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1.
J Steroid Biochem Mol Biol ; 205: 105765, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32991989

RESUMO

The aim of this project was to investigate the endocrine disrupting effects of three γ-aminobutyric acid type A receptor (GABAAR) agonists, diazepam (DZ), oxazepam (OX) and alprazolam (AL) using the steroidogenic in vitro H295R cell line assay, a recombinant CYP17A1 assay, qPCR analysis and computational modelling. Similar effects for DZ and OX on the steroidogenesis were observed in the H295R experiment at therapeutically relevant concentrations. Progestagens and corticosteroids were increased up to 10 fold and androgens were decreased indicating CYP17A1 lyase inhibition. For DZ the inhibition on both the hydroxylase and lyase was confirmed by the recombinant CYP17A1 assay, whereas OX did not appear to directly affect the recombinant CYP17A1 enzyme. Androgens were decreased when exposing the H295R cells to AL, indicating a CYP17A1 lyase inhibition. However, this was not confirmed by the recombinant CYP17A1 assay but a down-regulation in gene expression was observed for StAR and CYP17A1. The present study showed that the three investigated benzodiazepines (BZDs) are rather potent endocrine disruptors in vitro, exerting endocrine effects close the therapeutic Cmax. Both direct and indirect effects on steroidogenesis were observed, but molecular modelling indicated no direct interactions between the heme group in the steroidogenic CYP enzymes and the unique diazepin structure. In contrast, physicochemical properties such as high log P, structure and molecular weight similar to that of steroids appeared to influence the endocrine disrupting abilities of the investigated pharmaceuticals in vitro. Docking of the three BZDs in CYP17A1 and CYP21A2 confirmed that shape complementarity and hydrophobic effects seem to determine the binding modes.


Assuntos
Benzodiazepinas/química , Disruptores Endócrinos/química , Esteroide 17-alfa-Hidroxilase/química , Esteroide 21-Hidroxilase/química , Esteroides/biossíntese , Corticosteroides/química , Corticosteroides/farmacologia , Glândulas Suprarrenais/efeitos dos fármacos , Alprazolam/química , Alprazolam/farmacologia , Androgênios/genética , Benzodiazepinas/farmacologia , Diazepam/química , Diazepam/farmacologia , Disruptores Endócrinos/farmacologia , Humanos , Simulação de Acoplamento Molecular , Oxazepam/química , Oxazepam/farmacologia , Receptores Androgênicos/química , Receptores Androgênicos/genética , Receptores de GABA-A/química , Receptores de GABA-A/genética , Esteroide 17-alfa-Hidroxilase/antagonistas & inibidores , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 21-Hidroxilase/antagonistas & inibidores , Esteroide 21-Hidroxilase/genética , Esteroides/química
2.
Mol Pharmacol ; 99(1): 39-48, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33268553

RESUMO

The family of GABAA receptors is an important drug target group in the treatment of sleep disorders, anxiety, epileptic seizures, and many others. The most frequent GABAA receptor subtype is composed of two α-, two ß-, and one γ2-subunit, whereas the nature of the α-subunit critically determines the properties of the benzodiazepine binding site of those receptors. Nearly all of the clinically relevant drugs target all GABAA receptor subtypes equally. In the past years, however, drug development research has focused on studying α5-containing GABAA receptors. Beyond the central nervous system, α5-containing GABAA receptors in airway smooth muscles are considered as an emerging target for bronchial asthma. Here, we investigated a novel compound derived from the previously described imidazobenzodiazepine SH-053-2'F-R-CH3 (SH53d-ester). Although SH53d-ester is only moderately selective for α5-subunit-containing GABAA receptors, the derivative SH53d-acid shows superior (>40-fold) affinity selectivity and is a positive modulator. Using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes and radioligand displacement assays with human embryonic kidney 293 cells, we demonstrated that an acid group as substituent on the imidazobenzodiazepine scaffold leads to large improvements of functional and binding selectivity for α5ß3γ2 over other αxß3γ2 GABAA receptors. Atom level structural studies provide hypotheses for the improved affinity to this receptor subtype. Mutational analysis confirmed the hypotheses, indicating that loop C of the GABAA receptor α-subunit is the dominant molecular determinant of drug selectivity. Thus, we characterize a promising novel α5-subunit-selective drug candidate. SIGNIFICANCE STATEMENT: In the current study we present the detailed pharmacological characterization of a novel compound derived from the previously described imidazobenzodiazepine SH-053-2'F-R-CH3. We describe its superior (>40-fold) affinity selectivity for α5-containing GABAA receptors and show atom-level structure predictions to provide hypotheses for the improved affinity to this receptor subtype. Mutational analysis confirmed the hypotheses, indicating that loop C of the GABAA receptor α-subunit is the dominant molecular determinant of drug selectivity.


Assuntos
Benzodiazepinas/metabolismo , Moduladores GABAérgicos/metabolismo , Receptores de GABA-A/metabolismo , Animais , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Relação Dose-Resposta a Droga , Feminino , Flunitrazepam/química , Flunitrazepam/metabolismo , Flunitrazepam/farmacologia , Moduladores GABAérgicos/química , Moduladores GABAérgicos/farmacologia , Células HEK293 , Humanos , Ligantes , Simulação de Acoplamento Molecular/métodos , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Ratos , Receptores de GABA-A/química , Xenopus laevis
3.
Nature ; 585(7824): 303-308, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32879488

RESUMO

Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABAA) receptors to dampen neuronal activity in the brain1-5. However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABAA receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABAA receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABAA receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain.


Assuntos
Anestésicos Gerais/química , Anestésicos Gerais/farmacologia , Barbitúricos/química , Barbitúricos/farmacologia , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Microscopia Crioeletrônica , Receptores de GABA-A/química , Regulação Alostérica/efeitos dos fármacos , Anestésicos Gerais/metabolismo , Barbitúricos/metabolismo , Benzodiazepinas/metabolismo , Bicuculina/química , Bicuculina/metabolismo , Bicuculina/farmacologia , Sítios de Ligação , Ligação Competitiva/efeitos dos fármacos , Diazepam/química , Diazepam/metabolismo , Diazepam/farmacologia , Eletrofisiologia , Etomidato/química , Etomidato/metabolismo , Etomidato/farmacologia , Flumazenil/farmacologia , Antagonistas de Receptores de GABA-A/química , Antagonistas de Receptores de GABA-A/metabolismo , Antagonistas de Receptores de GABA-A/farmacologia , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Simulação de Dinâmica Molecular , Fenobarbital/química , Fenobarbital/metabolismo , Fenobarbital/farmacologia , Picrotoxina/química , Picrotoxina/metabolismo , Picrotoxina/farmacologia , Propofol/química , Propofol/metabolismo , Propofol/farmacologia , Receptores de GABA-A/metabolismo , Receptores de GABA-A/ultraestrutura , Ácido gama-Aminobutírico/química , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/farmacologia
4.
Eur J Med Chem ; 200: 112405, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32492595

RESUMO

A series of 4-phenyl-6H-imidazo[1,5-a]thieno[3,2-f][1,4]diazepine-7-carboxylate esters were synthesized and tested as central benzodiazepine receptor (CBR) ligands by the ability to displace [3H]flumazenil from rat cortical membranes. All the compounds showed high affinity with IC50 values ranging from 5.19 to 16.22 nM. In particular, compounds 12b (IC50 = 8.66 nM) and 12d (IC50 = 5.19 nM) appeared as the most effective ligands being their affinity values significantly lower than that of diazepam (IC50 = 18.52 nM). Compounds 12a-f were examined in vivo for their pharmacological effects in mice and five potential benzodiazepine (BDZ) actions were thus taken into consideration: anxiolytic, anticonvulsant, anti-amnesic, hypnotic, and locomotor activities. All the new synthesized compounds were able to induce a significant antianxiety effect and, among them, compound 12f protected pentylenetetrazole (PTZ)-induced convulsions in a dose-dependent manner reaching a 40% effect at 30 mg/kg. In addition, all the compounds were able to significantly prevent the memory impairment evoked by scopolamine, while none of them was able to interfere with pentobarbital-evoked sleep and influence motor coordination. Moreover, title compounds did not affect locomotor and exploratory activity at the same time and doses at which the anti-anxiety effect was observed. Finally, molecular docking simulations were carried out in order to assess the binding mode for compounds 12a-f. The obtained results demonstrated that these compounds bind the BDZ binding site in a similar fashion to flumazenil.


Assuntos
Ansiolíticos/síntese química , Benzodiazepinas/química , Desenho de Fármacos , Animais , Ansiolíticos/farmacologia , Anticonvulsivantes , Benzodiazepinas/metabolismo , Sítios de Ligação , Locomoção/efeitos dos fármacos , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/prevenção & controle , Camundongos , Simulação de Acoplamento Molecular , Ratos , Receptores de GABA-A/metabolismo
5.
J Med Chem ; 63(13): 6941-6958, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32515951

RESUMO

It is urgent to find new antibiotic classes with activity against multidrug-resistant (MDR) Gram-negative pathogens as the pipeline of antibiotics is essentially empty. Modified pyrrolobenzodiazepines with a C8-linked aliphatic heterocycle provide a new class of broad-spectrum antibacterial agents with activity against MDR Gram-negative bacteria, including WHO priority pathogens. The structure-activity relationship established that the third ring was particularly important for Gram-negative activity. Minimum inhibitory concentrations for the lead compounds ranged from 0.125 to 2 mg/L for MDR Gram-negative, excluding Pseudomonas aeruginosa, and between 0.03 and 1 mg/L for MDR Gram-positive species. The lead compounds were rapidly bactericidal with >5 log reduction in viable count within 4 h for Acinetobacter baumannii and Klebsiella pneumoniae. The lead compound inhibited DNA gyrase in gel-based assays, with an IC50 of 3.16 ± 1.36 mg/L. This study provides a new chemical scaffold for developing novel broad-spectrum antibiotics which can help replenish the pipeline of antibiotics.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Desenho de Fármacos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Antibacterianos/metabolismo , Benzodiazepinas/metabolismo , Linhagem Celular , DNA Girase/química , DNA Girase/metabolismo , Bactérias Gram-Negativas/enzimologia , Humanos , Simulação de Acoplamento Molecular , Conformação Proteica
6.
Life Sci ; 252: 117643, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32298738

RESUMO

AIMS: Non-peptide ligands of oxytocin receptor (OTR) have promising potentialities as therapeutic agents with improved pharmacological properties. WAY-267,464 is a non-peptide agonist which loses its agonist activity when its resorcinol moiety is methylated, yielding a partial antagonist (denoted here, WAY-Methylated). This study attempts to rationalize these opposing activities by comparative analyses of structural dynamicsof OTR in complex with these ligands. MAIN METHODS: Glide extra precision (XP) docking with and without positional constraints was employed to probe alternative binding poses of both WAY-267,464 and WAY-Methylated. The more preferred configuration of each system was subjected to an extended 2 µs MD simulation and the physics-based Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) binding energy was used to rank the complexes with improved accuracy, in addition to empirical-based Glide docking score. Network analysis was performed, and the identified critical residues were cross-referenced with the experimental mutagenesis data. KEY FINDINGS: The added methyl groups in the antagonist WAY-Methylated enhanced hydrophobicity, resulting in a flipped binding pose deeper in the binding pocket. Interestingly, OTR responded to the methylation by stabilizing the initial inactive conformation, decreasing fluctuations and increasing the overall secondary structural composition. Conversely, the agonist WAY-267,464 produced larger fluctuations to allow the receptor to change from the default inactive state to a state of partial activation. These transitions were further supported by the identified critical residues overlapping with experimental mutagenesis data. SIGNIFICANCE: These findings provide insights into the activation mechanism of OTR by WAY-267.464 and its antagonism by WAY-Methylated.


Assuntos
Benzodiazepinas/farmacologia , Simulação de Dinâmica Molecular , Pirazóis/farmacologia , Receptores de Ocitocina/agonistas , Benzodiazepinas/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Metilação , Simulação de Acoplamento Molecular , Ligação Proteica , Pirazóis/química , Receptores de Ocitocina/antagonistas & inibidores , Receptores de Ocitocina/metabolismo
7.
J Exp Clin Cancer Res ; 39(1): 50, 2020 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-32164732

RESUMO

BACKGROUND: Inhibition of ABC transporters is considered the most effective way to circumvent multidrug resistance (MDR). In the present study, we evaluated the MDR modulatory potential of ERK5-IN-1, a potent extracelluar signal regulated kinase 5 (ERK5) inhibitor. METHODS: The cytotoxicity and MDR reversal effect of ERK5-IN-1 were assessed by MTT assay. The KBv200-inoculated nude mice xenograft model was used for the in vivo study. Doxorubicin efflux and accumulation were measured by flow cytometry. The modulation of ABCB1 activity was measured by colorimetric ATPase assay and [125I]-iodoarylazidoprazosin (IAAP) photolabeling assay. Effect of ERK5-IN-1 on expression of ABCB1 and its downstream markers was measured by PCR and/or Western blot. Cell surface expression and subcellular localization of ABCB1 were tested by flow cytometry and immunofluorescence. RESULTS: Our results showed that ERK5-IN-1 significantly increased the sensitivity of vincristine, paclitaxel and doxorubicin in KBv200, MCF7/adr and HEK293/ABCB1 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Moreover, in vivo combination studies showed that ERK5-IN-1 effectively enhanced the antitumor activity of paclitaxel in KBv200 xenografts without causing addition toxicity. Mechanistically, ERK5-IN-1 increased intracellular accumulation of doxorubicin dose dependently by directly inhibiting the efflux function of ABCB1. ERK5-IN-1 stimulated the ABCB1 ATPase activity and inhibited the incorporation of [125I]-iodoarylazidoprazosin (IAAP) into ABCB1 in a concentration-dependent manner. In addition, ERK5-IN-1 treatment neither altered the expression level of ABCB1 nor blocked the phosphorylation of downstream Akt or Erk1/2. No significant reversal effect was observed on ABCG2-, ABCC1-, MRP7- and LRP-mediated drug resistance. CONCLUSIONS: Collectively, these results indicated that ERK5-IN-1 efficiently reversed ABCB1-mediated MDR by competitively inhibiting the ABCB1 drug efflux function. The use of ERK5-IN-1 to restore sensitivity to chemotherapy or to prevent resistance could be a potential treatment strategy for cancer patients.


Assuntos
Benzodiazepinas/administração & dosagem , Doxorrubicina/administração & dosagem , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Células HEK293 , Células HL-60 , Humanos , Camundongos , Camundongos Nus , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Neoplasias , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Org Lett ; 22(4): 1420-1425, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31951410

RESUMO

Catalyst-free and mild synthetic methods for the construction of hindered α-amino acid derivatives are presented herein. A wide range of hindered amino acid amides can be readily obtained from the reaction of α-halohydroxamates with a variety of amines, including anilines, primary amines, and secondary amines. Moreover, the aza/aza-[4+3] cycloaddition of in situ-generated aza-oxyallyl cations with 2-aminophenyl α,ß-unsaturated carbonyls to furnish seven-membered benzodiazepin-3-ones is reported for the first time.


Assuntos
Compostos Alílicos/química , Aminoácidos/síntese química , Compostos Aza/química , Benzodiazepinas/síntese química , Aminoácidos/química , Benzodiazepinas/química , Cátions/química , Estrutura Molecular , Estereoisomerismo
9.
J Am Chem Soc ; 142(7): 3440-3448, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-31944685

RESUMO

Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum-sensing molecule hormaomycin, and antimicrobial griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of l-tyrosine- or l-leucine-derived 4-alkyl-l-proline derivatives (APDs) in their structures. Here, we report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F420H2-dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosyntheses of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4-substituted Δ1-pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. Furthermore, the differences in the reaction specificity of the Apd6 reductases determine the formation of the fully saturated APD moiety of lincomycin versus the unsaturated APD moiety of PBDs, providing molecules with optimal shapes to bind their distinct biological targets. Moreover, the Apd6 reductases establish the first F420H2-dependent enzymes from the luciferase-like hydride transferase protein superfamily in the biosynthesis of bioactive molecules. Finally, our bioinformatics analysis demonstrates that Apd6 and their homologues, widely distributed within several bacterial phyla, play a role in the formation of novel yet unknown natural products with incorporated l-proline-like precursors and likely in the microbial central metabolism.


Assuntos
Benzodiazepinas/metabolismo , Lincomicina/biossíntese , Oxirredutases/metabolismo , Pirróis/metabolismo , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Catálise , Depsipeptídeos/biossíntese , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Lincomicina/química , Lincomicina/farmacologia , Modelos Moleculares , Oxirredutases/química , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Prolina/análogos & derivados , Prolina/metabolismo , Pirróis/química , Pirróis/farmacologia , Riboflavina/análogos & derivados , Riboflavina/química , Riboflavina/metabolismo , Especificidade por Substrato , Tirosina/análogos & derivados , Tirosina/metabolismo
10.
Int J Mol Sci ; 21(1)2020 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-31947863

RESUMO

Many allosteric binding sites that modulate gamma aminobutyric acid (GABA) effects have been described in heteropentameric GABA type A (GABAA) receptors, among them sites for benzodiazepines, pyrazoloquinolinones and etomidate. Diazepam not only binds at the high affinity extracellular "canonical" site, but also at sites in the transmembrane domain. Many ligands of the benzodiazepine binding site interact also with homologous sites in the extracellular domain, among them the pyrazoloquinolinones that exert modulation at extracellular α+/ß- sites. Additional interaction of this chemotype with the sites for etomidate has also been described. We have recently described a new indole-based scaffold with pharmacophore features highly similar to pyrazoloquinolinones as a novel class of GABAA receptor modulators. Contrary to what the pharmacophore overlap suggests, the ligand presented here behaves very differently from the identically substituted pyrazoloquinolinone. Structural evidence demonstrates that small changes in pharmacophore features can induce radical changes in ligand binding properties. Analysis of published data reveals that many chemotypes display a strong tendency to interact promiscuously with binding sites in the transmembrane domain and others in the extracellular domain of the same receptor. Further structural investigations of this phenomenon should enable a more targeted path to less promiscuous ligands, potentially reducing side effect liabilities.


Assuntos
Antagonistas de Receptores de GABA-A/química , Antagonistas de Receptores de GABA-A/farmacologia , Domínios Proteicos/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Sítios de Ligação/efeitos dos fármacos , Desenho de Fármacos , Humanos , Ligantes , Modelos Moleculares , Quinolonas/química , Quinolonas/farmacologia , Receptores de GABA-A/química , Ácido gama-Aminobutírico/metabolismo
11.
Mol Pharm ; 17(1): 50-58, 2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31742408

RESUMO

DNA-targeting indolinobenzodiazepine dimer (IGN) payloads are used in several clinical-stage antibody-drug conjugates. IGN drugs alkylate DNA through the single imine moiety present in the dimer in contrast to the pyrrolobenzodiazepine dimer drugs, such as talirine and tesirine, which contain two imine moieties per dimer and cross-link DNA. This study explored the mechanism of binding of IGN to DNA in cells and to synthetic duplex and hairpin oligonucleotides. New, highly sensitive IGN-DNA binding enzyme-linked immunosorbent assay methods were developed using biotinylated IGN analogues (monoimine, diimine, and diamine IGNs) and digoxigenin-labeled duplex oligonucleotides, which allowed the measurement of drug-DNA adducts in viable cells at concentrations below IC50. Furthermore, the release of free drug from the IGN-DNA adduct upon treatment with nuclease ex vivo was tested under physiological conditions. The monoimine IGN drug formed a highly stable adduct with DNA in cells, with stability similar to that of the diimine drug analogue. Both monoimine and diimine IGN-DNA adducts released free drugs upon DNA cleavage by nuclease at 37 °C, although more free drug was released from the monoimine compared to the diimine adduct, which presumably was partly cross-linked. The strong binding of the monoimine IGN drug to duplex DNA results from both the noncovalent IGN-DNA interaction and the covalent bond formation between the 2-amino group of a guanine residue and the imine moiety in IGN.


Assuntos
Antineoplásicos/química , Benzodiazepinas/química , Adutos de DNA/química , DNA/química , Imunoconjugados/farmacologia , Indóis/química , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Adutos de DNA/metabolismo , Dimerização , ELISPOT , Humanos , Iminas/química , Imunoconjugados/administração & dosagem , Oligonucleotídeos/química , Pirróis/química
12.
Nat Prod Res ; 34(13): 1884-1890, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30760040

RESUMO

(+) Benzomalvins E (1) and (-) Benzomalvins E (2), a pair of epimeric derivatives, together with three known benzomalvins (3-5), were isolated from solid cultures of a interrhizospheric fungus Penicillium sp. SYPF 8411. The planar structure of (+) Benzomalvins E (1) has been previously reported. While, the absolute configuration of compound 1 was established by X-ray crystallographic analysis for the first time. The planar structure of the new compound 2 were elucidated by detailed interpretation of their HR ESI-TOF MS and NMR spectroscopic data. The absolute configuration of compound 2 was established by Rh2(OCOCF3)4-induced CD spectral data and the electronic circular dichroic (ECD) method. Furthermore, the epimerization induced by pH, temperature and H2O was revealed. Benzomalvins (1-3, 5), a type of indoximod, enhanced the cytotoxic capability of 5-fluorouracil against A549.


Assuntos
Benzodiazepinas/isolamento & purificação , Codonopsis/microbiologia , Penicillium/química , Rizosfera , Células A549 , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Cristalografia por Raios X , Citotoxinas/isolamento & purificação , Citotoxinas/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Conformação Molecular , Solo/química
13.
Mol Divers ; 24(1): 179-189, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30895449

RESUMO

A novel series of 1,2,3-triazolo-benzodiazepine derivatives 6a-o has been synthesized and evaluated in vivo for their anticonvulsant activities using by pentylenetetrazole (PTZ)- and maximal electroshock (MES)-induced seizures in mice. The synthetic approach started with diazotizing 2-aminobenzoic acids 1 to produce 2-azidobenzoic acids 2. Next, reaction of the latter compounds with propargylamine 3, benzaldehyde 4, and isocyanides 5 led to the formation of the title compounds 6a-o, in good yields. All the synthesized compounds exhibited high anticonvulsant activity in the PTZ test, comparable to or better than the standard drug diazepam. Among the tested compounds, N-(tert-butyl)-2-(9-chloro-6-oxo-4H-[1,2,3]triazolo[1,5-a][1,4]benzodiazepin-5(6H)-yl)-2-(3-bromophenyl)acetamide 6h was the most potent compound in this assay. Moreover, compounds 6i and 6k showed excellent activity in MES test. Loss of the anticonvulsant effect of compound 6h in the presence of flumazenil in the PTZ test and appropriate interaction of this compound in the active site of benzodiazepine (BZD)-binding site of GABAA receptor confirm involvement of BZD receptors in the anticonvulsant activity of compound 6h. A novel series of 1,2,3-triazolo-benzodiazepine derivatives 6a-o have been synthesized and evaluated in vivo for their anticonvulsant activities using by pentylenetetrazole (PTZ)- and maximal electroshock (MES)-induced seizures in mice. All the synthesized compounds exhibited high anticonvulsant activity, comparable to or better than the standard drug diazepam in the PTZ test and compounds 6i and 6k showed excellent activity in MES test. Flumazenil test and in silico docking study confirm involvement of benzodiazepine receptors in the anticonvulsant activity of these compounds.


Assuntos
Anticonvulsivantes/química , Anticonvulsivantes/farmacologia , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Triazóis/química , Anticonvulsivantes/síntese química , Benzodiazepinas/síntese química , Sítios de Ligação , Técnicas de Química Sintética , Desenho de Fármacos , Antagonistas de Receptores de GABA-A/química , Antagonistas de Receptores de GABA-A/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Relação Quantitativa Estrutura-Atividade , Receptores de GABA-A/química , Convulsões/tratamento farmacológico , Convulsões/etiologia
14.
J Pharm Biomed Anal ; 179: 113027, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31830625

RESUMO

Antibody drug conjugates (ADCs) are heterogeneous biopharmaceutical products that demand extensive characterization to ensure batch consistency, safety, and efficacy. Hydrophobic interaction chromatography (HIC) is the state-of-the-art analytical tool to monitor conjugation-related critical quality attributes (CQAs) e.g. drug-load distribution and Drug-to-Antibody Ratio (DAR). For the next generation site-specific PBD-ADCs (PBD: pyrrolobenzodiazepine dimer), denaturing RP-HPLC (reverse-phase high-performance chromatography) is the current method to determine average DAR. In this manuscript, we have utilized native HIC for the first time to understand conjugation related CQAs in PBD-ADCs. In terms of the method development, the type of stationary phase and salt, coupled with reduction of the reactive imine in the PBD drug-linker to an amine form in the sample preparation, have played a key role in achieving the best HIC resolution for the drug-load variants. The established HIC conditions resolved DAR 0, DAR 1, and two DAR 2 peaks for PBD-ADCs. Extended characterization of the DAR 2 peaks confirmed that they have retained characteristically distinct antibody Fc N-glycan distributions (Fc = Fragment crystallization region). Therefore, the results support that the HIC conditions established for PBD-ADCs is valuable in not only determining DAR values but also other important attributes including native drug-load distribution and unique DAR 2 conformations existed as a result of the N-glycan heterogeneity.


Assuntos
Benzodiazepinas/análise , Imunoconjugados/análise , Pirróis/análise , Benzodiazepinas/química , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Dimerização , Interações Hidrofóbicas e Hidrofílicas , Imunoconjugados/química , Pirróis/química
15.
J Org Chem ; 84(24): 16338-16345, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31765150

RESUMO

The 5N-arylsulfonyl-1,5-benzodiazepin-2-ones with antiproliferative activity were prepared and successfully separated into the (a1R,a2R)- and (a1S,a2S)-atropisomers with extraordinary stability (ΔG⧧ = ∼130 kJ/mol) by freezing the conformation around the sp2-sp2 axis in an Ar-N(SO2) moiety with a C6-methyl group. Also, by introducing a C3-methyl group (central chirality) into the 1,5-benzodiazepine nucleus, the stereochemistry at the axis was biased to take solely one diastereomer with a relative stereochemistry of (a1R*,a2R*,3R*). The (a1S) stereochemistry was crucial for exerting the antiproliferative activity.


Assuntos
Antineoplásicos/farmacologia , Benzodiazepinas/farmacologia , Sulfonamidas/farmacologia , Células A549 , Antineoplásicos/síntese química , Antineoplásicos/química , Benzodiazepinas/síntese química , Benzodiazepinas/química , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Humanos , Modelos Moleculares , Conformação Molecular , Estereoisomerismo , Sulfonamidas/síntese química , Sulfonamidas/química , Termodinâmica
16.
Clin Cancer Res ; 25(23): 6986-6994, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31685491

RESUMO

PURPOSE: ADCT-402 (loncastuximab tesirine) is an antibody-drug conjugate comprising a CD19-targeting antibody and pyrrolobenzodiazepine dimers. A first-in-human study evaluated the safety and preliminary clinical activity of loncastuximab tesirine in patients with B-cell non-Hodgkin lymphoma (NHL). PATIENTS AND METHODS: A multicenter, phase I, dose-escalation and dose-expansion study enrolled patients ages ≥18 years with relapsed/refractory (R/R) B-cell NHL. Patients received loncastuximab tesirine every 3 weeks at doses assigned by a 3+3 dose-escalation design. Dose escalation was used to assess the safety and tolerability of loncastuximab tesirine to determine the dose for expansion. Secondary objectives evaluated clinical activity, characterized the pharmacokinetic profile, and evaluated antidrug antibodies. RESULTS: During dose escalation, 88 patients with R/R B-cell NHL were treated with loncastuximab tesirine at doses 15 to 200 µg/kg. Treatment-emergent adverse events (TEAEs) were experienced by 87/88 (98.9%) patients. Most common TEAEs (≥20% of patients) were hematologic abnormalities, fatigue, edema, liver test abnormalities, nausea, rash, and dyspnea. Grade ≥3 TEAEs (≥5% of patients) included hematologic abnormalities, liver test abnormalities, fatigue, and dyspnea. Overall response rate at doses ≥120 µg/kg was 59.4% (41 of 69 patients; 40.6% complete response; 18.8% partial response). Median duration of response, progression-free survival, and overall survival (all doses) were 4.8, 5.5, and 11.6 months, respectively. Drug exposure increased with increasing dose, showing moderate accumulation with multiple doses ≥150 µg/kg. There was no evidence of immunogenicity. CONCLUSIONS: Loncastuximab tesirine had promising activity with acceptable safety in this dose-escalation study. A phase II study with initial dosing at 150 µg/kg has been initiated based on these results.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos CD19/química , Antineoplásicos/uso terapêutico , Benzodiazepinas/química , Imunoconjugados/uso terapêutico , Linfoma de Células B/terapia , Recidiva Local de Neoplasia/terapia , Pirróis/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD19/imunologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Seguimentos , Humanos , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/patologia , Prognóstico , Terapia de Salvação , Taxa de Sobrevida , Adulto Jovem
17.
Sensors (Basel) ; 19(22)2019 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-31744128

RESUMO

The presented manuscript reports the simultaneous detection of a ternary mixture of the benzodiazepines diazepam, lorazepam, and flunitrazepam using an array of voltammetric sensors and the electronic tongue principle. The electrodes used in the array were selected from a set of differently modified graphite epoxy composite electrodes; specifically, six electrodes were used incorporating metallic nanoparticles of Cu and Pt, oxide nanoparticles of CuO and WO3, plus pristine electrodes of epoxy-graphite and metallic Pt disk. Cyclic voltammetry was the technique used to obtain the voltammetric responses. Multivariate examination using Principal Component Analysis (PCA) justified the choice of sensors in order to get the proper discrimination of the benzodiazepines. Next, a quantitative model to predict the concentrations of mixtures of the three benzodiazepines was built employing the set of voltammograms, and was first processed with the Discrete Wavelet Transform, which fed an artificial neural network response model. The developed model successfully predicted the concentration of the three compounds with a normalized root mean square error (NRMSE) of 0.034 and 0.106 for the training and test subsets, respectively, and coefficient of correlation R ≥ 0.938 in the predicted vs. expected concentrations comparison graph.


Assuntos
Benzodiazepinas/isolamento & purificação , Técnicas Biossensoriais , Técnicas Eletroquímicas , Benzodiazepinas/química , Grafite/química , Humanos , Nanopartículas Metálicas/química , Redes Neurais de Computação , Análise de Componente Principal , Análise de Ondaletas
18.
J Am Soc Mass Spectrom ; 30(12): 2795-2804, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31720974

RESUMO

Mass spectrometry (MS)-based protein footprinting, a valuable structural tool in mapping protein-ligand interaction, has been extensively applied to protein-protein complexes, showing success in mapping large interfaces. Here, we utilized an integrated footprinting strategy incorporating both hydrogen-deuterium exchange (HDX) and hydroxyl radical footprinting (i.e., fast photochemical oxidation of proteins (FPOP)) for molecular-level characterization of the interaction of human bromodomain-containing protein 4 (BRD4) with a hydrophobic benzodiazepine inhibitor. HDX does not provide strong evidence for the location of the binding interface, possibly because the shielding of solvent by the small molecule is not large. Instead, HDX suggests that BRD4 appears to be stabilized by showing a modest decrease in dynamics caused by binding. In contrast, FPOP points to a critical binding region in the hydrophobic cavity, also identified by crystallography, and, therefore, exhibits higher sensitivity than HDX in mapping the interaction of BRD4 with compound 1. In the absence or under low concentrations of the radical scavenger, FPOP modifications on Met residues show significant differences that reflect the minor change in protein conformation. This problem can be avoided by using a sufficient amount of proper scavenger, as suggested by the FPOP kinetics directed by a dosimeter of the hydroxyl radical.


Assuntos
Benzodiazepinas/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Espectrometria de Massas em Tandem/métodos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Benzodiazepinas/química , Proteínas de Ciclo Celular/química , Medição da Troca de Deutério/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Radical Hidroxila/análise , Radical Hidroxila/metabolismo , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Fatores de Transcrição/química
19.
Eur J Med Chem ; 182: 111670, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31499359

RESUMO

A series of novel structurally-related tubulin polymerization inhibitors based on benzodiazepine were designed, synthesized, and evaluated for anticancer activity. Extensive structure modifications were performed to investigate the detailed structure and activity relationships (SARs). Most compounds exhibited potent antiproliferative activity against a panel of cancer cell lines. Among these compounds, the optimal compound, 9a, possessed the most superior activity, including cytotoxicity against five cancer cell lines (IC50 = 6-15 nM) and inhibition of tubulin polymerization (IC50 = 1.65 ±â€¯0.11 µM). Mechanistic studies revealed that 9a could disrupt intracellular microtubule organization, arrest cell cycle at the G2/M phase and eventually induce cell apoptosis. Compound 9a exhibited good metabolic stability with a t1/2 of 161.2 min, which was much better than the reference compound CA-4. Moreover, the disodium salt of 9a, 9a-P, exhibited excellent in vivo antitumor activity in xenograft mice model with inhibitory rate of 89.3%, which was better than the reference compounds CA-4P (inhibitory rate: 52.8%) and Y-01P (inhibitory rate: 77.7%). Altogether, 9a could serve as a promising lead compound for the development of highly efficient anticancer agents.


Assuntos
Antineoplásicos/farmacologia , Benzodiazepinas/farmacologia , Desenho de Fármacos , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Benzodiazepinas/química , Benzodiazepinas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Polimerização/efeitos dos fármacos , Relação Estrutura-Atividade , Moduladores de Tubulina/química , Moduladores de Tubulina/metabolismo
20.
Chem Commun (Camb) ; 55(68): 10128-10131, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31386708

RESUMO

Fueled by the therapeutic potential of the epigenetic machinery, BET bromodomains have seen high interest as drug targets. Herein, we introduce different linkers to a BET bromodomain benzodiazepine ligand (I-BET762) to gauge its implications in the development of hybrid drugs, imaging probes and small molecule drug conjugates. Biophysical studies confirmed minimal disruption to binding of the BRD4 cavity by the synthesized entities, which includes imaging probes. Target engagement was confirmed in a cellular context, but poor membrane diffusion was found despite efficient localization in the nuclei after membrane disruption. Our study highlights challenges and opportunities for the successful design of benzodiazepine-derived drug-delivery systems.


Assuntos
Benzodiazepinas/farmacologia , Fluoresceínas/farmacologia , Corantes Fluorescentes/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Benzodiazepinas/síntese química , Benzodiazepinas/química , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Desenho de Fármacos , Fluoresceínas/síntese química , Fluoresceínas/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Ligantes , Estrutura Molecular , Proteínas Nucleares/química , Domínios Proteicos
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