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1.
Nat Chem Biol ; 15(11): 1085-1092, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31451761

RESUMO

Sensory photoreceptor proteins underpin light-dependent adaptations in nature and enable the optogenetic control of organismal behavior and physiology. We identified the bacterial light-oxygen-voltage (LOV) photoreceptor PAL that sequence-specifically binds short RNA stem loops with around 20 nM affinity in blue light and weaker than 1 µM in darkness. A crystal structure rationalizes the unusual receptor architecture of PAL with C-terminal LOV photosensor and N-terminal effector units. The light-activated PAL-RNA interaction can be harnessed to regulate gene expression at the RNA level as a function of light in both bacteria and mammalian cells. The present results elucidate a new signal-transduction paradigm in LOV receptors and conjoin RNA biology with optogenetic regulation, thereby paving the way toward hitherto inaccessible optoribogenetic modalities.


Assuntos
Luz , Biossíntese de Proteínas , RNA/metabolismo , Proteínas de Bactérias/metabolismo , Ligação Proteica , Transdução de Sinais
2.
J Struct Biol ; 211(2): 107534, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32454240

RESUMO

In many organisms, the ubiquitous second messenger cAMP is formed by at least one member of the adenylyl cyclase (AC) Class III. These ACs feature a conserved dimeric catalytic core architecture, either through homodimerization or through pseudo-heterodimerization of a tandem of two homologous catalytic domains, C1 and C2, on a single protein chain. The symmetric core features two active sites, but in the C1-C2 tandem one site degenerated into a regulatory center. Analyzing bacterial AC sequences, we identified a Pseudomonas aeruginosa AC-like protein (PaAClp) that shows a surprising swap of the catalytic domains, resulting in an unusual C2-C1 arrangement. We cloned and recombinantly produced PaAClp. The protein bound nucleotides but showed no AC or guanylyl cyclase activity, even in presence of a variety of stimulating ligands of other ACs. Solving the crystal structure of PaAClp revealed an overall structure resembling active class III ACs but pronounced shifts of essential catalytic residues and structural elements. The structure contains a tightly bound ATP, but in a binding mode not suitable for cAMP formation or ATP hydrolysis, suggesting that PaAClp acts as an ATP-binding protein.


Assuntos
Adenilil Ciclases/ultraestrutura , Proteínas de Bactérias/ultraestrutura , Proteínas de Transporte/ultraestrutura , Pseudomonas aeruginosa/ultraestrutura , Trifosfato de Adenosina/genética , Adenilil Ciclases/genética , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Domínio Catalítico/genética , Cristalografia por Raios X , AMP Cíclico/genética , Cinética , Ligantes , Modelos Moleculares , Pseudomonas aeruginosa/enzimologia
3.
PLoS Biol ; 15(3): e2000374, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28257421

RESUMO

Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with age-associated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanisms involved remain unclear. Here, we provide mechanistic insight into the interplay between sirtuin 2 and α-synuclein, the major component of the pathognomonic protein inclusions in PD and other synucleinopathies. We found that α-synuclein is acetylated on lysines 6 and 10 and that these residues are deacetylated by sirtuin 2. Genetic manipulation of sirtuin 2 levels in vitro and in vivo modulates the levels of α-synuclein acetylation, its aggregation, and autophagy. Strikingly, mutants blocking acetylation exacerbate α-synuclein toxicity in vivo, in the substantia nigra of rats. Our study identifies α-synuclein acetylation as a key regulatory mechanism governing α-synuclein aggregation and toxicity, demonstrating the potential therapeutic value of sirtuin 2 inhibition in synucleinopathies.


Assuntos
Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Sirtuína 2/metabolismo , alfa-Sinucleína/toxicidade , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Acetilação/efeitos dos fármacos , Animais , Autofagia/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Córtex Cerebral/patologia , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Deleção de Genes , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Lisina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Neuroproteção/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , Ligação Proteica
5.
Proc Natl Acad Sci U S A ; 114(23): E4676-E4685, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28533375

RESUMO

The activity of the transcription factor nuclear factor-erythroid 2 p45-derived factor 2 (NRF2) is orchestrated and amplified through enhanced transcription of antioxidant and antiinflammatory target genes. The present study has characterized a triazole-containing inducer of NRF2 and elucidated the mechanism by which this molecule activates NRF2 signaling. In a highly selective manner, the compound covalently modifies a critical stress-sensor cysteine (C151) of the E3 ligase substrate adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), the primary negative regulator of NRF2. We further used this inducer to probe the functional consequences of selective activation of NRF2 signaling in Huntington's disease (HD) mouse and human model systems. Surprisingly, we discovered a muted NRF2 activation response in human HD neural stem cells, which was restored by genetic correction of the disease-causing mutation. In contrast, selective activation of NRF2 signaling potently repressed the release of the proinflammatory cytokine IL-6 in primary mouse HD and WT microglia and astrocytes. Moreover, in primary monocytes from HD patients and healthy subjects, NRF2 induction repressed expression of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα. Together, our results demonstrate a multifaceted protective potential of NRF2 signaling in key cell types relevant to HD pathology.


Assuntos
Doença de Huntington/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Adulto , Idoso , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Doença de Huntington/genética , Proteína 1 Associada a ECH Semelhante a Kelch/química , Intoxicação por MPTP/metabolismo , Intoxicação por MPTP/prevenção & controle , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Pessoa de Meia-Idade , Fator 2 Relacionado a NF-E2/química , Células-Tronco Neurais/metabolismo , Fármacos Neuroprotetores/farmacologia , Conformação Proteica/efeitos dos fármacos , Ratos , Transdução de Sinais
6.
Proc Natl Acad Sci U S A ; 111(10): 3727-32, 2014 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-24567411

RESUMO

cAMP is an evolutionary conserved, prototypic second messenger regulating numerous cellular functions. In mammals, cAMP is synthesized by one of 10 homologous adenylyl cyclases (ACs): nine transmembrane enzymes and one soluble AC (sAC). Among these, only sAC is directly activated by bicarbonate (HCO3(-)); it thereby serves as a cellular sensor for HCO3(-), carbon dioxide (CO2), and pH in physiological functions, such as sperm activation, aqueous humor formation, and metabolic regulation. Here, we describe crystal structures of human sAC catalytic domains in the apo state and in complex with substrate analog, products, and regulators. The activator HCO3(-) binds adjacent to Arg176, which acts as a switch that enables formation of the catalytic cation sites. An anionic inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, inhibits sAC through binding to the active site entrance, which blocks HCO3(-) activation through steric hindrance and trapping of the Arg176 side chain. Finally, product complexes reveal small, local rearrangements that facilitate catalysis. Our results provide a molecular mechanism for sAC catalysis and cellular HCO3(-) sensing and a basis for targeting this system with drugs.


Assuntos
Adenilil Ciclases/química , Ativação Enzimática/fisiologia , Modelos Moleculares , Conformação Proteica , Transdução de Sinais/genética , Bicarbonato de Sódio/metabolismo , Catálise , Clonagem Molecular , Cristalização , Ativação Enzimática/genética , Humanos , Ligação Proteica
7.
J Struct Biol ; 182(2): 136-43, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23454361

RESUMO

Sirtuins are NAD(+)-dependent protein deacetylases that regulate metabolism and aging-related processes. Sirt2 is the only cytoplasmic isoform among the seven mamalian Sirtuins (Sirt1-7) and structural information concerning this isoform is limited. We crystallized Sirt2 in complex with a product analog, ADP-ribose, and solved this first crystal structure of a Sirt2 ligand complex at 2.3Å resolution. Additionally, we re-refined the structure of the Sirt2 apoform and analyzed the conformational changes associated with ligand binding to derive insights into the dynamics of the enzyme. Our analyses also provide information on Sirt2 peptide substrate binding and structural states of a Sirt2-specific protein region, and our insights and the novel Sirt2 crystal form provide helpful tools for the development of Sirt2 specific inhibitors.


Assuntos
Adenosina Difosfato Ribose/química , Modelos Moleculares , Conformação Proteica , Sirtuína 2/química , Cristalização , Humanos
8.
J Biol Chem ; 287(17): 13656-65, 2012 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-22343627

RESUMO

Sialic acids are essential components of membrane glycoconjugates. They are responsible for the interaction, structure, and functionality of all deuterostome cells and have major functions in cellular processes in health and diseases. The key enzyme of the biosynthesis of sialic acid is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase that transforms UDP-N-acetylglucosamine to N-acetylmannosamine (ManNAc) followed by its phosphorylation to ManNAc 6-phosphate and has a direct impact on the sialylation of cell surface components. Here, we present the crystal structures of the human N-acetylmannosamine kinase (MNK) domain of UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase in complexes with ManNAc at 1.64 Å resolution, MNK·ManNAc·ADP (1.82 Å) and MNK·ManNAc 6-phosphate · ADP (2.10 Å). Our findings offer detailed insights in the active center of MNK and serve as a structural basis to design inhibitors. We synthesized a novel inhibitor, 6-O-acetyl-ManNAc, which is more potent than those previously tested. Specific inhibitors of sialic acid biosynthesis may serve to further study biological functions of sialic acid.


Assuntos
Hexosaminas/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Ácido Aspártico/química , Sítios de Ligação , Membrana Celular/metabolismo , Cristalografia por Raios X/métodos , Dimerização , Inibidores Enzimáticos/química , Escherichia coli/metabolismo , Glicoconjugados/química , Glicoproteínas/química , Humanos , Ácido N-Acetilneuramínico/química , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Zinco/química
9.
ACS Sens ; 8(11): 4014-4019, 2023 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-37856082

RESUMO

We report here the development of two different sensing strategies based on the use of antigen-conjugated nucleic acid strands for the detection of a bispecific antibody against the tumor-related proteins Mucin1 and epidermal growth factor receptor. Both approaches work well in serum samples (nanomolar sensitivity), show high specificity against the two monospecific antibodies, and are rapid. The results presented here demonstrate the versatility of DNA-based platforms for the detection of bispecific antibodies and could represent a versatile alternative to other more reagent-intensive and time-consuming analytical approaches.


Assuntos
Anticorpos Biespecíficos , Anticorpos Biespecíficos/metabolismo
10.
Artigo em Inglês | MEDLINE | ID: mdl-22949189

RESUMO

The opportunistic bacterial pathogen Pseudomonas aeruginosa employs three transcriptional regulators, LasR, RhlR and PqsR, to control the transcription of a large subset of its genes in a cell-density-dependent process known as quorum sensing. Here, the recombinant production, crystallization and structure solution of the ligand-binding domain of PqsR (MvfR), the LysR-type transcription factor that responds to the Pseudomonas quinolone signal (PQS), a quinolone-based quorum-sensing signal that is unique to P. aeruginosa and possibly a small number of other bacteria, is reported. PqsR regulates the expression of many virulence genes and may therefore be an interesting drug target. The ligand-binding domain (residues 91-319) was produced as a fusion with SUMO, and hexagonal-shaped crystals of purified PqsR_91-319 were obtained using the vapour-diffusion method. Crystallization in the presence of a PQS precursor allowed data collection to 3.25 Å resolution on a synchrotron beamline, and initial phases have been obtained using single-wavelength anomalous diffraction data from seleno-L-methionine-labelled crystals, revealing the space group to be P6(5)22, with unit-cell parameters a = b = 116-120, c = 115-117 Å.


Assuntos
Proteínas de Bactérias/química , Pseudomonas aeruginosa/química , Fatores de Transcrição/química , Cristalização , Ligantes , Modelos Moleculares , Estrutura Terciária de Proteína , Percepção de Quorum
11.
J Am Chem Soc ; 131(22): 7879-86, 2009 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-19445459

RESUMO

PfluDING is a bacterial protein isolated from Pseudomonas fluorescens that belongs to the DING protein family, which is ubiquitous in eukaryotes and extends to prokaryotes. DING proteins and PfluDING have very similar topologies to phosphate Solute Binding Proteins (SBPs). The three-dimensional structure of PfluDING was obtained at subangstrom resolution (0.88 and 0.98 A) at two different pH's (4.5 and 8.5), allowing us to discuss the hydrogen bond network that sequesters the phosphate ion in the binding site. From this high resolution data, we experimentally elucidated the molecular basis of phosphate binding in phosphate SBPs. The phosphate ion is tightly bound to the protein via 12 hydrogen bonds between phosphate oxygen atoms and OH and NH groups of the protein. The proton on one oxygen atom of the phosphate dianion forms a 2.5 A low barrier hydrogen bond with an aspartate, with the energy released by forming this strong bond ensuring the specificity for the dianion even at pH 4.5. In particular, contrary to previous theories on phosphate SBPs, accurate electrostatic potential calculations show that the binding cleft is positively charged. PfluDING structures reveal that only dibasic phosphate binds to the protein at both acidic and basic phosphate, suggesting that the protein binding site environment stabilizes the HPO(4)(2-) form of phosphate.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a Fosfato/química , Fosfatos/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Proteínas de Ligação a Fosfato/metabolismo , Fosfatos/metabolismo , Ligação Proteica , Pseudomonas fluorescens/química , Pseudomonas fluorescens/metabolismo , Eletricidade Estática
12.
ChemistryOpen ; 7(11): 858-864, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30397576

RESUMO

Natural and synthetic electrophilic compounds have been shown to activate the antioxidant protective Nrf2 (nuclear factor erythroid 2-related factor 2)/heme oxygenase-1 (HO-1) axis in cells and tissues. Here, we tested the ability of different isoxazoline-based electrophiles to up-regulate Nrf2/HO-1. The potency of activation is dependent on the leaving group at the 3-position of the isoxazoline nucleus, and an additional ring on the molecule limits the Nrf2/HO-1 activating properties. Among the synthetized compounds, we identified 3-bromo-5-phenyl-4,5-dihydroisoxazole 1 as the derivative with best activating properties in THP-1 human monocytic cells. We have confirmed that the target of our compounds is the Cys151 of the BTB domain of Keap1 by using mass spectrometry analyses and X-ray crystallography. Our findings demonstrate that these compounds affect the Nrf2/HO-1 axis and highlight a positive activity that can be of relevance from a therapeutic perspective in inflammation and infection.

13.
FEBS Lett ; 581(18): 3455-60, 2007 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-17612529

RESUMO

A recombinant DING protein from Pseudomonas fluorescens has been previously shown to have a phosphate-binding site, and to be mitogenic for human cells. Here we report the three-dimensional structure of the protein, confirming a close similarity to the "Venus flytrap" structure seen in other human and bacterial phosphate-binding proteins. Site-directed mutagenesis confirms the role of a key residue involved in phosphate binding, and that the mitogenic activity is not dependent on this property. Deletion of one of the two hinged domains that constitute the Venus flytrap also eliminates phosphate binding whilst enhancing mitogenic activity.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Células Cultivadas , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Fosfatos/química , Fosfatos/metabolismo , Dobramento de Proteína , Estrutura Terciária de Proteína , Pseudomonas fluorescens/química , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína
14.
Artigo em Inglês | MEDLINE | ID: mdl-17620718

RESUMO

PfluDING is a phosphate-binding protein expressed in Pseudomonas fluorescens. This protein is clearly distinct from the bacterial ABC transporter soluble phosphate-binding protein PstS and is more homologous to eukaryotic DING proteins. Interestingly, bacterial DING proteins have only been detected in certain Pseudomonas species. Although DING proteins seem to be ubiquitous in eukaryotes, they are systematically absent from eukaryotic genomic databases and thus are still quite mysterious and poorly characterized. PfluDING displays mitogenic activity towards human cells and binds various ligands such as inorganic phosphate, pyrophosphate, nucleotide triphosphates and cotinine. Here, the crystallization of PfluDING is reported in a monoclinic space group (P2(1)), with typical unit-cell parameters a = 36.7, b = 123.7, c = 40.8 A, alpha = 90, beta = 116.7, gamma = 90 degrees. Preliminary crystallographic analysis reveals good diffraction quality for these crystals and a 1.43 A resolution data set has been collected.


Assuntos
Proteínas de Bactérias/química , Proteínas de Escherichia coli/química , Pseudomonas fluorescens/química , Proteínas de Bactérias/isolamento & purificação , Cristalização , Cristalografia por Raios X , Proteínas de Escherichia coli/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Difração de Raios X
15.
J Med Chem ; 60(5): 1928-1945, 2017 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-28135086

RESUMO

Sirtuins (SIRTs) are NAD-dependent deacylases, known to be involved in a variety of pathophysiological processes and thus remain promising therapeutic targets for further validation. Previously, we reported a novel thienopyrimidinone SIRT2 inhibitor with good potency and excellent selectivity for SIRT2. Herein, we report an extensive SAR study of this chemical series and identify the key pharmacophoric elements and physiochemical properties that underpin the excellent activity observed. New analogues have been identified with submicromolar SIRT2 inhibtory activity and good to excellent SIRT2 subtype-selectivity. Importantly, we report a cocrystal structure of one of our compounds (29c) bound to SIRT2. This reveals our series to induce the formation of a previously reported selectivity pocket but to bind in an inverted fashion to what might be intuitively expected. We believe these findings will contribute significantly to an understanding of the mechanism of action of SIRT2 inhibitors and to the identification of refined, second generation inhibitors.


Assuntos
Sirtuína 2/antagonistas & inibidores , Tienopiridinas/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Ligantes , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Tienopiridinas/química
16.
J Med Chem ; 60(6): 2344-2360, 2017 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-28240897

RESUMO

Sirt2 is a target for the treatment of neurological, metabolic, and age-related diseases including cancer. Here we report a series of Sirt2 inhibitors based on the 1,2,4-oxadiazole scaffold. These compounds are potent Sirt2 inhibitors active at single-digit µM level by using the Sirt2 substrate α-tubulin-acetylLys40 peptide and inactive up to 100 µM against Sirt1, -3, and -5 (deacetylase and desuccinylase activities). Their mechanism of inhibition is uncompetitive toward both the peptide substrate and NAD+, and the crystal structure of a 1,2,4-oxadiazole analog in complex with Sirt2 and ADP-ribose reveals its orientation in a still unexplored subcavity useful for further inhibitor development. Tested in leukemia cell lines, 35 and 39 induced apoptosis and/or showed antiproliferative effects at 10 or 25 µM after 48 h. Western blot analyses confirmed the involvement of Sirt2 inhibition for their effects in NB4 and in U937 cells. Our results provide novel Sirt2 inhibitors with a compact scaffold and structural insights for further inhibitor improvement.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Oxidiazóis/química , Oxidiazóis/farmacologia , Sirtuína 2/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Sirtuína 2/química , Sirtuína 2/metabolismo , Relação Estrutura-Atividade
17.
Science ; 355(6331): 1312-1317, 2017 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-28336669

RESUMO

DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD+ (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD+ to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD+ concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD+ Thus, NAD+ directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Envelhecimento/metabolismo , Reparo do DNA , NAD/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Envelhecimento/genética , Animais , Sequência Conservada , Dano ao DNA/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Paraquat/farmacologia , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/genética , Domínios e Motivos de Interação entre Proteínas , RNA Interferente Pequeno/genética , Tolerância a Radiação/genética , Homologia de Sequência do Ácido Nucleico
18.
J Med Chem ; 59(4): 1471-91, 2016 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-26689352

RESUMO

Modulators of sirtuins are considered promising therapeutic targets for the treatment of cancer, cardiovascular, metabolic, inflammatory, and neurodegenerative diseases. Here we prepared new 1,4-dihydropyridines (DHPs) bearing changes at the C2/C6, C3/C5, C4, or N1 position. Tested with the SIRTainty procedure, some of them displayed increased SIRT1 activation with respect to the prototype 3a, high NO release in HaCat cells, and ameliorated skin repair in a mouse model of wound healing. In C2C12 myoblasts, two of them improved mitochondrial density and functions. All the effects were reverted by coadministration of compound C (9), an AMPK inhibitor, or of EX-527 (10), a SIRT1 inhibitor, highlighting the involvement of the SIRT1/AMPK pathway in the action of DHPs. Finally, tested in a panel of cancer cells, the water-soluble form of 3a, compound 8, displayed antiproliferative effects in the range of 8-35 µM and increased H4K16 deacetylation, suggesting a possible role for SIRT1 activators in cancer therapy.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Antineoplásicos/farmacologia , Di-Hidropiridinas/farmacologia , Mitocôndrias/efeitos dos fármacos , Sirtuína 1/metabolismo , Pele/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Animais , Antineoplásicos/química , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Di-Hidropiridinas/química , Ativação Enzimática/efeitos dos fármacos , Humanos , Masculino , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais/efeitos dos fármacos , Pele/metabolismo , Pele/patologia
19.
Cell Chem Biol ; 23(7): 849-861, 2016 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-27427231

RESUMO

There are currently no disease-modifying therapies for the neurodegenerative disorder Huntington's disease (HD). This study identified novel thiazole-containing inhibitors of the deacetylase sirtuin-2 (SIRT2) with neuroprotective activity in ex vivo brain slice and Drosophila models of HD. A systems biology approach revealed an additional SIRT2-independent property of the lead-compound, MIND4, as an inducer of cytoprotective NRF2 (nuclear factor-erythroid 2 p45-derived factor 2) activity. Structure-activity relationship studies further identified a potent NRF2 activator (MIND4-17) lacking SIRT2 inhibitory activity. MIND compounds induced NRF2 activation responses in neuronal and non-neuronal cells and reduced production of reactive oxygen species and nitrogen intermediates. These drug-like thiazole-containing compounds represent an exciting opportunity for development of multi-targeted agents with potentially synergistic therapeutic benefits in HD and related disorders.


Assuntos
Modelos Animais de Doenças , Doença de Huntington/tratamento farmacológico , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fármacos Neuroprotetores/farmacologia , Sirtuína 2/antagonistas & inibidores , Tiazóis/farmacologia , Tiazóis/uso terapêutico , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Drosophila , Doença de Huntington/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/uso terapêutico , Ratos , Sirtuína 2/deficiência , Sirtuína 2/metabolismo , Relação Estrutura-Atividade , Tiazóis/química
20.
ChemMedChem ; 10(1): 69-82, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25395356

RESUMO

Sirtuins, NAD(+) -dependent histone deacetylases (HDACs), have recently emerged as potential therapeutic targets for the treatment of a variety of diseases. The discovery of potent and isoform-selective inhibitors of this enzyme family should provide chemical tools to help determine the roles of these targets and validate their therapeutic value. Herein, we report the discovery of a novel class of highly selective SIRT2 inhibitors, identified by pharmacophore screening. We report the identification and validation of 3-((2-methoxynaphthalen-1-yl)methyl)-7-((pyridin-3-ylmethyl)amino)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (ICL-SIRT078), a substrate-competitive SIRT2 inhibitor with a Ki value of 0.62 ± 0.15 µM and more than 50-fold selectivity against SIRT1, 3 and 5. Treatment of MCF-7 breast cancer cells with ICL-SIRT078 results in hyperacetylation of α-tubulin, an established SIRT2 biomarker, at doses comparable with the biochemical IC50 data, while suppressing MCF-7 proliferation at higher concentrations. In concordance with the recent reports that suggest SIRT2 inhibition is a potential strategy for the treatment of Parkinson's disease, we find that compound ICL-SIRT078 has a significant neuroprotective effect in a lactacystin-induced model of Parkinsonian neuronal cell death in the N27 cell line. These results encourage further investigation into the effects of ICL-SIRT078, or an optimised derivative thereof, as a candidate neuroprotective agent in in vivo models of Parkinson's disease.


Assuntos
Inibidores de Histona Desacetilases/química , Fármacos Neuroprotetores/química , Pirimidinonas/química , Sirtuína 2/antagonistas & inibidores , Tiofenos/química , Animais , Sítios de Ligação , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Avaliação Pré-Clínica de Medicamentos , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Células MCF-7 , Simulação de Acoplamento Molecular , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Ligação Proteica , Estrutura Terciária de Proteína , Pirimidinonas/farmacologia , Pirimidinonas/uso terapêutico , Ratos , Sirtuína 2/metabolismo , Relação Estrutura-Atividade , Tiofenos/farmacologia , Tiofenos/uso terapêutico
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