RESUMEN
In the emerging context of gut-brain control of multiple sclerosis (MS), developing therapeutics targeting proinflammatory proteins controlling the gut-brain immunomodulation is welcoming. One such immunomodulator is glia maturation factor-ß (GMF-ß). GMF-ß activation following GMF-ß-ser-83 phosphorylation upregulates proinflammatory responses and exacerbates experimental autoimmune encephalomyelitis (EAE). Notably, GMF-ß-/- mice exhibited no EAE symptoms. Thus, we identified 1H-indazole-4-yl-methanol (GMFBI.1) inhibitor which blocked GMF-ß-ser-83 phosphorylation critical in EAE suppression. To establish gut GMF-ß's role in EAE in the context of gut-brain involvement in neurodegenerative diseases, we altered gut GMFBI.1 bioavailability as an index of EAE suppression. At first, we identified Miglyol 812N as a suitable biocompatible GMFBI.1 carrier compared to other FDA-approved carriers using in silico molecular docking analysis. GMFBI.1 administration in Miglyol 812N enhanced its retention/brain permeability. Subsequently, we administered GMFBI.1-Miglyol 812N by subcutaneous/oral routes at different doses with differential GMFBI.1 bioavailability in gut and brain to assess the role of local GMFBI.1 bioavailability in EAE reversal by a pharmacokinetic approach. Deprival of gut GMFBI.1 bioavailability led to partial EAE suppression despite having sufficient GMFBI.1 in circulation to inhibit brain GMF-ß activity. Restoration of gut GMFBI.1 bioavailability led to complete EAE reversal. Molecular pathology behind partial/full EAE reversal was associated with differential GMF-ß-Ser-83 phosphorylation/GM-CSF expression levels in enteric glial cells owing to GMFBI.1 bioavailability. In addition, we observed leaky gut reversal, tight junction protein ZO-1 restoration, beneficial gut microbiome repopulation, recovery from gut dysbiosis, and upregulation of Treg cells. GMFBI.1's dual gut/brain targeting of GMF-ß has therapeutical/translational potential in controlling autoimmunity in MS.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Animales , Ratones , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/metabolismo , Factor de Maduración de la Glia/metabolismo , Citocinas/metabolismo , Metanol , Simulación del Acoplamiento Molecular , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Neuroglía/metabolismo , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Bacterial surface proteins act as potential adhesins or invasins. The GroEL is a signal peptide-free surface expressed protein that aids adhesion in Escherichia coli by binding to LOX-1 receptor of the host cells. Mycobacterium tuberculosis (Mtb) expresses GroEL2 protein, having high level sequence identity with E. coli GroEL. This study investigates the interaction mechanism of GroEL2 protein of Mtb with LOX-1 of macrophages using integrated computational and experimental approach. METHODS: Mtb GroEL2 protein was purified as histidine tagged protein using Ni-NTA chromatography. Confocal and scanning electron microscopies were used to study the uptake of GroEL2 coated fluorescent latex beads through the LOX-1 receptor in RAW264.7 macrophage cell line. Docking studies were performed to understand the interaction between the GroEL2 and LOX-1 proteins. Polyinosinic acid (PIA) was used as a LOX-1 inhibitor in both in silico and in vitro experiments. RESULTS: GroEL2 protein coating enhances uptake of latex beads into macrophages through LOX-1 receptor. LOX-1 inhibitor PIA decreased the uptake of GroEL2 coated latex beads. GroEL2 interacts with the key ligand binding regions of the LOX-1 receptor, such as the basic spine and the saddle hydrophobic patch. PIA molecule destabilized the LOX-1-GroEL2 docked complex. CONCLUSION: Surface associated GroEL2 protein of Mtb is a potential ligand for macrophage LOX-1 receptor. Interaction between GroEL2 and LOX-1 receptor may be utilized by Mtb to gain its intracellular access. GENERAL SIGNIFICANCE: Surface associated GroEL2 of Mtb may bind to the macrophage LOX-1 receptor, enabling the internalization of the bacteria and progression of the infection.
Asunto(s)
Chaperonina 60/metabolismo , Interacciones Huésped-Patógeno , Macrófagos/metabolismo , Mycobacterium tuberculosis/fisiología , Receptores Depuradores de Clase E/metabolismo , Tuberculosis/metabolismo , Animales , Macrófagos/microbiología , Ratones , Simulación del Acoplamiento Molecular , Unión Proteica , Células RAW 264.7 , Tuberculosis/microbiologíaRESUMEN
The concept of substrate inhibition to prevent its phosphorylation has potential in drug discovery and is envisioned to treat the autoimmune disorder multiple sclerosis (MS). Glia maturation factor-ß (GMF-ß) Ser83 phosphorylation by protein kinase A (PKA) is pivotal in the activation of GMF-ß-p38MAPK-NFκB biochemical pathway towards proinflammatory response induction in experimental autoimmune encephalomyelitis (EAE). Using structure-based drug design, we identified the small molecule inhibitor 1-H-indazole-4yl methanol (GMFBI.1) that specifically blocked Ser83 phosphorylation site on GMF-ß substrate. Using in vitro and in vivo techniques, molecular mechanism of action of GMFBI.1's direct interaction with GMF-ß substrate and prevention of its Ser83 phosphorylation was established. GMFBI.1 down regulated p38MAPK phosphorylation and NFκB expression essential for proinflammatory response. Further, GMFBI.1 administration at peak of EAE reversed clinical symptoms, immunopathology, proinflammatory cytokine response and up regulated the anti-inflammatory cytokines. Present strategy of substrate inhibition against the key immunomodulatory target has immense therapeutic potential in MS.
Asunto(s)
Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Factor de Maduración de la Glia/metabolismo , Esclerosis Múltiple/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Secuencias de Aminoácidos , Animales , Diseño de Fármacos , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Factor de Maduración de la Glia/química , Humanos , Factores Inmunológicos/química , Factores Inmunológicos/metabolismo , Ratones Endogámicos C57BL , Esclerosis Múltiple/metabolismo , Fosforilación/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Effective therapeutic regimens for the treatment of tuberculosis (TB) are limited. They are comprised of multiple drugs that inhibit the essential cellular pathways in Mycobacterium tuberculosis (Mtb). The present study investigates an approach which enables a combination of Amoxicillin-Clavulanic acid (AMC) and a repurposed drug for its synergistic effect towards TB treatment. We identified Diosmin (DIO), by targeting the active site residues of L,D-transpeptidase (Ldt) enzymes involved in Mtb cell wall biosynthesis by using a structure-based drug design method. DIO is rapidly converted into aglycone form Diosmetin (DMT) after oral administration. Binding of DIO or DMT towards Ldt enzymes was studied using molecular docking and bioassay techniques. Combination of DIO (or DMT) and AMC exhibited higher mycobactericidal activity against Mycobacterium marinum as compared to individual drugs. Scanning electron microscopy study of M. marinum treated with AMC-DIO and AMC-DMT showed marked cellular leakage. M. marinum infected Drosophila melanogaster fly model showed an increased fly survival of ~60% upon treatment with a combination of AMC and DIO (or DMT). Finally, the enhanced in vitro antimicrobial activity of AMC-DIO was validated against Mtb H37Ra and a MDR clinical isolate. Our results demonstrate the potential for AMC and DIO (or DMT) as a synergistic combination for the treatment of TB.
Asunto(s)
Combinación Amoxicilina-Clavulanato de Potasio/farmacología , Antituberculosos/farmacología , Diosmina/farmacología , Drosophila melanogaster/crecimiento & desarrollo , Reposicionamiento de Medicamentos/métodos , Mycobacterium tuberculosis/crecimiento & desarrollo , Tuberculosis/tratamiento farmacológico , Secuencia de Aminoácidos , Animales , Antituberculosos/química , Proteínas Bacterianas/metabolismo , Drosophila melanogaster/efectos de los fármacos , Diseño de Fármacos , Quimioterapia Combinada , Masculino , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Homología de Secuencia , Tuberculosis/metabolismo , Tuberculosis/microbiologíaRESUMEN
Lactobacillus species possesses surface exposed Mucin Binding Protein (MucBP) which plays a role in adhesion to gastrointestinal mucin. MucBP contains one or more mucin binding domain (MBD), the functionality of which has yet not been characterized thoroughly. Here, we have characterized a 93-amino acid MBD (MBD93) of MucBP (LAF_0673) from Lactobacillus fermentum. Multiple sequence alignment of L. fermentum MBD93 exhibited â¼60% sequence homology with MBDs from other Lactobacillus species. Further, we cloned, expressed and purified MBD93 from Escherichia coli as N-terminal histidine-tagged protein (6X His-MBD93). The purified MBD93 was able to bind to mucin and showed strong affinity towards the terminally expressed mucin glycans viz. N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), Galactose (Gal), and Sialic acid (N-acetylneuraminic acid; Neu5Ac). In silico experiments further confirmed the interaction between homology modeled MBD93 to mucin glycans through hydrogen-bonding with its surface amino acid residues Ser57, Pro58, Ile60, Tyr63 and Ala65. We also have demonstrated that MBD93 was able to inhibit the adhesion of enteric pathogens, including E. coli, Salmonella Paratyphi A, Shigella sonnei and Proteus vulgaris to mucin. Our results suggested that L. fermentum MBD93 is a functionally sufficient unit to act as an adhesin and to protect from invading enteric pathogens.
Asunto(s)
Adhesinas Bacterianas , Adhesión Bacteriana/efectos de los fármacos , Limosilactobacillus fermentum , Mucinas/metabolismo , Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Adhesinas Bacterianas/farmacología , Animales , Escherichia coli Enteropatógena/metabolismo , Escherichia coli Enteropatógena/patogenicidad , Limosilactobacillus fermentum/química , Limosilactobacillus fermentum/genética , Limosilactobacillus fermentum/metabolismo , Dominios Proteicos , Proteus vulgaris/metabolismo , Proteus vulgaris/patogenicidad , Salmonella paratyphi A/metabolismo , Salmonella paratyphi A/patogenicidad , Shigella sonnei/metabolismo , Shigella sonnei/patogenicidad , PorcinosRESUMEN
Epidermal growth factor receptor (EGFR), upregulated in gastric cancer patients, is an oncogene of interest in the development of targeted cancer nanomedicines. This study demonstrates in silico modeling of monoclonal antibody cetuximab (CET MAb)-conjugated docetaxel (DOCT)-loaded poly(γ-glutamic acid) (γ-PGA) nanoparticles (Nps) and evaluates the in vitro/in vivo effects on EGFR-overexpressing gastric cancer cells (MKN-28). Nontargeted DOCT-γ-PGA Nps (NT Nps: 110±40 nm) and targeted CET MAb-DOCT-γ-PGA Nps (T Nps: 200±20 nm) were prepared using ionic gelation followed by 1-Ethyl-3-(3-dimethyl aminopropyl)carbodiimide-N-Hydoxysuccinimide (EDC-NSH) chemistry. Increased uptake correlated with enhanced cytotoxicity induced by targeted Nps to EGFR +ve MKN-28 compared with nontargeted Nps as evident from MTT and flow cytometric assays. Nanoformulated DOCT showed a superior pharmacokinetic profile to that of free DOCT in Swiss albino mice, indicating the possibility of improved therapeutic effect in the disease model. Qualitative in vivo imaging showed early and enhanced tumor targeted accumulation of CET MAb-DOCT-γ-PGA Nps in EGFR +ve MKN-28-based gastric cancer xenograft, which exhibited efficient arrest of tumor growth compared with nontargeted Nps and free DOCT. Thus, actively targeted CET MAb-DOCT-γ-PGA Nps could be developed as a substitute to conventional nonspecific chemotherapy, and hence could become a feasible strategy for cancer therapy for EGFR-overexpressing gastric tumors.
Asunto(s)
Cetuximab/uso terapéutico , Receptores ErbB/metabolismo , Nanomedicina/métodos , Ácido Poliglutámico/análogos & derivados , Neoplasias Gástricas/tratamiento farmacológico , Taxoides/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Cetuximab/farmacocinética , Cetuximab/farmacología , Cromatografía Líquida de Alta Presión , Simulación por Computador , Docetaxel , Endocitosis/efectos de los fármacos , Femenino , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Nanopartículas , Ácido Poliglutámico/química , Neoplasias Gástricas/sangre , Neoplasias Gástricas/patología , Taxoides/sangre , Taxoides/farmacocinética , Taxoides/farmacología , Distribución Tisular/efectos de los fármacosRESUMEN
Human nucleotide-binding oligomerization domain proteins, hNOD1 and hNOD2, are host intracellular receptors with C-terminal leucine-rich repeat (LRR) domains, which recognize specific bacterial peptidoglycan (PG) fragments as their ligands. The specificity of this recognition is dependent on the third amino acid of the stem peptide of the PG ligand, which is usually meso-diaminopimelic acid (mesoDAP) or l-lysine (l-Lys). Since the LRR domains of hNOD receptors had been experimentally shown to confer the PG ligand-sensing specificity, we developed three-dimensional structures of hNOD1-LRR and the hNOD2-LRR to understand the mechanism of differential recognition of muramyl peptide ligands by hNOD receptors. The hNOD1-LRR and hNOD2-LRR receptor models exhibited right-handed curved solenoid shape. The hot-spot residues experimentally proved to be critical for ligand recognition were located in the concavity of the NOD-LRR and formed the recognition site. Our molecular docking analyses and molecular electrostatic potential mapping studies explain the activation of hNOD-LRRs, in response to effective molecular interactions of PG ligands at the recognition site; and conversely, the inability of certain PG ligands to activate hNOD-LRRs, by deviations from the recognition site. Based on molecular docking studies using PG ligands, we propose few residues - G825, D826 and N850 in hNOD1-LRR and L904, G905, W931, L932 and S933 in hNOD2-LRR, evolutionarily conserved across different host species, which may play a major role in ligand recognition. Thus, our integrated experimental and computational approach elucidates the molecular basis underlying the differential recognition of PG ligands by hNOD receptors.
Asunto(s)
Acetilmuramil-Alanil-Isoglutamina/química , Simulación del Acoplamiento Molecular , Proteína Adaptadora de Señalización NOD1/química , Proteína Adaptadora de Señalización NOD2/química , Acetilmuramil-Alanil-Isoglutamina/genética , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Células HEK293 , Humanos , Ligandos , Proteína Adaptadora de Señalización NOD1/genética , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD2/genética , Proteína Adaptadora de Señalización NOD2/metabolismo , Dominios ProteicosRESUMEN
Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) is an intracellular pattern recognition receptor that recognizes bacterial peptidoglycan (PG) containing meso-diaminopimelic acid (mesoDAP) and activates the innate immune system. Interestingly, a few pathogenic and commensal bacteria modify their PG stem peptide by amidation of mesoDAP (mesoDAPNH2). In the present study, NOD1 stimulation assays were performed using bacterial PG containing mesoDAP (PGDAP) and mesoDAPNH2 (PGDAPNH2) to understand the differences in their biomolecular recognition mechanism. PGDAP was effectively recognized, whereas PGDAPNH2 showed reduced recognition by the NOD1 receptor. Restimulation of the NOD1 receptor, which was initially stimulated with PGDAP using PGDAPNH2, did not show any further NOD1 activation levels than with PGDAP alone. But the NOD1 receptor initially stimulated with PGDAPNH2 responded effectively to restimulation with PGDAP The biomolecular structure-recognition relationship of the ligand-sensing leucine-rich repeat (LRR) domain of human NOD1 (NOD1-LRR) with PGDAP and PGDAPNH2 was studied by different computational techniques to further understand the molecular basis of our experimental observations. The d-Glu-mesoDAP motif of GMTPDAP, which is the minimum essential motif for NOD1 activation, was found involved in specific interactions at the recognition site, but the interactions of the corresponding d-Glu-mesoDAP motif of PGDAPNH2 occur away from the recognition site of the NOD1 receptor. Hot-spot residues identified for effective PG recognition by NOD1-LRR include W820, G821, D826 and N850, which are evolutionarily conserved across different host species. These integrated results thus successfully provided the atomic level and biochemical insights on how PGs containing mesoDAPNH2 evade NOD1-LRR receptor recognition.
Asunto(s)
Ácido Diaminopimélico/química , Ácido Diaminopimélico/metabolismo , Proteína Adaptadora de Señalización NOD1/química , Proteína Adaptadora de Señalización NOD1/metabolismo , Peptidoglicano/química , Peptidoglicano/metabolismo , Secuencia de Aminoácidos , Humanos , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
Computational tools for predicting toxicity have been envisioned to have the potential to broadly impact up on the attrition rate of compounds in pre-clinical drug discovery and development. An integrated approach of computer-assisted, predictive, and physico-chemical properties of a compound, along with its in vitro and in vivo analysis, needs to be routinely exercised in the lead identification and lead optimization processes. Starting with a good lead can save a lot of money and it can significantly reduce the entire drug discovery process. The journey towards triple R's- reduce, replace and refine, further proves to be successful in predicting adverse drug reactions in patients (or animals) enrolled in clinical trials. However, the impact of predictive toxicity analysis was modest and relatively narrow in scope, due to the limited domain knowledge in this field. It is important to note that advances within medical science and newer approaches in drug development will require predictive toxicology applications to be viable. The field of computational toxicology has been heading in a direction more relevant to human diseases by reducing the adverse drug reactions. Therefore, efforts must be directed to integrating these tools relevant to the goal of preventing undesired toxicity in pre-clinical trials followed by different phases of clinical trials.
Asunto(s)
Simulación por Computador , Descubrimiento de Drogas , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Preparaciones Farmacéuticas/química , Animales , Biología Computacional , HumanosRESUMEN
CCR3, a G protein-coupled receptor, plays a central role in allergic inflammation and is an important drug target for inflammatory diseases. To understand the structure-function relationship of CCR3 receptor, different computational techniques were employed, which mainly include: (i) homology modeling of CCR3 receptor, (ii) 3D-quantitative pharmacophore model of CCR3 antagonists, (iii) virtual screening of small compound databases, and (iv) finally, molecular docking at the binding site of the CCR3 receptor homology model. Pharmacophore model was developed for the first time, on a training data set of 22 CCR3 antagonists, using CATALYST HypoRefine program. Best hypothesis (Hypo1) has three different chemical features: two hydrogen-bond acceptors, one hydrophobic, and one ring aromatic. Hypo1 model was further validated using (i) 87 test set CCR3 antagonists, (ii) Cat Scramble randomization technique, and (iii) Decoy data set. Molecular docking studies were performed on modeled CCR3 receptor using 303 virtually screened hits, obtained from small compound database virtual screening. Finally, five hits were identified as potential leads against CCR3 receptor, which exhibited good estimated activities, favorable binding interactions, and high docking scores. These studies provided useful information on the structurally vital residues of CCR3 receptor involved in the antagonist binding, and their unexplored potential for the future development of potent CCR3 receptor antagonists.
Asunto(s)
Receptores CCR3/antagonistas & inhibidores , Receptores CCR3/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células CHO , Bovinos , Quimiocina CCL11/química , Quimiocina CCL11/metabolismo , Quimiocina CCL11/farmacología , Cricetinae , Cricetulus , Bases de Datos Factuales , Ensayos Analíticos de Alto Rendimiento , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Hipersensibilidad/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Unión Proteica/efectos de los fármacos , Receptores CCR3/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Alineación de Secuencia , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-ActividadRESUMEN
Series of pyrazole ester prodrugs analogues have been synthesized and found to contain highly potent inhibitors of the cyclooxygenase-2 (COX-2) enzyme. The paper describes synthesis of the target pyrazole analogues. The structure of the synthesized mutual ester prodrugs (6-8c) were confirmed by (1)H-, (13)C-NMR mass spectroscopy (MS) and their purity were ascertained by TLC and elemental analyses. The biological in vivo evaluation of these compounds in experimental models (carrageenan-induced oedema) proved the presence of anti-inflammatory activity. Docking studies into the catalytic site of COX-2 were used to identify potential anti-inflammatory lead compounds. One lead derivative was chosen endowed with good binding energies.