RESUMO
A series of novel hydroxamic acid derivatives was designed and synthesized, and their growth inhibitory activity against bloodstream form Trypanosoma brucei was evaluated. These compounds are based on conformationally constrained, lipophilic, spiro carbocyclic 2,6-diketopiperazine (2,6-DKP) scaffolds and bear a side pharmacophoric functionality that contains an acetohydroxamic acid moiety (CH2CONHOH) linked with the imidic nitrogen atom of the 2,6-DKP ring via an acetamido portion [CH2CON(R), R = H, CH3]. Most of these analogues were active in the midnanomolar to low micromolar range against T. brucei. (S)-Isobutyl- or (S)-benzyl-substitution on the methylene carbon located between the amine nitrogen atom and carbonyl of the 2,6-DKP ring was studied. The effect of the methyl-substitution on the nitrogen atom of the acetamido portion in the side pharmacophoric functionality was also examined. Compounds 22 and 23, bearing an isobutyl- or benzyl-substituent, respectively, and concurrently a methyl-substituent, were found to be the most potent hydroxamates of this series (IC50 = 34 and 53 nM, respectively). Both had promising selectivity over the parasite compared to mammalian cells (SI = 940 and 470, respectively). Moreover, an E/Z conformational behavior study on hydroxamic acid 18 and its methyl-substituted counterpart 21 was undertaken using NMR spectroscopy and theoretical calculations.
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The delta opioid receptor (δOR or DOR) is a G protein-coupled receptor (GPCR) showing a promising profile as a drug target for nociception and analgesia. Herein, we design and synthesize new fluorescent antagonist probes with high δOR selectivity that are ideally suited for single-molecule microscopy (SMM) applications in unmodified, untagged receptors. Using our new probes, we investigated wild-type δOR localization and mobility at low physiological receptor densities for the first time. Furthermore, we investigate the potential formation of δOR homodimers, as such a receptor organization might exhibit distinct pharmacological activity, potentially paving the way for innovative pharmacological therapies. Our findings indicate that the majority of δORs labeled with these probes exist as freely diffusing monomers on the cell surface in a simple cell model. This discovery advances our understanding of OR behavior and offers potential implications for future therapeutic research.
Assuntos
Desenho de Fármacos , Corantes Fluorescentes , Receptores Opioides delta , Receptores Opioides delta/metabolismo , Receptores Opioides delta/antagonistas & inibidores , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Humanos , Imagem Individual de Molécula/métodos , Células HEK293 , Animais , Microscopia de FluorescênciaRESUMO
A new cyclopeptide alkaloid, spinachristene A (1), along with two previously described, sanjoinenine (2) and oxyphylline C (3), were isolated from the fruits of Paliurus spina-christi Mill. All three metabolites are being isolated for the first time from the genus Paliurus. A model for the in silico binding affinity of compounds 1-3 to Dipeptidyl Peptidase IV (DPP4), which is related to type 2 diabetes (T2D), was developed. According to our model, compounds 1-3 were ranked in positions 9/12, 11/12 and 8/12, respectively and are predicted to exhibit significant affinity to DPP4, in the range of low 2-digit µΜ.
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Chronic hepatitis C virus (HCV) infections are a worldwide medical problem responsible for diverse types of liver diseases. The NS5B polymerase enzyme has become a very interesting target for the development of anti-HCV drugs owing to its fundamental role in viral replication. Here we report the synthesis of a novel series of 1-substituted phenyl-4(1H)-quinazolinone and 2-methyl-1-substituted phenyl-4(1H)-quinazolinone derivatives and evaluate their activity against HCV in HCV subgenomic replicon assays. The biological data revealed that compound 11a showed the highest activity against HCV GT1b at a micromolar concentration (EC50 = 0.984 µM) followed by compound 11b (EC50 = 1.38 µM). Both compounds 11a and 11b had high selectivity indices (SI = CC50/EC50), 160.71 and 71.75, respectively, which make them very interesting candidates for further development of more potent and selective anti-HCV agents.
Assuntos
Antivirais , Inibidores Enzimáticos , Hepacivirus , Humanos , Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica , Quinazolinonas/farmacologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais , Replicação ViralRESUMO
Opioid receptors (ORs) represent one of the most significant groups of G-protein coupled receptor (GPCR) drug targets and also act as prototypical models for GPCR function. In a constant effort to develop drugs with less side effects, and tools to explore the ORs nature and function, various (poly)pharmacological ligand design approaches have been performed. That is, besides classical ligands, a great number of bivalent ligands (i. e. aiming on two distinct OR subtypes), univalent heteromer-selective ligands and bitopic and allosteric ligands have been synthesized for the ORs. The scope of our review is to present the most important of the aforementioned ligands, highlight their properties and exhibit the current state-of-the-art pallet of promising drug candidates or useful molecular tools for the ORs.
Assuntos
Analgésicos Opioides , Receptores Opioides , Regulação Alostérica , Sítio Alostérico , Analgésicos Opioides/farmacologia , Sítios de Ligação , Ligantes , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Tumor necrosis factor (TNF) is a regulator of several chronic inflammatory diseases, such as rheumatoid arthritis. Although anti-TNF biologics have been used in clinic, they render several drawbacks, such as patients' progressive immunodeficiency and loss of response, high cost, and intravenous administration. In order to find new potential anti-TNF small molecule inhibitors, we employed an in silico approach, aiming to find natural products, analogs of Ampelopsin H, a compound that blocks the formation of TNF active trimer. Two out of nine commercially available compounds tested, Nepalensinol B and Miyabenol A, efficiently reduced TNF-induced cytotoxicity in L929 cells and production of chemokines in mice joints' synovial fibroblasts, while Nepalensinol B also abolished TNF-TNFR1 binding in non-toxic concentrations. The binding mode of the compounds was further investigated by molecular dynamics and free energy calculation studies, using and advancing the Enalos Asclepios pipeline. Conclusively, we propose that Nepalensinol B, characterized by the lowest free energy of binding and by a higher number of hydrogen bonds with TNF, qualifies as a potential lead compound for TNF inhibitors' drug development. Finally, the upgraded Enalos Asclepios pipeline can be used for improved identification of new therapeutics against TNF-mediated chronic inflammatory diseases, providing state-of-the-art insight on their binding mode.
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Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Descoberta de Drogas/métodos , Inibidores do Fator de Necrose Tumoral/química , Inibidores do Fator de Necrose Tumoral/farmacologia , Animais , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Simulação por Computador , Desenho de Fármacos , Fibroblastos/efeitos dos fármacos , Camundongos , Cultura Primária de Células , Líquido Sinovial/efeitos dos fármacos , Fator de Necrose Tumoral alfa/toxicidadeRESUMO
Opioid receptors (ORs) are classified among the oldest and best investigated drug targets due to their fundamental role in the treatment of pain and related disorders. ORs are divided in three conventional subtypes (µ, κ, δ) and the non-classical nocicepetin receptor. All ORs are family A G protein-coupled receptors (GPCRs), and are located on the cell surface. Modern biophysical methods use light to investigate physiological processes at organismal, cellular and subcellular level. Many of these methods rely on fluorescent ligands, thus highlighting their importance. This review addresses the advancements in the development of opioid fluorescent ligands and their use in biological, pharmacological and imaging applications.
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Corantes Fluorescentes/química , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , Animais , Humanos , Ligantes , Microscopia Confocal , Microscopia de Fluorescência , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismoRESUMO
Opioid receptors (ORs) are among the best-studied G protein-coupled receptors due to their involvement in neurological disorders and important role in pain treatment. Contrary to the classical monomeric model, indirect evidence suggests that ORs might form dimers, which could be endowed with a distinct pharmacological profile, and, thus, be targeted to develop innovative pharmacological therapies. However, direct evidence for the spontaneous formation of OR dimers in living cells under physiological conditions is missing. Despite a growing interest in the κ opioid receptor (KOR), KOR-selective fluorescent probes are particularly scarce in the literature. Herein, we present the first set of fluorescent KOR-selective probes with antagonistic properties. Two of these were employed in single-molecule microscopy (SMM) experiments to investigate KOR homodimerization, localization, and trafficking. Our findings indicate that most KORs labeled with the new fluorescent probes are present as apparently freely diffusing monomers on the surface of a simple cell model.
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Corantes Fluorescentes/farmacologia , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Multimerização Proteica/efeitos dos fármacos , Receptores Opioides kappa/antagonistas & inibidores , Animais , Células CHO , Cricetulus , Corantes Fluorescentes/síntese química , Células HEK293 , Humanos , Ligantes , Naltrexona/síntese química , Receptores Opioides kappa/metabolismo , Imagem Individual de MoléculaRESUMO
µ-Opioid receptors (µ-ORs) play a critical role in the modulation of pain and mediate the effects of the most powerful analgesic drugs. Despite extensive efforts, it remains insufficiently understood how µ-ORs produce specific effects in living cells. We developed new fluorescent ligands based on the µ-OR antagonist E-p-nitrocinnamoylamino-dihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved single-molecule imaging of µ-ORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of µ-ORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that µ-ORs interact with each other to form short-lived homodimers on the plasma membrane. This approach provides a new strategy to investigate µ-OR pharmacology at single-molecule level.
Assuntos
Corantes Fluorescentes/química , Hidrocodona/química , Multimerização Proteica , Receptores Opioides mu/química , Imagem Individual de Molécula/métodos , Difusão , Corantes Fluorescentes/farmacologia , Hidrocodona/farmacologia , Ligantes , Estrutura Quaternária de Proteína , Receptores Opioides mu/antagonistas & inibidores , Receptores Opioides mu/metabolismoRESUMO
Recently, the binding kinetics of a ligand-target interaction, such as the residence time of a small molecule on its protein target, are seen as increasingly important for drug efficacy. Here, we investigate these concepts to explain binding and proton blockage of rimantadine variants bearing progressively larger alkyl groups to influenza A virus M2 wild type (WT) and M2 S31N protein proton channel. We showed that resistance of M2 S31N to rimantadine analogues compared to M2 WT resulted from their higher koff rates compared to the kon rates according to electrophysiology (EP) measurements. This is due to the fact that, in M2 S31N, the loss of the V27 pocket for the adamantyl cage resulted in low residence time inside the M2 pore. Both rimantadine enantiomers have similar channel blockage and binding kon and koff against M2 WT. To compare the potency between the rimantadine variants against M2, we applied approaches using different mimicry of M2, i.e., isothermal titration calorimetry and molecular dynamics simulation, EP, and antiviral assays. It was also shown that a small change in an amino acid at site 28 of M2 WT, which does not line the pore, seriously affects M2 WT blockage kinetics.
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The alkaloid rutaecarpine and its derivatives have been described as cytotoxic and hold potential as antitumor agents. Nevertheless, their synthesis is demanding and compounds display poor water solubility. Herein, we describe the synthesis of two sets of rutaecarpine derivatives with amine functions to improve solubility. Using a classic shake-flask experiment and a potentiometric titration platform, the water solubility of the compounds was determined. Solubility improved significantly with the amine functions connected over the indole-N atom. Reduction of metabolic activity and cell viability on HeLa cells was in the same range or better for these derivatives compared to the chemically unaltered parent compounds prepared in a new synthetic procedure established in our group.
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Antineoplásicos/química , Alcaloides Indólicos/química , Quinazolinas/química , Alcaloides/química , Alcaloides/toxicidade , Antineoplásicos/síntese química , Antineoplásicos/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Alcaloides Indólicos/síntese química , Alcaloides Indólicos/toxicidade , Quinazolinas/síntese química , Quinazolinas/toxicidade , Solubilidade , Relação Estrutura-AtividadeRESUMO
Recent findings from solid state NMR (ssNMR) studies suggested that the (R)-enantiomer of rimantadine binds to the full M2 protein with higher affinity than the (S)-enantiomer. Intrigued by these findings, we applied functional assays, such as antiviral assay and electrophysiology (EP), to evaluate the binding affinity of rimantadine enantiomers to the M2 protein channel. Unexpectedly, no significant difference was found between the two enantiomers. Our experimental data based on the full M2 protein function were further supported by alchemical free energy calculations and isothermal titration calorimetry (ITC) allowing an evaluation of the binding affinity of rimantadine enantiomers to the M2TM pore. Both enantiomers have similar channel blockage, affinity, and antiviral potency.
RESUMO
Adamantane derivatives, such as amantadine and rimantadine, have been reported to block the transmembrane domain (TM) of the M2 protein of influenza A virus (A/M2) but their clinical use has been discontinued due to evolved resistance in humans. Although experiments and simulations have provided adequate information about the binding interaction of amantadine or rimantadine to the M2 protein, methods for predicting binding affinities of whole series of M2 inhibitors have so far been scarcely applied. Such methods could assist in the development of novel potent inhibitors that overcome A/M2 resistance. Here we show that alchemical free energy calculations of ligand binding using the Bennett acceptance ratio (BAR) method are valuable for determining the relative binding potency of A/M2 inhibitors of the aminoadamantane type covering a binding affinity range of only â¼2 kcal mol(-1). Their binding affinities measured by isothermal titration calorimetry (ITC) against the A/M2TM tetramer from the Udorn strain in its closed form at pH 8 were used as experimental probes. The binding constants of rimantadine enantiomers against M2TMUdorn were measured for the first time and found to be equal. Two series of alchemical free energy calculations were performed using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids to mimic the membrane environment. A fair correlation was found for DPPC that was significantly improved using DMPC, which resembles more closely the DPC lipids used in the ITC experiments. This demonstrates that binding free energy calculations by the BAR approach can be used to predict relative binding affinities of aminoadamantane derivatives toward M2TM with good accuracy.
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Adamantano/química , Adamantano/metabolismo , Membrana Celular/metabolismo , Temperatura , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo , Sequência de Aminoácidos , Calorimetria , Entropia , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos , Prótons , EstereoisomerismoRESUMO
A pharmacophore model for butyrylcholinesterase (BChE) inhibitors was applied to a human cannabinoid subtypeâ 2 receptor (hCB2 R) agonist and verified it as a first-generation lead for respective dual-acting compounds. The design, synthesis, and pharmacological evaluation of various derivatives led to the identification of aminobenzimidazoles as second-generation leads with micro- or sub-micromolar activities at both targets and excellent selectivity over hCB1 and AChE, respectively. Computational studies of the first- and second-generation lead structures by applying molecular dynamics (MD) on the active hCB2 R model, along with docking and MD on hBChE, has enabled an explanation of their binding profiles at the protein levels and opened the way for further optimization. Dual-acting compounds with "balanced" affinities and excellent selectivities could be obtained that represent leads for treatment of both cognitive and pathophysiological impairment occurring in neurodegenerative disorders.
Assuntos
Benzimidazóis/química , Butirilcolinesterase/química , Inibidores da Colinesterase/uso terapêutico , Doenças Neurodegenerativas/tratamento farmacológico , Receptor CB2 de Canabinoide/antagonistas & inibidores , Sequência de Aminoácidos , Benzimidazóis/metabolismo , Benzimidazóis/uso terapêutico , Sítios de Ligação , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/metabolismo , Desenho de Fármacos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Receptor CB1 de Canabinoide/química , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Alinhamento de Sequência , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Background: Cyclic aminals are core features of natural products, drug molecules and important synthetic intermediates. Despite their relevance, systematic investigations into their stability towards hydrolysis depending on the pH value are lacking. Results: A set of cyclic aminals was synthesized and their stability quantified by kinetic measurements. Steric and electronic effects were investigated by choosing appropriate groups. Both molecular mechanics (MM) and density functional theory (DFT) based studies were applied to support and explain the results obtained. Rapid decomposition is observed in acidic aqueous media for all cyclic aminals which occurs as a reversible reaction. Electronic effects do not seem relevant with regard to stability, but the magnitude of the conformational energy of the ring system and pKa values of the N-3 nitrogen atom. Conclusion: Cyclic aminals are stable compounds when not exposed to acidic media and their stability is mainly dependent on the conformational energy of the ring system. Therefore, for the preparation and work-up of these valuable synthetic intermediates and natural products, appropriate conditions have to be chosen and for application as drug molecules their sensitivity towards hydrolysis has to be taken into account.