RESUMO
Outer membrane vesicles (OMVs) have been widely explored to develop vaccine candidates for bacterial pathogens due to their ability to combine adjuvant properties with immunogenic activity. OMV expresses a variety of proteins and carbohydrate antigens on their surfaces. For this reason, there is an analytical need to thoroughly characterize the species expressed at their surface: we here present a simple and accurate reversed-phase ultrahigh-performance liquid chromatography (RP-UPLC) method developed according to quality by design principles. This work provides an analytical alternative to the classical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) characterization. The higher selectivity and sensitivity of the RP-UHPLC assay allow for the identification of additional protein species with respect to SDS-PAGE and facilitate its precise relative abundance quantification. According to validation results, the assay showed high accuracy, linearity, precision, repeatability, and a limit of quantification of 1% for less abundant proteins. This performance paves the way for improved production campaign consistency while also being analytically simple (no sample pretreatment required), making it suitable for routine quality control testing. In addition, the applicability of the assay to a wider range of vesicle classes (GMMA) was demonstrated.
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GlaxoSmithKline (GSK) is currently developing a fully liquid presentation to ease the administration of the licensed quadrivalent conjugate vaccine (Menveo) against meningococcal serogroup A, C, W, and Y (MenACWY) infections. Herein, we report a new method for determining the free saccharide (FS) content of CRM197-MenACWY conjugated antigens, with the aim of improving accuracy and reproducibility. Mathematical models have been used to support technical knowledge in reducing the need for experimental development. This results in an improved, faster, and platform-based technique for FS separation with one single pretreatment applicable to all antigens of the multivalent meningococcal vaccine.
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A series of 4-quinolylhydrazones was synthesized and tested in vitro against Mycobacterium tuberculosis. At a concentration of 6.25microg/mL, most of the newly synthesized compounds displayed 100% inhibitory activity against M. tuberculosis in cellular assays. Further screening allowed the identification of very potent antitubercular agents. Compound 4c was also tested in a time-course experiment and against mtb clinical isolates, displaying interesting results.
Assuntos
Antituberculosos/química , Hidrazonas/química , Animais , Antituberculosos/síntese química , Antituberculosos/farmacologia , Linhagem Celular , Chlorocebus aethiops , Hidrazonas/síntese química , Hidrazonas/farmacologia , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Relação Estrutura-Atividade , Células VeroRESUMO
We describe herein the design, synthesis, biological evaluation, and structure-activity relationship (SAR) studies of an innovative class of antimalarial agents based on a polyaromatic pharmacophore structurally related to clotrimazole and easy to synthesize by low-cost synthetic procedures. SAR studies delineated a number of structural features able to modulate the in vitro and in vivo antimalarial activity. A selected set of antimalarials was further biologically investigated and displayed low in vitro toxicity on a panel of human and murine cell lines. In vitro, the novel compounds proved to be selective for free heme, as demonstrated in the beta-hematin inhibitory activity assay, and did not show inhibitory activity against 14-alpha-lanosterol demethylase (a fungal P450 cytochrome). Compounds 2, 4e, and 4n exhibited in vivo activity against P. chabaudi after oral administration and thus represent promising antimalarial agents for further preclinical development.
Assuntos
Antimaláricos/síntese química , Clotrimazol/análogos & derivados , Clotrimazol/síntese química , Animais , Antifúngicos/síntese química , Antifúngicos/farmacologia , Antifúngicos/toxicidade , Antimaláricos/farmacologia , Antimaláricos/toxicidade , Linhagem Celular , Clotrimazol/farmacologia , Clotrimazol/toxicidade , Inibidores das Enzimas do Citocromo P-450 , Desenho de Fármacos , Feminino , Compostos Férricos/química , Heme/química , Humanos , Técnicas In Vitro , Camundongos , Modelos Moleculares , Oxirredutases/antagonistas & inibidores , Testes de Sensibilidade Parasitária , Plasmodium berghei/efeitos dos fármacos , Plasmodium chabaudi/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Protoporfirinas/química , Estereoisomerismo , Esterol 14-Desmetilase , Relação Estrutura-AtividadeRESUMO
A promising way to interfere with biological processes is through the modulation of protein-protein interactions by means of small molecules acting as peptidomimetics. The 1,4-benzodiazepine scaffold has been widely reported as a peptide-mimicking, pharmacogenic system. While several synthetic pathways to C6-8 substituted benzodiazepines have been disclosed, few 1,4-benzodiazepines substituted at C9 have been reported. Herein, we describe a versatile approach to introduce cyclic, protonatable functionality at C8/C9. Introduction of the piperazine system at C8 and C9 gave access to a unique functionalization of the versatile benzodiazepine skeleton, broadening tailoring options on the benzofused side of the molecule, and the possibility of discovering novel peptidomimetics potentially able to modulate protein-protein interactions. Coupling of activated amino acids with poorly reactive anilines under mild conditions, while avoiding racemization, gave easy access to these compounds. Efficient amino acid activation was obtained by exploiting the rapid formation of acid chlorides under low temperature and acid/base free conditions, using triphenylphosphine and hexachloroacetone. This procedure successfully resulted in high reaction yields, did not produce racemization (ee > 98%, as demonstrated by using chiral solvating agents), and was compatible with the acid sensitive protecting groups present in the substrates.
Assuntos
Benzodiazepinas/síntese química , Mimetismo Molecular , Peptídeos/química , Benzodiazepinas/química , Piperazina , Piperazinas/química , Ligação Proteica , EstereoisomerismoRESUMO
Tacrine based reversible inhibitors of cholinesterases (ChEIs) containing peptidic tethers were synthesized to interact with specific regions at the gorge level, and their potency was determined with human (h) acetylcholinesterase and butyrylcholinesterase. Analogues 3i,j and 3l,m were identified as promising hits and may pave the way for the development of a new series of tacrine based enzyme selective hChEIs.
Assuntos
Inibidores da Colinesterase , Peptídeos/síntese química , Peptídeos/farmacologia , Tacrina , Acetilcolinesterase/metabolismo , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Técnicas de Química Combinatória , Desenho de Fármacos , Humanos , Estrutura Molecular , Peptídeos/química , Relação Estrutura-Atividade , Tacrina/análogos & derivados , Tacrina/síntese química , Tacrina/química , Tacrina/farmacologiaRESUMO
Here we report the synthesis and evaluation of antiplasmodial activity of a novel series of bicyclic peroxides inspired by the marine natural compound dihydroplakortin. We developed a synthetic strategy leading to the dihydroplakortin-related peroxides in only a few steps. The in vitro antiplasmodial potency of the peroxides was similar to, or greater than, that of the reference natural compound, and structure-activity relationship studies revealed several key structural requirements for activity and potency.
Assuntos
Antimaláricos/síntese química , Antimaláricos/farmacologia , Dioxanos/síntese química , Dioxanos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Animais , Antimaláricos/química , Dioxanos/química , Relação Dose-Resposta a Droga , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Peróxidos/síntese química , Peróxidos/química , Peróxidos/farmacologia , Plasmodium falciparum/crescimento & desenvolvimento , Relação Estrutura-AtividadeRESUMO
The physiological function of kainate receptors (GluK1-GluK5) in the central nervous system is not fully understood yet. With the aim of developing potent and selective GluK1 ligands, we have synthesized a series of new thiophene-based GluK1 agonists (6a-c) and antagonists (7a-d). Pharmacological evaluation revealed that they are selective for the GluK1 subunit, with 7b being the most subtype-selective ligand reported to date (GluK1 vs GluK3). The antagonist 7a was cocrystallized with the GluK1 ligand binding domain, and an X-ray crystallographic analysis revealed the largest flexibility in GluK1 ligand binding domain opening upon binding of a ligand seen to date. The results provide new insights into the molecular mechanism of GluK1 receptor ligand binding and pave the way to the development of new tool compounds for studying kainate receptor function.
Assuntos
Modelos Moleculares , Pirimidinas/síntese química , Receptores de Ácido Caínico/agonistas , Receptores de Ácido Caínico/antagonistas & inibidores , Tiofenos/síntese química , Animais , Linhagem Celular , Cricetinae , Cristalografia por Raios X , Feminino , Ligantes , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Técnicas de Patch-Clamp , Conformação Proteica , Pirimidinas/química , Pirimidinas/farmacologia , Ratos , Receptores de Ácido Caínico/química , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Estereoisomerismo , Relação Estrutura-Atividade , Tiofenos/química , Tiofenos/farmacologia , Xenopus laevisRESUMO
Starting from the prototypic compound 4, we describe new, potent, and broad-spectrum pyrrolobenzo(pyrido)oxazepinones antivirals. A biochemical and enzymological investigation was performed for defining their mechanism of inhibition at either recombinant HIV-1 RT wild type and non-nucleoside reverse transcriptase inhibitors (NNRTIs)-resistant mutants. For the novel compounds (S)-(+)-5 and (S)-(-)-7, a clear-cut stereoselective mechanism of enzyme inhibition was found. Molecular modeling studies were performed for revealing the underpinnings of this behavior.
Assuntos
Fármacos Anti-HIV/química , Resistência a Medicamentos/efeitos dos fármacos , Transcriptase Reversa do HIV/antagonistas & inibidores , Inibidores da Transcriptase Reversa/química , Fármacos Anti-HIV/farmacologia , Sequência Conservada , Resistência a Medicamentos/genética , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , HIV-1/genética , Humanos , Modelos Moleculares , Mutação , Inibidores da Transcriptase Reversa/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The synthesis and the biological characterization of novel highly selective pyrroloquinoxaline 5-HT(3) receptor (5-HT(3)R) ligands are described. In functional and in vivo biological studies the novel quinoxalines modulated cardiac parameters by direct interaction with myocardial 5-HT(3)Rs. The potent 5-HT(3)R ligands 4h and 4n modulate chronotropy (right atrium) but not inotropy (left atrium) at the cardiac level, being antagonist and partial agonist, respectively. Preliminary pharmacokinetic studies indicate that (S)-4n and 4a, representatives of the novel 5-HT(3)R ligands, possess poor blood-brain barrier permeability, being the prototypes of peripherally acting 5-HT(3)R modulators endowed with a clear-cut pharmacological activity at the cardiac level. The unique properties of 4h and 4n, compared to their previously described centrally active N-methyl analogue 5a, are mainly due to the hydrophilic groups at the distal piperazine nitrogen. These analogues represent novel pharmacological tools for investigating the role of peripheral 5-HT(3)R in the modulation of cardiac parameters.
Assuntos
Coração/efeitos dos fármacos , Miocárdio/metabolismo , Pirróis/síntese química , Pirróis/farmacologia , Quinoxalinas/síntese química , Quinoxalinas/farmacologia , Receptores 5-HT3 de Serotonina/efeitos dos fármacos , Receptores 5-HT3 de Serotonina/metabolismo , Animais , Barorreflexo/efeitos dos fármacos , Fenômenos Químicos , Feminino , Cobaias , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/metabolismo , Masculino , Camundongos , Modelos Moleculares , Pirróis/farmacocinética , Quinoxalinas/farmacocinética , Quipazina/análogos & derivados , Quipazina/síntese química , Ratos , Ratos Sprague-Dawley , Receptores de Serotonina/metabolismo , Agonistas do Receptor 5-HT3 de Serotonina , Relação Estrutura-AtividadeRESUMO
Dopamine D(3) antagonism combined with serotonin 5-HT(1A) and 5-HT(2A) receptor occupancy may represent a novel paradigm for developing innovative antipsychotics. The unique pharmacological features of 5i are a high affinity for dopamine D(3), serotonin 5-HT(1A) and 5-HT(2A) receptors, together with a low affinity for dopamine D(2) receptors (to minimize extrapyramidal side effects), serotonin 5-HT(2C) receptors (to reduce the risk of obesity under chronic treatment), and for hERG channels (to reduce incidence of torsade des pointes). Pharmacological and biochemical data, including specific c-fos expression in mesocorticolimbic areas, confirmed an atypical antipsychotic profile of 5i in vivo, characterized by the absence of catalepsy at antipsychotic dose.
Assuntos
Antipsicóticos/síntese química , Antipsicóticos/farmacologia , Comportamento Animal/efeitos dos fármacos , Desenho de Fármacos , Receptor 5-HT1A de Serotonina/metabolismo , Receptor 5-HT2A de Serotonina/metabolismo , Receptores de Dopamina D3/metabolismo , Animais , Antipsicóticos/química , Antipsicóticos/classificação , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligantes , Camundongos , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Antimalarial agents structurally based on novel pharmacophores, synthesized by low-cost synthetic procedures and characterized by low potential for developing resistance are urgently needed. Recently, we developed an innovative class of antimalarials based on a polyaromatic pharmacophore. Hybridizing the 4-aminoquinoline or the 9-aminoacridine system of known antimalarials with the clotrimazole-like pharmacophore, characterized by a polyarylmethyl group, we describe herein the development of a unique class (4a-l and 5a-c) of antimalarials selectively interacting with free heme and interfering with Plasmodium falciparum (Pf) heme metabolism. Combination of the polyarylmethyl system, able to form and stabilize radical intermediates, with the iron-complexing and conjugation-mediated electron transfer properties of the 4(9)-aminoquinoline(acridine) system led to potent antimalarials in vitro against chloroquine sensitive and resistant Pf strains. Among the compounds synthesized, 4g was active in vivo against P. chabaudi and P. berghei after oral administration and, possessing promising pharmacokinetic properties, it is a candidate for further preclinical development.
Assuntos
Aminoquinolinas/farmacologia , Antimaláricos/farmacologia , Clotrimazol/farmacologia , Aminoquinolinas/química , Aminoquinolinas/farmacocinética , Animais , Antimaláricos/síntese química , Antimaláricos/farmacocinética , Linhagem Celular , Clotrimazol/química , Clotrimazol/farmacocinética , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Plasmodium/efeitos dos fármacos , Ratos , Especificidade da Espécie , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
(S)-CPW399 ((S)-1) is a potent and excitotoxic AMPA receptor partial agonist. Modifying the cyclopentane ring of (S)-1, we developed two of the most potent and selective functional antagonists (5 and 7) for kainate receptor (KA-R) subunit iGluR5. Derivatives 5 and 7, with their unique pharmacological profile, may lead to a better understanding of the different roles and modes of action of iGluR1-5 subunits, paving the way for the synthesis of new potent, subunit selective iGluR5 modulators.