modeling strategies by the SO values revealed that the quality of the models, i.e. the similarity to the template was greatly increased in the presence of the prosthetic groups, modeling multiple protein chains together, enforcing symmetrical chains and applying additional loop modeling. For the best homology models achieved this way, the SO values express similar evolutionary relationships between the proteins modeled and the templates that were previously established by phylogenetic analysis. In three out of the four catalases of R. oryzae the highest quality models, the active center, i.e. the heme molecule and the surrounding amino acids showed a spatial arrangement identical to that observed experimentally in other catalases. The remaining protein is missing an 11 residue long fragment and has mutated residues within the active center. SIGNIFICANCE: Better homology models can be obtained with templates chosen by phylogenetic relationship, although building an accurate model needs structural constraints too. Calculating the structural overlap between the models and the templates may also help to find the appropriate templates.


Assuntos
Catalase/química , Catalase/metabolismo , Rhizopus/metabolismo , Algoritmos , Sequência de Aminoácidos , Animais , Teorema de Bayes , Bovinos , Genoma , Heme/química , Humanos , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Mutação , Filogenia , Conformação Proteica , Reação em Cadeia da Polimerase em Tempo Real/métodos , Rhizopus/enzimologia , Homologia de Sequência de Aminoácidos , Fatores de Tempo
17.
Neurochem Int ; 61(3): 378-88, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22613132

RESUMO

Increasing number of publications shows that cannabinoid receptor 1 (CB(1)) specific compounds might act in a CB(1) independent manner, including rimonabant, a potent CB(1) receptor antagonist. Opioids, cannabinoids and their receptors are well known for their overlapping pharmacological properties. We have previously reported a prominent decrease in µ-opioid receptor (MOR) activity when animals were acutely treated with the putative endocannabinoid noladin ether (NE). In this study, we clarified whether the decreased MOR activation caused by NE could be reversed by rimonabant in CB(1) receptor deficient mice. In functional [(35)S]GTPγS binding assays, we have elucidated that 0.1mg/kg of intraperitoneal (i.p.) rimonabant treatment prior to that of NE treatment caused further attenuation on the maximal stimulation of Tyr-d-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO), which is a highly specific MOR agonist. Similar inhibitory effects were observed when rimonabant was injected i.p. alone and when it was directly applied to forebrain membranes. These findings are cannabinoid receptor independent as rimonabant caused inhibition in both CB(1) single knockout and CB(1)/CB(2) double knockout mice. In radioligand competition binding assays we highlighted that rimonabant fails to displace effectively [(3)H]DAMGO from MOR in low concentrations and is highly unspecific on the receptor at high concentrations in CB(1) knockout forebrain and in their wild-type controls. Surprisingly, docking computational studies showed a favorable binding position of rimonabant to the inactive conformational state of MOR, indicating that rimonabant might behave as an antagonist at MOR. These findings were confirmed by radioligand competition binding assays in Chinese hamster ovary cells stably transfected with MOR, where a higher affinity binding site was measured in the displacement of the tritiated opioid receptor antagonist naloxone. However, based on our in vivo data we suggest that other, yet unidentified mechanisms are additionally involved in the observed effects.


Assuntos
Antagonistas de Receptores de Canabinoides/farmacologia , Piperidinas/farmacologia , Prosencéfalo/efeitos dos fármacos , Pirazóis/farmacologia , Receptores de Canabinoides/metabolismo , Receptores Opioides mu/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Células CHO , Cricetinae , Cricetulus , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Camundongos , Camundongos Knockout , Prosencéfalo/metabolismo , Receptores Opioides mu/metabolismo , Rimonabanto
18.
PLoS One ; 2(6): e545, 2007 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-17579717

RESUMO

Ca(2+)-loaded calmodulin normally inhibits multiple Ca(2+)-channels upon dangerous elevation of intracellular Ca(2+) and protects cells from Ca(2+)-cytotoxicity, so blocking of calmodulin should theoretically lead to uncontrolled elevation of intracellular Ca(2+). Paradoxically, classical anti-psychotic, anti-calmodulin drugs were noted here to inhibit Ca(2+)-uptake via the vanilloid inducible Ca(2+)-channel/inflamatory pain receptor 1 (TRPV1), which suggests that calmodulin inhibitors may block pore formation and Ca(2+) entry. Functional assays on TRPV1 expressing cells support direct, dose-dependent inhibition of vanilloid-induced (45)Ca(2+)-uptake at microM concentrations: calmidazolium (broad range) > or = trifluoperazine (narrow range) chlorpromazine/amitriptyline>fluphenazine>>W-7 and W-13 (only partially). Most likely a short acidic domain at the pore loop of the channel orifice functions as binding site either for Ca(2+) or anti-calmodulin drugs. Camstatin, a selective peptide blocker of calmodulin, inhibits vanilloid-induced Ca(2+)-uptake in intact TRPV1(+) cells, and suggests an extracellular site of inhibition. TRPV1(+), inflammatory pain-conferring nociceptive neurons from sensory ganglia, were blocked by various anti-psychotic and anti-calmodulin drugs. Among them, calmidazolium, the most effective calmodulin agonist, blocked Ca(2+)-entry by a non-competitive kinetics, affecting the TRPV1 at a different site than the vanilloid binding pocket. Data suggest that various calmodulin antagonists dock to an extracellular site, not found in other Ca(2+)-channels. Calmodulin antagonist-evoked inhibition of TRPV1 and NMDA receptors/Ca(2+)-channels was validated by microiontophoresis of calmidazolium to laminectomised rat monitored with extracellular single unit recordings in vivo. These unexpected findings may explain empirically noted efficacy of clinical pain adjuvant therapy that justify efforts to develop hits into painkillers, selective to sensory Ca(2+)-channels but not affecting motoneurons.


Assuntos
Antidepressivos Tricíclicos/farmacologia , Cálcio/metabolismo , Calmodulina/antagonistas & inibidores , Ativação do Canal Iônico/efeitos dos fármacos , Dor/tratamento farmacológico , Canais de Cátion TRPV/antagonistas & inibidores , Animais , Antipsicóticos/farmacologia , Capsaicina/farmacologia , Células Cultivadas , Quimioterapia Adjuvante , Clorpromazina/farmacologia , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Imidazóis/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Modelos Moleculares , Células NIH 3T3 , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Dor/metabolismo , Ratos , Ratos Wistar , Fármacos do Sistema Sensorial/farmacologia
19.
J Chem Inf Model ; 46(3): 1431-8, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16711763

RESUMO

Multidimensional QSAR methodologies can be used to predict the active conformation encoded in the conformational preferences of molecules in an active series by utilizing conformational sampling. In the 3+3D-QSAR approach, the conformational free energy loss is modeled with internal coordinate-based flexibility descriptors. While the pharmacophore point pair distance descriptors introduced earlier proved useful in the construction of QSAR models and in the prediction of important features of the active conformation, they are inherently incapable of describing the chiral arrangement of the pharmacophores. As an improvement, a chirality-sensitive flexibility (CSF) descriptor is now introduced, which is based on the distance between a pharmacophore point and a plane defined by three pharmacophore points. The performance of the CSF descriptor was tested on two active series: 37 endomorphin analogues with opiate activity and 38 PGF2alpha analogues with antinidatory activity. The newly devised descriptor resulted in improved QSAR models in terms of both prediction accuracy and precision of the chiral geometric features of the predicted active conformations.


Assuntos
Desenho de Fármacos , Modelos Moleculares , Relação Quantitativa Estrutura-Atividade , Estereoisomerismo
20.
J Med Chem ; 48(9): 3239-50, 2005 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-15857130

RESUMO

A conceptionally new 3D molecular descriptor type and methodology are deduced by simple statistical thermodynamic reasoning, based on the free energy change encountered during a transformation of a conformational ensemble of the ligand to an active conformation. The performance of the descriptor was first tested on 37 endomorphin analogues with mu-opiate activity. The method resulted in predictive 3D-QSAR models, and the active conformation was also predicted. Generally, the methodology can be combined with the traditional 3D-QSAR techniques in a 3+3D-QSAR manner. This feature was tested on a series of 38 PGF2alphaprostaglandin analogues with antinidatory activity; the extent to which the molecular flexibility explains the variation in the biological activity was estimated and the active conformation was predicted. The novel descriptors in combination with the grid-based SOMFA descriptors resulted in 3+3D-QSAR models with good levels of predictivity leading to the approach of separation of the effect of the molecular interaction field of the active conformation and the effect of the conformational free energy loss.


Assuntos
Desenho de Fármacos , Oligopeptídeos/química , Relação Quantitativa Estrutura-Atividade , Aminoácidos/química , Ligantes , Modelos Moleculares , Conformação Molecular , Estatística como Assunto , Estereoisomerismo , Termodinâmica
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