RESUMO
The cell membrane is a restrictive biological barrier, especially for large, charged molecules, such as proteins. The use of cell-penetrating peptides (CPPs) can facilitate the delivery of proteins, protein complexes, and peptides across the membrane by a variety of mechanisms that are all limited by endosomal sequestration. To improve CPP-mediated delivery, we previously reported the rapid and effective cytosolic delivery of proteins in vitro and in vivo by their coadministration with the peptide S10, which combines a CPP and an endosomal leakage domain. Amphiphilic peptides with hydrophobic properties, such as S10, can interact with lipids to destabilize the cell membrane, thus promoting cargo internalization or escape from endosomal entrapment. However, acute membrane destabilization can result in a dose-limiting cytotoxicity. In this context, the partial or transient deactivation of S10 by modification with methoxy poly(ethylene glycol) (mPEG; i.e., PEGylation) may provide the means to alter membrane destabilization kinetics, thereby attenuating the impact of acute permeabilization on cell viability. This study investigates the influence of PEGylation parameters (molecular weight, architecture, and conjugation chemistry) on the delivery efficiency of a green fluorescent protein tagged with a nuclear localization signal (GFP-NLS) and cytotoxicity on cells in vitro. Results suggest that PEGylation mostly interferes with adsorption and secondary structure formation of S10 at the cell membrane, and this effect is exacerbated by the mPEG molecular weight. This effect can be compensated for by increasing the concentration of conjugates prepared with lower molecular weight mPEG (5 to â¼20 kDa) but not for conjugates prepared with higher molecular weight mPEG (40 kDa). For conjugates prepared with moderate-to-high molecular weight mPEG (10 to 20 kDa), partial compensation of inactivation could be achieved by the inclusion of a reducible disulfide bond, which provides a mechanism to liberate the S10 from the polymer. Grafting multiple copies of S10 to a high-molecular-weight multiarmed PEG (40 kDa) improved GFP-NLS delivery efficiency. However, these constructs were more cytotoxic than the native peptide. Considering that PEGylation could be harnessed for altering the pharmacokinetics and biodistribution profiles of peptide-based delivery agents in vivo, the trends observed herein provide new perspectives on how to manipulate the membrane permeabilization process, which is an important variable for achieving delivery.
Assuntos
Peptídeos Penetradores de Células , Polietilenoglicóis , Distribuição Tecidual , Polietilenoglicóis/química , Polímeros/química , Peptídeos Penetradores de Células/farmacologia , Sinais de Localização NuclearRESUMO
Steroid sulfatase (STS) is an important enzyme regulating the conversion of sulfated steroids into their active hydroxylated forms. Notably, the inhibition of STS has been shown to decrease the levels of active estrogens and was translated into clinical trials for the treatment of breast cancer. Based on quantitative structure-activity relationship (QSAR) and molecular modeling studies, we herein report the design of fluorescent inhibitors of STS by adding a dansyl group on an estrane scaffold. Synthesis of 17α-dansylaminomethyl-estradiol (7) and its sulfamoylated analog 8 were achieved from estrone in 5 and 6 steps, respectively. Inhibition assays on HEK-293 cells expressing exogenous STS revealed a high level of inhibition for compound 7 (IC50 = 69 nM), a value close to the QSAR model prediction (IC50 = 46 nM). As an irreversible inhibitor, sulfamate 8 led to an even more potent inhibition in the low nanomolar value (IC50 = 2.1 nM). In addition, we show that the potent STS inhibitor 8 can be employed as an optical imaging tool to investigate intracellular enzyme sub-localization as well as inhibitory behavior. As a result, confocal microscopy analysis confirmed good penetration of the STS fluorescent inhibitor 8 in cells and its localization in the endoplasmic reticulum where STS is localized.
Assuntos
Estradiol/análogos & derivados , Esteril-Sulfatase/antagonistas & inibidores , Estradiol/síntese química , Estrona/química , Células HEK293 , Humanos , Microscopia Confocal , Modelos Moleculares , Estrutura Molecular , Imagem Óptica , Relação Quantitativa Estrutura-AtividadeRESUMO
Understanding the function of cellular systems requires describing how proteins assemble with each other into transient and stable complexes and to determine their spatial relationships. Among the tools available to perform these analyses on a large scale is Protein-fragment Complementation Assay based on the dihydrofolate reductase (DHFR PCA). Here we test how longer linkers between the fusion proteins and the reporter fragments affect the performance of this assay. We investigate the architecture of the RNA polymerases, the proteasome and the conserved oligomeric Golgi (COG) complexes in living cells and performed large-scale screens with these extended linkers. We show that longer linkers significantly improve the detection of protein-protein interactions and allow to measure interactions further in space than the standard ones. We identify new interactions, for instance between the retromer complex and proteins related to autophagy and endocytosis. Longer linkers thus contribute an enhanced additional tool to the existing toolsets for the detection and measurements of protein-protein interactions and protein proximity in living cells.
Assuntos
Mapeamento de Interação de Proteínas/métodos , Tetra-Hidrofolato Desidrogenase/metabolismo , Bioensaio , Escherichia coli/genética , Tetra-Hidrofolato Desidrogenase/genética , Leveduras/genéticaRESUMO
Duchenne muscular dystrophy (DMD), a severe hereditary disease affecting 1 in 3,500 boys, mainly results from the deletion of exon(s), leading to a reading frameshift of the DMD gene that abrogates dystrophin protein synthesis. Pairs of sgRNAs for the Cas9 of Staphylococcus aureus were meticulously chosen to restore a normal reading frame and also produce a dystrophin protein with normally phased spectrin-like repeats (SLRs), which is not usually obtained by skipping or by deletion of complete exons. This can, however, be obtained in rare instances where the exon and intron borders of the beginning and the end of the complete deletion (patient deletion plus CRISPR-induced deletion) are at similar positions in the SLR. We used pairs of sgRNAs targeting exons 47 and 58, and a normal reading frame was restored in myoblasts derived from muscle biopsies of 4 DMD patients with different exon deletions. Restoration of the DMD reading frame and restoration of dystrophin expression were also obtained in vivo in the heart of the del52hDMD/mdx. Our results provide a proof of principle that SaCas9 could be used to edit the human DMD gene and could be considered for further development of a therapy for DMD.
Assuntos
Sistemas CRISPR-Cas/genética , Distrofina/genética , Terapia Genética , Distrofia Muscular de Duchenne/genética , Animais , Proteína 9 Associada à CRISPR/genética , Modelos Animais de Doenças , Distrofina/uso terapêutico , Éxons/genética , Mutação da Fase de Leitura/genética , Edição de Genes , Regulação da Expressão Gênica , Humanos , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/terapia , Mioblastos , Deleção de Sequência , Staphylococcus aureus/enzimologiaRESUMO
Herein we report the anti-inflammatory activity of lobaric acid and pseudodepsidones isolated from the nordic lichen Stereocaulon paschale. Lobaric acid (1) and three compounds (2, 7 and 9) were found to inhibit the NF-κB activation and the secretion of pro-inflammatory cytokines (IL-1ß and TNF-α) in LPS-stimulated macrophages. Inhibition and docking simulation experiments provided evidence that lobaric acid and pseudodepsidones bind to PPAR-γ between helix H3 and the beta sheet, similarly to partial PPAR-γ agonists. These findings suggest that lobaric acid and pseudodepsidones reduce the expression of pro-inflammatory cytokines by blocking the NF-κB pathway via the activation of PPAR-γ.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Depsídeos/farmacologia , Lactonas/farmacologia , Líquens/química , NF-kappa B/antagonistas & inibidores , PPAR gama/agonistas , Salicilatos/farmacologia , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Depsídeos/química , Depsídeos/isolamento & purificação , Relação Dose-Resposta a Droga , Humanos , Lactonas/química , Lactonas/isolamento & purificação , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Simulação de Acoplamento Molecular , Estrutura Molecular , NF-kappa B/metabolismo , PPAR gama/metabolismo , Salicilatos/química , Salicilatos/isolamento & purificação , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Células U937RESUMO
Aeromonas salmonicida subsp. salmonicida is a fish pathogen known to have a rich plasmidome. In the present study, we discovered an isolate of this bacterium bearing an additional unidentified small plasmid. After having sequenced the DNA of that isolate by next-generation sequencing, it appeared that the new small plasmid is a ColE1-type replicon plasmid, named here pAsa7. This plasmid bears a functional chloramphenicol-acetyltransferase-encoding gene (cat-pAsa7) previously unknown in A. salmonicida and responsible for resistance to chloramphenicol. A comparison of pAsa7 with pAsa2, the only known ColE1-type replicon plasmid usually found in A. salmonicida subsp. salmonicida, revealed that even if both plasmids share a high structural similarity, it is still unclear if pAsa7 is a derivative of pAsa2 since they showed several mutations at the nucleotide level. Transcriptomic analysis revealed that the cat-pAsa4 gene, another chloramphenicol-acetyltransferase-encoding gene, found on the large plasmid pAsa4, was significantly more transcribed than cat-pAsa7. This was correlated with a higher chloramphenicol resistance for isolates bearing pAsa4 compared with the one having pAsa7. Finally, a phylogenetic analysis showed that both CAT-pAsa4 and CAT-pAsa7 proteins were in different clusters. The clustering was supported by the identity of residues involved in the catalytic site. In addition, to give a better understanding of the large drug-resistance panel of A. salmonicida, this study reinforces the hypothesis that A. salmonicida subsp. salmonicida is a considerable reservoir for mobile genetic elements such as plasmids.
Assuntos
Aeromonas salmonicida/genética , Proteínas de Bactérias/genética , Cloranfenicol O-Acetiltransferase/genética , Farmacorresistência Bacteriana/genética , Plasmídeos/genética , Aeromonas salmonicida/efeitos dos fármacos , Aeromonas salmonicida/isolamento & purificação , Animais , Sequência de Bases , Cloranfenicol/farmacologia , Peixes/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala , Testes de Sensibilidade Microbiana , Filogenia , Replicon/genética , Análise de Sequência de DNARESUMO
The bacterium Aeromonas salmonicida, a fish pathogen, uses the type three secretion system (TTSS) to inject effector proteins into host cells to promote the infection. The study of the genome of A. salmonicida has revealed the existence of Ati2, a potential TTSS effector protein. In the present study, a structure-function analysis of Ati2 has been done to determine its role in the virulence of A. salmonicida. Biochemical assays revealed that Ati2 is secreted into the medium in a TTSS-dependent manner. Protein sequence analyses, molecular modelling and biochemical assays demonstrated that Ati2 is an inositol polyphosphate 5-phosphatase, which hydrolyses PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in a way similar to VPA0450, a protein from Vibrio parahaemolyticus having high sequence similarity with Ati2. Mutants of Ati2 with altered amino acids at two different locations in the catalytic site displayed no phosphatase activity. Wild-type and mutant forms of Ati2 were cloned into expression systems for Dictyostelium discoideum, a soil amoeba used as an alternative host to study A. salmonicida virulence. Expression tests allowed us to demonstrate that Ati2 is toxic for the host cell in a catalytic-dependent manner. Finally, this study demonstrated the existence of a new TTSS effector protein in A. salmonicida.
Assuntos
Aeromonas salmonicida/enzimologia , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Monoéster Fosfórico Hidrolases/metabolismo , Aeromonas salmonicida/química , Aeromonas salmonicida/genética , Aeromonas salmonicida/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Inositol Polifosfato 5-Fosfatases , Dados de Sequência Molecular , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Estrutura Terciária de Proteína , Transporte Proteico , Alinhamento de SequênciaRESUMO
The delivery of biologic cargoes to airway epithelial cells is challenging due to the formidable barriers imposed by its specialized and differentiated cells. Among cargoes, recombinant proteins offer therapeutic promise but the lack of effective delivery methods limits their development. Here, we achieve protein and SpCas9 or AsCas12a ribonucleoprotein (RNP) delivery to cultured human well-differentiated airway epithelial cells and mouse lungs with engineered amphiphilic peptides. These shuttle peptides, non-covalently combined with GFP protein or CRISPR-associated nuclease (Cas) RNP, allow rapid entry into cultured human ciliated and non-ciliated epithelial cells and mouse airway epithelia. Instillation of shuttle peptides combined with SpCas9 or AsCas12a RNP achieves editing of loxP sites in airway epithelia of ROSAmT/mG mice. We observe no evidence of short-term toxicity with a widespread distribution restricted to the respiratory tract. This peptide-based technology advances potential therapeutic avenues for protein and Cas RNP delivery to refractory airway epithelial cells.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Endonucleases/metabolismo , Células Epiteliais/metabolismo , Pneumopatias/terapia , Pulmão/metabolismo , Peptídeos/genética , Animais , Proteínas de Bactérias/genética , Brônquios/citologia , Brônquios/metabolismo , Endonucleases/genética , Terapia Genética , Humanos , Pneumopatias/genética , Pneumopatias/metabolismo , Camundongos , Peptídeos/administração & dosagem , Peptídeos/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , SuínosRESUMO
17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is a promising therapeutic target known to play a pivotal role in the progression of estrogen-dependent diseases such as breast cancer, and endometriosis. This enzyme is responsible for the last step in the biosynthesis of the most potent estrogen, estradiol (E2) and its inhibition would prevent the growth of estrogen-sensitive tumors. Based on molecular modeling with docking experiments, we identified two promising C3-oxiranyl/oxiranylmethyl-estrane derivatives that would bind competitively and irreversibly in the catalytic site of 17ß-HSD1. They have been synthesized in a short and efficient route and their inhibitory activities over 17ß-HSD1 have been assessed by an enzymatic assay. Compound 15, with an oxiranylmethyl group at position C3, was more likely to bind the catalytic site and showed an interesting, but weak, inhibitory activity with an IC50 value of 1.3⯵M (for the reduction of estrone into E2 in T-47D cells). Compound 11, with an oxiranyl at position C3, produced a lower inhibition rate, and the IC50 value cannot be determined. When tested in estrogen-sensitive T-47D cells, both compounds were also slightly estrogenic, although much less than the estrogenic hormone E2.
Assuntos
17-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 17-Hidroxiesteroide Desidrogenases/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Estranos/síntese química , Estranos/farmacologia , Simulação de Acoplamento Molecular , 17-Hidroxiesteroide Desidrogenases/química , Linhagem Celular Tumoral , Técnicas de Química Sintética , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Estranos/química , Estranos/metabolismo , Humanos , Conformação ProteicaRESUMO
The ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) was recently shown to promote mineralization of the aortic valve, hence, its inhibition represents a significant target. A quinazoline-4-piperidine sulfamide compound (QPS1) has been described as a specific and non-competitive inhibitor of NPP1. We report herein the synthesis and in vitro inhibition studies of novel quinazoline-4-piperidine sulfamide analogues using QPS1 as the lead compound. Of the 26 derivatives prepared, four compounds were found to have Kiâ¯<â¯105â¯nM against human NPP1.
Assuntos
Amidas/farmacologia , Inibidores Enzimáticos/farmacologia , Piperidinas/farmacologia , Pirofosfatases/antagonistas & inibidores , Quinazolinas/farmacologia , Amidas/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Diester Fosfórico Hidrolases/metabolismo , Piperidinas/química , Pirofosfatases/metabolismo , Quinazolinas/química , Relação Estrutura-AtividadeRESUMO
Delivery of recombinant proteins to therapeutic cells is limited by a lack of efficient methods. This hinders the use of transcription factors or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleoproteins to develop cell therapies. Here, we report a soluble peptide designed for the direct delivery of proteins to mammalian cells including human stem cells, hard-to-modify primary natural killer (NK) cells, and cancer cell models. This peptide is composed of a 6x histidine-rich domain fused to the endosomolytic peptide CM18 and the cell penetrating peptide PTD4. A less than two-minute co-incubation of 6His-CM18-PTD4 peptide with spCas9 and/or asCpf1 CRISPR ribonucleoproteins achieves robust gene editing. The same procedure, co-incubating with the transcription factor HoxB4, achieves transcriptional regulation. The broad applicability and flexibility of this DNA- and chemical-free method across different cell types, particularly hard-to-transfect cells, opens the way for a direct use of proteins for biomedical research and cell therapy manufacturing.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Fatores de Transcrição/administração & dosagem , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Cricetulus , Citosol/metabolismo , Endocitose , Escherichia coli , Humanos , Camundongos , Mioblastos/citologia , Mioblastos/metabolismo , Ratos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismoRESUMO
We describe an efficient epoxidation process leading to chiral epoxyketones using the reusable homo-oligopeptide poly-l-leucine (PLL) in pure water, without any organic co-solvent. A range of substituted epoxyketones can be accessed with good conversions and high enantioselectivities. Based on the experimental results and computational studies, we propose a mechanism that demonstrates the importance of both the α-helical structure and the presence of a hydrophobic groove of the homo-oligopeptide catalyst for reactivity and selectivity.
RESUMO
The ectonucleotide phosphodiesterase/pyrophosphatase-1 (NPP1) is a type II transmembrane glycoprotein that regulates extracellular inorganic purine nucleotide and inorganic diphosphate levels through the hydrolysis of ATP into AMP and diphosphate. NPP1 is a promising drug target as it plays a role in several disorders. In the present work, we report the 3D structure modeling and extensive molecular dynamics simulations of NPP1-h, both in its free and ATP-bound forms. We identified the key residues involved in the binding of the ATP and the binding modes. The simulations suggest that NPP1-h is a rigid enzyme except for specific residues or segments, with the most mobile residues located in the unstructure "lasso loop" (LSO) domain. The binding of ATP significantly affected the dynamics of NPP1-h, with a rigidification of the phosphodiesterase (PDE) catalytic domain and an increase in mobility for the residues of the Nuclease-like (NUC) and the LSO domains. A dynamical network analysis identified that the most prevalent edges of the networks were located between the PDE and the NUC domains. In presence of ATP the networks became scattered through the PDE domain while the networks converged into a specific path that stretched from the PDE-NUC interdomain up to the middle of the LSO loop throughout the NUC domain. We suggest that these sections of the dynamical network may host potential allosteric inhibition sites. These results provide an improved understanding of the structure and dynamics of NPP1-h and will contribute to the rational design of NPP1-h inhibitors.
Assuntos
Modelos Moleculares , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/química , Pirofosfatases/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , HumanosRESUMO
Emergence of pan neuraminidase inhibitor (NAI)-resistant variants constitutes a serious clinical concern. An influenza A(H1N1)pdm09 variant containing the I427T/Q313R neuraminidase (NA) substitutions was previously identified in a surveillance study. Although these changes are not part of the NA active site, the variant showed reduced susceptibility to many NAIs. In this study, we investigated the mechanism of resistance for the I427T/Q313R substitution and its impact on the NA enzyme and viral fitness. Recombinant wild-type (WT), I427T/Q313R and I427T A(H1N1)pdm09 viruses were generated by reverse genetics and tested for their drug susceptibilities, enzymatic properties and replication kinetics in vitro as well as their virulence in mice. Molecular dynamics (MD) simulations were performed for NA structural analysis. The I427T substitution, which was responsible for the resistance phenotype observed in the double (I427T/Q313R) mutant, induced 17-, 56-, 7-, and 14-fold increases in IC50 values against oseltamivir, zanamivir, peramivir and laninamivir, respectively. The I427T substitution alone or combined to Q313R significantly reduced NA affinity. The I427T/Q313R and to a lesser extent I427T recombinant viruses displayed reduced viral titers vs WT in vitro. In experimentally-infected mice, the mortality rates were 62.5%, 0% and 14.3% for the WT, I417T/Q313R and I427T viruses, respectively. There were about 2.5- and 2-Log reductions in mean lung viral titers on day 5 post-infection for the I427T/Q313R and I427T mutants, respectively, compared to WT. Results from simulations revealed that the I427T change indirectly altered the stability of the catalytic R368 residue of the NA enzyme causing its reduced binding to the substrate/inhibitor. This study demonstrates that the I427T/Q313R mutant, not only alters NAI susceptibility but also compromises NA properties and viral fitness, which could explain its infrequent detection in clinic.
Assuntos
Antivirais/farmacologia , Aptidão Genética , Vírus da Influenza A Subtipo H1N1/genética , Neuraminidase/química , Neuraminidase/genética , Ácidos Carbocíclicos , Substituição de Aminoácidos , Animais , Domínio Catalítico , Simulação por Computador , Ciclopentanos/farmacologia , Farmacorresistência Viral , Inibidores Enzimáticos/farmacologia , Guanidinas/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/enzimologia , Concentração Inibidora 50 , Camundongos , Simulação de Dinâmica Molecular , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/virologia , Oseltamivir/farmacologia , Genética ReversaRESUMO
17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is involved in the biosynthesis of estradiol, the major bioactive endogenous estrogen in mammals, and constitutes an interesting therapeutic target for estrogen-dependent diseases. A steroidal derivative, 3-{[(16ß,17ß)-3-(2-bromoethyl)-17-hydroxyestra-1,3,5(10)-trien-16-yl]methyl} benzamide (PBRM), has recently been described as a non-estrogenic, irreversible inhibitor of 17ß-HSD1. However, the mode of action of this inhibitor and its selectivity profile have not yet been elucidated. We assessed PBRM potency via in vitro kinetic measurements. The mechanism of enzyme inactivation was also investigated using interspecies (human, mouse, pig and monkey) comparisons via both in vitro assays and in silico analysis. Mouse and human plasma protein binding of PBRM was determined, whereas its selectivity of action was studied using a wide range of potential off-targets (e.g. GPCR, hERG, CYPs, etc.). The affinity constant (Ki=368nM) and the enzyme inactivation rate (kinact=0.087min-1) values for PBRM were determined with purified 17ß-HSD1. PBRM was found to be covalently linked to the enzyme. A long delay period (i.e. 3-5days) is required to recover 17ß-HSD1 activity following a pretreatment of breast and placenta cell lines with PBRM. Mechanistic analyses showed important interspecies differences of 17ß-HSD1 inhibition which support the importance of inactivation for PBRM effect. Evidences of the potency and selectivity of action presented herein for this first non-estrogenic and steroidal covalent irreversible inhibitor of 17ß-HSD1 warrant its further development as a potential drug candidate for estrogen-dependent disorders.
Assuntos
Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Estradiol Desidrogenases/antagonistas & inibidores , Estradiol Desidrogenases/metabolismo , Animais , Benzamidas/química , Benzamidas/metabolismo , Benzamidas/farmacologia , Callithrix , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Feminino , Células HEK293 , Haplorrinos , Humanos , Camundongos , Ligação Proteica/fisiologia , SuínosRESUMO
Steroid sulfatase (STS), the enzyme which converts inactive sulfated steroid precursors into active hormones, is a promising therapeutic target for the treatment of estrogen-sensitive breast cancer. We report herein the synthesis and in vitro study of dual-action STS inhibitors with selective estrogen-receptor modulator (SERM) effects. A library of tetrahydroisoquinoline-N-substituted derivatives (phenolic compounds) was synthesized by solid-phase chemistry and tested on estrogen-sensitive breast cancer T-47D cells. Three phenolic compounds devoid of estrogenic activity and toxicity emerged from this screening. Their sulfamate analogs were then synthesized, tested in STS-transfected HEK-293 cells, and found to be potent inhibitors of the enzyme (IC50 of 3.9, 8.9, and 16.6 nM). When tested in T-47D cells they showed no estrogenic activity and produced a moderate antiestrogenic activity. The compounds were further tested on osteoblast-like Saos-2 cells and found to significantly stimulate their proliferation as well as their alkaline phosphatase activity, thus suggesting a SERM activity. These results are supported by molecular docking experiments.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Esteril-Sulfatase/antagonistas & inibidores , Ácidos Sulfônicos/farmacologia , Fosfatase Alcalina/metabolismo , Aminas/química , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Conformação Proteica , Moduladores Seletivos de Receptor Estrogênico/química , Moduladores Seletivos de Receptor Estrogênico/metabolismo , Esteril-Sulfatase/química , Esteril-Sulfatase/metabolismo , Ácidos Sulfônicos/química , Ácidos Sulfônicos/metabolismoRESUMO
The chemical synthesis of four stereoisomers (compounds 5a-d) of 16ß-(m-carbamoylbenzyl)-estradiol, a potent reversible inhibitor of 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1), and two intermediates (compounds 3a and b) was performed. Assignment of all nuclear magnetic resonance signals confirmed the stereochemistry at positions 13, 16 and 17. Nuclear overhauser effects showed clear correlations supporting a C-ring chair conformation for 5a and b and a C-ring boat conformation for 5c and d. These compounds were tested as 17ß-HSD1 inhibitors and to assess their proliferative activity on estrogen-sensitive breast cancer cells (T-47D) and androgen-sensitive prostate cancer cells (LAPC-4). Steroid derivative 5a showed the best inhibitory activity for the transformation of estrone to estradiol (95, 82 and 27%, at 10, 1 and 0.1µM, respectively), but like the other isomers 5c and d, it was found to be estrogenic. The intermediate 3a, however, was weakly estrogenic at 1µM, not at all at 0.1µM, and showed an interesting inhibitory potency on 17ß-HSD1 (90, 59 and 22%, at 10, 1 and 0.1µM, respectively). As expected, no compound showed an androgenic activity. The binding modes for compounds 3a and b, 5a-d and CC-156 were evaluated from molecular modeling. While the non-polar interactions were conserved for all the inhibitors in their binding to 17ß-HSD1, differences in polar interactions and in binding conformational energies correlated to the inhibitory potencies.
Assuntos
17-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Estradiol/química , Estradiol/farmacologia , Estrogênios/química , Estrogênios/farmacologia , 17-Hidroxiesteroide Desidrogenases/metabolismo , Linhagem Celular Tumoral , Estrona/metabolismo , Humanos , Modelos Moleculares , EstereoisomerismoRESUMO
The CRISPR/Cas9 system is a great revolution in biology. This technology allows the modification of genes in vitro and in vivo in a wide variety of living organisms. In most Duchenne muscular dystrophy (DMD) patients, expression of dystrophin (DYS) protein is disrupted because exon deletions result in a frame shift. We present here the CRISPR-induced deletion (CinDel), a new promising genome-editing technology to correct the DMD gene. This strategy is based on the use of two gRNAs targeting specifically exons that precede and follow the patient deletion in the DMD gene. This pair of gRNAs induced a precise large additional deletion leading to fusion of the targeted exons. Using an adequate pair of gRNAs, the deletion of parts of these exons and the intron separating them restored the DMD reading frame in 62% of the hybrid exons in vitro in DMD myoblasts and in vivo in electroporated hDMD/mdx mice. Moreover, adequate pairs of gRNAs also restored the normal spectrin-like repeat of the dystrophin rod domain; such restoration is not obtained by exon skipping or deletion of complete exons. The expression of an internally deleted DYS protein was detected following the formation of myotubes by the unselected, treated DMD myoblasts. Given that CinDel induces permanent reparation of the DMD gene, this treatment would not have to be repeated as it is the case for exon skipping induced by oligonucleotides.
RESUMO
A new family of cyclic carbamate-estradiol derivatives has been designed to remove the intrinsic estrogenic activity of a parent acyclic compound reported as a potent inhibitor of 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1). The synthesis of two series of fused 16ß,17ß-oxazinone-estradiol derivatives, saturated compounds 7a-d and unsaturated compounds 10a-d, led to the identification of 10b, a 17ß-HSD1 inhibitor (IC50 = 1.4 µM) without estrogenic activity in estrogen-sensitive T-47D cells. Interestingly, this compound was found selective over 17ß-HSD2 and 17ß-HSD12. A computational analysis of inhibitors into 17ß-HSD1 by molecular docking also revealed interesting structure-activity relationships that could be helpful in the design of new generation of 16ß,17ß-oxazinone-estradiol analogs.