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1.
Phys Chem Chem Phys ; 21(45): 25290-25301, 2019 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-31701097

RESUMO

The voltage-gated potassium channel Kv4.3 plays a vital role in shaping the timing, frequency, and backpropagation of electrical signals in the brain and heart by generating fast transient currents at subthreshold membrane potentials in repetitive firing neurons. To achieve its physiological function, Kv4.3 is assisted by auxiliary ß-subunits that become integral parts of the native A-type potassium channels, among which there are the Kv channel-interacting proteins (KChIPs). KChIPs are a family of cytosolic proteins that, when coexpressed with Kv4, lead to higher current density, modulation of channel inactivation and faster recovery from inactivation, while the loss of KChIP function may lead to severe pathological states. Recently, the structural basis of the KChIP1-Kv4.3 interaction was reported by using two similar X-ray crystallographic structures, which supported a crucial role for KChIP1 in enhancing the stability of the Kv4.3 tetrameric assembly, thus helping the trafficking of the channel to the plasma membrane. Here, we investigate through fully atomistic simulations the structure and stability of the human Kv4.3 tetramerization (T1) domain in complex with KChIP1 upon specific mutations located in the first and second interfaces of the complex, as compared to the wild-type (WT). Our results nicely complement the available structural and biophysical information collected so far on these complex variants. In particular, the degree of structural deviations and energetic instability, from small to substantial, observed in these variants with respect to the WT model seems to parallel well the level of channel dysfunction known from electrophysiology data. Our simulations provide an octameric structure of the WT KChIP1-Kv4.3 assembly very similar to the known crystal structures, and, at the same time, highlight the importance of a previously overlooked site of interaction between KChIP1 and the Kv4.3 T1 domain.


Assuntos
Simulação por Computador , Proteínas Interatuantes com Canais de Kv/química , Canais de Potássio Shal/química , Cristalografia por Raios X , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Modelos Moleculares , Mutação , Canais de Potássio Shal/genética
2.
Nucleic Acids Res ; 47(16): 8874-8887, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31616952

RESUMO

Localized arrays of proteins cooperatively assemble onto chromosomes to control DNA activity in many contexts. Binding cooperativity is often mediated by specific protein-protein interactions, but cooperativity through DNA structure is becoming increasingly recognized as an additional mechanism. During the site-specific DNA recombination reaction that excises phage λ from the chromosome, the bacterial DNA architectural protein Fis recruits multiple λ-encoded Xis proteins to the attR recombination site. Here, we report X-ray crystal structures of DNA complexes containing Fis + Xis, which show little, if any, contacts between the two proteins. Comparisons with structures of DNA complexes containing only Fis or Xis, together with mutant protein and DNA binding studies, support a mechanism for cooperative protein binding solely by DNA allostery. Fis binding both molds the minor groove to potentiate insertion of the Xis ß-hairpin wing motif and bends the DNA to facilitate Xis-DNA contacts within the major groove. The Fis-structured minor groove shape that is optimized for Xis binding requires a precisely positioned pyrimidine-purine base-pair step, whose location has been shown to modulate minor groove widths in Fis-bound complexes to different DNA targets.


Assuntos
Bacteriófago lambda/genética , Cromossomos Bacterianos/química , DNA Nucleotidiltransferases/química , DNA Bacteriano/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Fator Proteico para Inversão de Estimulação/química , Proteínas Virais/química , Sítio Alostérico , Bacteriófago lambda/metabolismo , Sequência de Bases , Sítios de Ligação , Cromossomos Bacterianos/metabolismo , Clonagem Molecular , Cristalografia por Raios X , DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator Proteico para Inversão de Estimulação/genética , Fator Proteico para Inversão de Estimulação/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reparo de DNA por Recombinação , Alinhamento de Sequência , Termodinâmica , Proteínas Virais/genética , Proteínas Virais/metabolismo
3.
Protein Pept Lett ; 26(10): 792-797, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31618172

RESUMO

BACKGROUND: Head-to-tail polymerising domains generating heterogeneous aggregates are generally difficult to crystallise. DIX domains, exclusively found in the Wnt signalling pathway, are polymerising factors following this head-to-tail arrangement; moreover, they are considered to play a key role in the heterotypic interaction between Dishevelled (Dvl) and Axin, which are cytoplasmic proteins also positively and negatively regulating the canonical Wnt/ß- catenin signalling pathway, but this interaction mechanism is still unknown. OBJECTIVE: This study mainly aimed to clarify whether the Dvl2 and Axin-DIX domains (Dvl2-DIX and Axin-DIX, respectively) form a helical polymer in a head-to-tail way during complexation. METHODS: Axin-DIX (DAX) and Dvl2-DIX (DIX), carrying polymerisation-blocking mutations, were expressed as a fusion protein by using a flexible peptide linker to fuse the C-terminal of the former to the N-terminal of the latter, enforcing a defined 1:1 stoichiometry between them. RESULTS: The crystal of the DAX-DIX fusion protein diffracted to a resolution of about 0.3 nm and a data set was collected at a 0.309 nm resolution. The structure was solved via the molecular replacement method by using the DIX and DAX structures. A packing analysis of the crystal revealed the formation of a tandem heterodimer in a head-to-tail way, as predicted by the Wntsignalosome model. CONCLUSION: The results demonstrated that the combination of polymerisation-blocking mutations and a fusion protein of two head-to-tail polymerising domains is effective especially for crystallising complexes among heterologous polymerising proteins or domains.


Assuntos
Proteína Axina/química , Proteína Axina/genética , Proteínas Desgrenhadas/química , Domínios Proteicos/genética , Sequência de Aminoácidos , Cristalização , Cristalografia por Raios X/métodos , Proteínas Desgrenhadas/genética , Escherichia coli , Regulação da Expressão Gênica , Humanos , Ligação Proteica , Via de Sinalização Wnt
4.
Chem Commun (Camb) ; 55(86): 12932-12935, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31599282

RESUMO

Here, we report the development of novel PET radiotracer ([11C]CW22) of BET proteins. In vivo imaging results in rodents and nonhuman primates (NHP) demonstrate that [11C]CW22 has excellent brain uptake, good specificity, good selectivity, suitable metabolism, appropriate kinetics and distribution in the brain. Our studies demonstrated that [11C]CW22 exhibits ideal properties as a PET imaging probe of BET proteins for further validation.


Assuntos
Encéfalo/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/química , Animais , Barreira Hematotesticular/metabolismo , Encéfalo/metabolismo , Radioisótopos de Carbono/química , Cristalografia por Raios X , Cinética , Macaca , Camundongos , Conformação Molecular , Proteínas do Tecido Nervoso/química , Neurônios , Domínios Proteicos , Compostos Radiofarmacêuticos/metabolismo , Receptores de Superfície Celular/química
5.
Inorg Chem ; 58(20): 13933-13944, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31566371

RESUMO

Density functional vibrational frequency calculations have been performed on eight geometry optimized cytochrome c oxidase (CcO) dinuclear center (DNC) reaction cycle intermediates and on the oxymyoglobin (oxyMb) active site. The calculated Fe-O and O-O stretching modes and their frequency shifts along the reaction cycle have been compared with the available resonance Raman (rR) measurements. The calculations support the proposal that in state A[Fea33+-O2-•···CuB+] of CcO, O2 binds with Fea32+ in a similar bent end-on geometry to that in oxyMb. The calculations show that the observed 20 cm-1 shift of the Fea3-O stretching mode from the PR to F state is caused by the protonation of the OH- ligand on CuB2+ (PR[Fea34+═O2-···HO--CuB2+] → F[Fea34+═O2-···H2O-CuB2+]), and that the H2O ligand is still on the CuB2+ site in the rR identified F[Fea34+═O2-···H2O-CuB2+] state. Further, the observed rR band at 356 cm-1 between states PR and F is likely an O-Fea3-porphyrin bending mode. The observed 450 cm-1 low Fea3-O frequency mode for the OH active oxidized state has been reproduced by our calculations on a nearly symmetrically bridged Fea33+-OH-CuB2+ structure with a relatively long Fea3-O distance near 2 Å. Based on Badger's rule, the calculated Fea3-O distances correlate well with the calculated νFe-O-2/3 (νFe-O is the Fea3-O stretching frequency) with correlation coefficient R = 0.973.


Assuntos
Teoria da Densidade Funcional , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Ferro/química , Oxigênio/química , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Complexo IV da Cadeia de Transporte de Elétrons/química , Ferro/metabolismo , Ligantes , Modelos Moleculares , Oxigênio/metabolismo , Vibração
6.
Chem Pharm Bull (Tokyo) ; 67(10): 1061-1071, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31582626

RESUMO

The activation of epidermal growth factor receptor (EGFR) involves the geometrical conversion of the extracellular domain (ECD) from the tethered to the extended forms with the dynamic rearrangement of the relative positions of four subdomains (SDs); however, this conversion process has not yet been thoroughly understood. We compare the two different forms of the X-ray crystal structures of ECD and simulate the ECD conversion process using adiabatic mapping that combines normal mode analysis of the elastic network model (ENM-NMA) and energy optimization. A comparison of the crystal structures reveals the rigidity of the intradomain geometry of the SD-I and -III backbone regardless of the form. The forward mapping from the tethered to the extended forms retains the intradomain geometry of the SD-I and -III backbone and reveals the trends to rearrange the relative positions of SD-I and -III and to dissociate the C-terminal tail of SD-IV from the hairpin loop in SD-II. The reverse mapping from the extended to the tethered forms complements the promotion of ECD conversion in the presence of epidermal growth factor (EGF).


Assuntos
Modelos Moleculares , Mapas de Interação de Proteínas , Cristalografia por Raios X , Elasticidade , Receptores ErbB/química , Receptores ErbB/metabolismo , Humanos
7.
Nature ; 574(7779): 581-585, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645725

RESUMO

The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation3-5. Because SUCNR1 senses succinate as an immunological danger signal6-which has relevance for diseases including ulcerative colitis, liver fibrosis7, diabetes and rheumatoid arthritis3,8-it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo.


Assuntos
Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Piperazinas/química , Piperazinas/farmacologia , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/química , Animais , Apoproteínas/antagonistas & inibidores , Apoproteínas/química , Apoproteínas/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ratos , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Purinérgicos P2Y1/química , Transdução de Sinais , Anticorpos de Domínio Único/química , Especificidade da Espécie , Ácido Succínico/metabolismo
8.
Nature ; 574(7779): 565-570, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645726

RESUMO

Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer1,2. Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies3. However, many of the other axes by which tumours become inhospitable to T cells are not fully understood. Here we report that V-domain immunoglobulin suppressor of T cell activation (VISTA) engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments. Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding to the adhesion and co-inhibitory receptor P-selectin glycoprotein ligand-1 (PSGL-1). Antibodies engineered to selectively bind and block this interaction in acidic environments were sufficient to reverse VISTA-mediated immune suppression in vivo. These findings identify a mechanism by which VISTA may engender resistance to anti-tumour immune responses, as well as an unexpectedly determinative role for pH in immune co-receptor engagement.


Assuntos
Antígenos B7/química , Antígenos B7/metabolismo , Glicoproteínas de Membrana/metabolismo , Linfócitos T/metabolismo , Animais , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/farmacologia , Antígenos B7/antagonistas & inibidores , Antígenos B7/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Cristalografia por Raios X , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Feminino , Histidina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Masculino , Glicoproteínas de Membrana/imunologia , Camundongos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Linfócitos T/citologia , Linfócitos T/imunologia , Microambiente Tumoral/imunologia
9.
Chem Commun (Camb) ; 55(90): 13546-13549, 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31647068

RESUMO

Drugs containing amine groups react with CO2 to form crystalline ammonium carbamates or carbamic acids. In this approach, both the cation and anion of the salt, or the neutral CO2 adduct, are derived from the parent drug, generating new crystalline versions in a 'masked' or prodrug form. It is proposed that this approach may serve as a valuable new tool in engineering the physical properties of drugs for formulation purposes.


Assuntos
Aminas/química , Compostos de Amônio/química , Carbamatos/química , Dióxido de Carbono/química , Ânions/química , Cátions/química , Cristalografia por Raios X , Modelos Moleculares , Estrutura Molecular , Sais/química
10.
Inorg Chem ; 58(19): 12843-12853, 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31502453

RESUMO

A series of five compounds TpMesMFla (TpMes = hydrotris(3-mesityl)pyrazolylborate; M = Mn, Fe, Co, Ni, Zn; Fla = 3-hydroxyflavonolate) has been synthesized as models for the 2,4-quercetin dioxygenase, QueD. The structures have been determined and the complexes proved to be isomorphous. Considering the structures more closely revealed that they differ in the degree of delocalization in the chelate ring formed through the binding of the two O donors of the flavonolate to the metal center, which is also supported by the results of UV-vis and IR spectroscopic investigations. The resulting trend (Zn/Fe > Co > Mn > Ni) is, however, not in line with the one that was found investigating the redox properties of the complexes by cyclic voltammetry (Zn > Fe > Ni > Co > Mn). Notably, from CV clear-cut information could be derived, as the complexes exhibited exceptionally well-behaved quasi-reversible redox transitions, indicating that the Tp ligand stabilizes the flavonolate radical formed in the oxidation process rather well. The fact that the rates, with which the complexes react with O2 in DMF solution, correlate with the position of the flavonolate redox couples, suggest that these reactions proceed via the initial electron transfer from the flavonolate to O2. After the O2 reaction, salicylic acid was identified as one of the products, the formation of which can be explained by the hydrolysis of the depside that should form upon a dioxygenation similar to the QueD enzyme-catalyzed reaction. 18O labeling experiments confirmed the presence of O2 derived O atoms. Mechanistic inferences based on the above results are discussed.


Assuntos
Bactérias/enzimologia , Complexos de Coordenação/química , Dioxigenases/química , Flavonóis/química , Pirazóis/química , Bactérias/química , Materiais Biomiméticos/química , Boratos/química , Catálise , Cristalografia por Raios X , Ligantes , Modelos Moleculares
11.
Chem Pharm Bull (Tokyo) ; 67(9): 935-939, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31474732

RESUMO

Chafuroside A and chafuroside B are flavone C-glycosides isolated from oolong tea leaves. They have a number of beneficial pharmacological activities related to antiinflammation at various concentrations. However, no crystallographic study of chafurosides has yet been reported. In the present study, the crystal structures of chafuroside A and chafuroside B were investigated using single-crystal X-ray diffraction. The asymmetric unit of the chafuroside A crystal consists of one chafuroside A and two water molecules, and that of chafuroside B contains one chafuroside B and one water molecule. The flavone moiety of chafuroside A is curved, i.e., the angle between the best-fit planes of the chromene and phenyl rings is 18.9°, whereas the chafuroside B flavone moiety is relatively flat. A comparison of the curvatures of the flavone moieties of various C-glycosides showed that the curvature of chafuroside A is significantly larger than those of the others. This structural feature might contribute to the differences between the strengths of the pharmacological activities of chafurosides A and B.


Assuntos
Flavonas/química , Glicosídeos/química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Chá/química , Camellia sinensis/química , Camellia sinensis/metabolismo , Cristalografia por Raios X , Conformação Molecular , Folhas de Planta/química , Folhas de Planta/metabolismo
12.
Chem Pharm Bull (Tokyo) ; 67(9): 953-958, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31474735

RESUMO

Asymmetric total syntheses of dihydropyran containing natural products, (+)-eurotiumide F and (+)-eurotiumide G have been described. These total syntheses revealed the absolute configuration of eurotiumide F and G, and confirmed the reported structure of eurotiumide F and revised the reported structure of eurotiumide G. Highlight of these syntheses is thermal rearrangement with 4-methoxyisochroman-1-one derivative having propargyl ether on phenolic ether under thermal condition to construct dihydropyran ring. X-Ray crystallographic analysis of (+)-eurotiumide G clarified the stereochemistry at the C1-position.


Assuntos
Produtos Biológicos/química , Piranos/química , Produtos Biológicos/síntese química , Cristalografia por Raios X , Conformação Molecular , Piranos/síntese química , Solventes/química , Estereoisomerismo
13.
J Chem Theory Comput ; 15(10): 5659-5673, 2019 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-31476125

RESUMO

Human heterochromatin protein 1 (HP1) is a key factor in heterochromatin formation and maintenance. Its chromo-shadow domain (CSD) homodimerizes, and the HP1 dimer acts as a hub, transiently interacting with diverse binding partner (BP) proteins. We analyze atomistic details of interactions of the HP1γ(CSD) dimer with one of its targets, the histone H3 N-terminal tail, using molecular dynamics (MD) simulations. The goal is to complement the available X-ray crystallography data and unravel potential dynamic effects in the molecular recognition. Our results suggest that HP1(CSD)-BP recognition involves structural dynamics of both partners, including structural communication between adjacent binding pockets that may fine-tune the sequence recognition. For example, HP1 Trp174 sidechain substates may help in distinguishing residues bound in the conserved HP1(CSD) ±2 hydrophobic pockets. Further, there is intricate competition between the binding of negatively charged HP1 C-terminal extension and solvent anions near the ±2 hydrophobic pockets, which is also influenced by the BP sequence. Phosphorylated H3 Y41 can interact with the same site. We also analyze the ability of several pair-additive force fields to describe the protein-protein interface. ff14SB and ff99SB-ILDN* provide the closest correspondence with the crystallographic model. The ff15ipq local dynamics are somewhat less consistent with details of the experimental structure, while larger perturbations of the interface commonly occur in CHARMM36m simulations. The balance of some interactions, mainly around the anion binding site, also depends on the ion parameters. Some differences between the simulated and experimental structures are attributable to crystal packing.


Assuntos
Proteínas Cromossômicas não Histona/química , Histonas/química , Cristalografia por Raios X , Dimerização , Histonas/metabolismo , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica
14.
Adv Exp Med Biol ; 1148: 81-103, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482495

RESUMO

Enzymes are key biological macromolecules that support life by accelerating the conversion of target molecules to desired products in many biochemical reactions. Enzymes are characterized by high affinity, specificity and great catalytic efficiency. Owing to their unique characteristics, enzymes have attracted significant attention for use in therapeutic settings as a distinct class of drugs different from other types of medicines. Enzyme-based therapies are currently in use for the treatment of a wide range of diseases, including leukemia, metabolic disorders, inflammation and cardiovascular disease. However, several challenges, such as immunogenicity and stability, remain. X-ray crystallography has provided key structural insights into the understanding of the molecular basis of diseases and development of enzyme-based therapies. Here, the role of X-ray crystallography in the development of therapeutic enzymes is examined and several examples are provided.


Assuntos
Cristalografia por Raios X , Enzimas/química , Enzimas/farmacologia , Relação Estrutura-Atividade
15.
Acta Crystallogr C Struct Chem ; 75(Pt 9): 1250-1258, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31484813

RESUMO

A drug-drug anhydrous pharmaceutical salt containing tolbutamide {systematic name: 3-butyl-1-[(4-methylbenzene)sulfonyl]urea, TOL, C12H18N2O3S} and metformin (systematic name: 1-carbamimidamido-N,N-dimethylmethanimidamide, MET, C4H11N5) was created based on antidiabetic drug combinations to overcome the poor pharmaceutical properties of the parent drugs. Proton transfer and the proportion of the two components were confirmed by 1H NMR spectroscopy and single-crystal X-ray diffraction analysis. Comprehensive characterization of the new pharmaceutical salt crystal, 2-[(dimethylamino)(iminiumyl)methyl]guanidine (butylcarbamoyl)[(4-methylbenzene)sulfonyl]azanide, C4H12N5+·C12H17N2O3S-, was performed and showed enhancement of the pharmaceutical properties, such as lower hygroscopicity and greater accelerated stability than the parent drug MET, and higher solubility and dissolution rate than TOL. The property alterations were correlated with the crystal packing features and potential hydrogen-bonding sites through observed changes in the crystal structures.


Assuntos
Hipoglicemiantes/farmacologia , Metformina/farmacologia , Tolbutamida/farmacologia , Cristalografia por Raios X , Combinação de Medicamentos , Ligações de Hidrogênio , Hipoglicemiantes/síntese química , Hipoglicemiantes/química , Metformina/síntese química , Metformina/química , Estrutura Molecular , Solubilidade , Tolbutamida/síntese química , Tolbutamida/química
16.
Inorg Chem ; 58(19): 13150-13160, 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31539237

RESUMO

Mitochondrial DNA (mtDNA) is an attractive cellular target for anticancer agents in addition to nuclear DNA (nDNA). The cationic platinum(II) complex cis-[Pt(NP)(NH3)2Cl]NO3 (PtNP, NP = N-(2-ethylpyridine)-1,8-naphthalimide) bearing the DNA-intercalating moiety NP was designed. The structure of PtNP was fully characterized by single-crystal X-ray crystallography, NMR, and HRMS. PtNP is superior to cisplatin in both in vitro and in vivo anticancer activities with low systemic toxicity. The interaction of PtNP with CT-DNA demonstrated that PtNP could effectively bind to DNA through both covalent and noncovalent double binding modes. In addition to causing significant damage to nDNA and remarkable inhibition to DNA damage repair, PtNP also distributed in mitochondria, inducing mtDNA damage and affecting the downstream transcriptional level of mitochondrion-encoded genes. In addition, PtNP disturbed the physiological processes of mitochondria by reducing the mitochondrial membrane potential and promoting the generation of reactive oxygen species. Mechanistic studies demonstrate that PtNP induced apoptosis via mitochondrial pathways by upregulating Bax and Puma and downregulating Bcl-2 proteins, leading to the release of cytochrome c and activation of caspase-3 and caspase-9. As a dual-DNA-damage agent, PtNP is able to improve the anticancer activity by damaging both nuclear and mitochondrial DNA, thus providing a new anticancer mechanism of action for the naphthalimide monofunctional platinum(II) complexes.


Assuntos
Antineoplásicos/farmacologia , Dano ao DNA/efeitos dos fármacos , Substâncias Intercalantes/farmacologia , Compostos Organoplatínicos/farmacologia , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Substâncias Intercalantes/química , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos ICR , Modelos Moleculares , Naftalimidas/química , Naftalimidas/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/genética , Compostos Organoplatínicos/química , Piridinas/química , Piridinas/farmacologia
17.
Inorg Chem ; 58(21): 14522-14531, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31550141

RESUMO

Herein we report the synthesis, characterization, and cellular internalization properties of two visible-light active luminescent Mn-based photoCORMs. The enhanced membrane permeability of the photoactive Mn carbonyl complex (photoCORM) derived from a designed lipophilic ligand namely, [Mn(CO)3(Imdansyl)(L1)](CF3SO3) (1) (where L1 = a diazabutadiene-based ligand containing two highly lipophilic adamantyl motifs, Imdansyl = dansylimidazole) promoted rapid internalization within human colorectal adenocarcinoma (HT-29) cells compared to [Mn(CO)3(Imdansyl)(L2)](CF3SO3) (2) (where L2 = a diazabutadiene ligand bearing two hydrophilic 1,3,5-triazaadamantyl group). Colocalization experiments using membrane stain indicate different extents of localization of the two CO complexes within the cellular matrix. Visible-light triggered CO release from the lipophilic photoCORM induced caspase-3/7 activation on HT-29 cells, which was detected using confocal microscopy. The rapid accumulation of the lipophilic photoCORM 1 in the cellular membrane resulted in more efficient CO-induced cell death compared to the hydrophilic analogue 2.


Assuntos
Complexos de Coordenação/farmacologia , Luz , Substâncias Luminescentes/farmacologia , Morte Celular/efeitos dos fármacos , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Cristalografia por Raios X , Células HT29 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Substâncias Luminescentes/síntese química , Substâncias Luminescentes/química , Modelos Moleculares , Neoplasias/tratamento farmacológico , Solubilidade
18.
Inorg Chem ; 58(19): 12954-12963, 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31550148

RESUMO

The luminescent chalcone gold(I) conjugates [Au(PPh3)(AN3E)]PF6(1) and [Au(SIMes)(AN3E)]PF6 (2) (AN3E = (E)-3-(9-anthracenyl)-1-(4-pyridyl)propenone; SIMes = N,N'-dimesitylimidazolidin-2-ylidene; Mes = 2,4,6-trimethylphenyl)) were prepared and characterized; complex 1 was also characterized by X-ray crystallography. In MTT assays against a panel of three human colon, a melanoma and a breast cancer cell lines both complexes were antiproliferative with low micromolar IC50 values. It is noteworthy that HCT116p53-/- colon carcinoma cells lacking functional p53 (a vital tumor suppressor) were more susceptible to them than the wildtype parent cell line. In flow cytometry analyses, the gold conjugates induced a significant arrest in G2/M phase primarily. Complexes 1 and 2 quickly increased the production of reactive oxygen species (ROS) and induced mitochondrial membrane potential depolarization, higher ROS values being obtained after coadministration with enzymatic inhibitors. The free chalcone AN3E and its gold(I) complex conjugates located in the cell mitochondria according to confocal microscopy. In addition, complexes 1 and 2 showed in vivo antivascular effects on the chorioallantoic membrane (CAM) of fertilized specific-pathogen-free (SPF) chicken eggs.


Assuntos
Inibidores da Angiogênese/farmacologia , Antracenos/farmacologia , Antineoplásicos/farmacologia , Chalcona/farmacologia , Neoplasias do Colo/tratamento farmacológico , Compostos Organoáuricos/farmacologia , Inibidores da Angiogênese/química , Animais , Antracenos/química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Chalcona/análogos & derivados , Galinhas , Cristalografia por Raios X , Células HCT116 , Humanos , Modelos Moleculares , Compostos Organoáuricos/química
19.
Nature ; 574(7776): 132-136, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31554965

RESUMO

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics1. They are widely present in bacteria, archaea, algae and algal viruses2,3. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the ß-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.


Assuntos
Rodopsinas Microbianas/química , Thermoplasmales/química , Bacteriorodopsinas/química , Sítios de Ligação , Cristalografia por Raios X , Microscopia de Força Atômica , Modelos Moleculares , Dobramento de Proteína , Multimerização Proteica , Retinaldeído/química , Rodopsinas Microbianas/ultraestrutura
20.
Plant Mol Biol ; 101(4-5): 455-469, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31541388

RESUMO

Regulation of abscisic acid (ABA) signaling is crucial in balancing responses to abiotic stresses and retaining growth in planta. An ABA receptor (PYL/RCAR) and a protein phosphatase (PP2C), a co-receptor, form a complex upon binding to ABA. Previously we reported that the second and fourth positions in the VxGΦL motif of PP2Cs from Oryza sativa are critical in the interaction of PP2Cs with PYL/RCARs. Considering substantial effects of the VxGΦL motif on ABA signaling outputs, further comprehensive characterization of residues in the second and fourth positions are required. Here we surveyed the second and fourth positions of the VxGΦL motif by combination of biochemical, structural and physiological analyses. We found that the fourth position of the VxGΦL motif, highly conserved to small hydrophobic residues, was a key determinant of the OsPP2C50:OsPYL/RCAR interactions across subfamilies. Large hydrophobic or any hydrophilic residues in the fourth position abrogated ABA responsiveness. Analysis of crystal structures of OsPP2C50 mutants, S265L/I267V ("LV"), I267L ("SL") and I267W ("SW"), in complex with ABA and OsPYL/RCAR3, along with energy calculation of the complexes, uncovered that a bulky hydrophobic residue in the fourth position of the VxGΦL motif pushed away side chains of nearby residues, conferring side-chain rotameric energy stress. Hydrophilic residues in this position imposed solvation energy stress to the PP2C:PYL/RCAR complex. Germination and gene expression analyses corroborated that OsPP2C50 AS and AK mutants modulated ABA responsiveness in Arabidopsis. Our results suggest that ABA responsiveness could be fine-tuned by the fourth position of the VxGΦL motif on PP2Cs. KEY MESSAGE: We comprehensively surveyed the VxGΦL motif to find that the fourth position, highly conserved to small hydrophobic residues, was critical in regulating ABA responsiveness.


Assuntos
Motivos de Aminoácidos , Oryza/fisiologia , Fosfoproteínas Fosfatases/química , Proteínas de Plantas/química , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Arabidopsis/genética , Clonagem Molecular , Cristalografia por Raios X , Oryza/genética , Oryza/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Reguladores de Crescimento de Planta/farmacologia , Plantas Geneticamente Modificadas/metabolismo , Transdução de Sinais
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