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1.
Genes Dev ; 38(11-12): 528-535, 2024 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-38960718

RESUMO

As part of the efforts to understand nuclear IκB function in NF-κB-dependent gene expression, we report an X-ray crystal structure of the IκBζ ankyrin repeat domain in complex with the dimerization domain of the NF-κB p50 homodimer. IκBζ possesses an N-terminal α helix that conveys domain folding stability. Affinity and specificity of the complex depend on a small portion of p50 at the nuclear localization signal. The model suggests that only one p50 subunit supports binding with IκBζ, and biochemical experiments confirm that IκBζ associates with DNA-bound NF-κB p50:RelA heterodimers. Comparisons of IκBζ:p50 and p50:κB DNA complex crystallographic models indicate that structural rearrangement is necessary for ternary complex formation of IκBζ and p50 with DNA.


Assuntos
Modelos Moleculares , Subunidade p50 de NF-kappa B , Ligação Proteica , Multimerização Proteica , Subunidade p50 de NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B/química , Subunidade p50 de NF-kappa B/genética , Humanos , Cristalografia por Raios X , DNA/metabolismo , DNA/química , Proteínas I-kappa B/metabolismo , Proteínas I-kappa B/química , Proteínas I-kappa B/genética , Animais , Sequência de Aminoácidos , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelA/química , Fator de Transcrição RelA/genética , Núcleo Celular/metabolismo , Camundongos
2.
Biomolecules ; 14(4)2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38672458

RESUMO

While cytochrome P450 (CYP; P450) enzymes are commonly associated with the metabolism of organic xenobiotics and drugs or the biosynthesis of organic signaling molecules, they are also impacted by a variety of inorganic species. Metallic nanoparticles, clusters, ions, and complexes can alter CYP expression, modify enzyme interactions with reductase partners, and serve as direct inhibitors. This commonly overlooked topic is reviewed here, with an emphasis on understanding the structural and physiochemical basis for these interactions. Intriguingly, while both organometallic and coordination compounds can act as potent CYP inhibitors, there is little evidence for the metabolism of inorganic compounds by CYPs, suggesting a potential alternative approach to evading issues associated with rapid modification and elimination of medically useful compounds.


Assuntos
Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Inibidores das Enzimas do Citocromo P-450/farmacologia , Inibidores das Enzimas do Citocromo P-450/química , Nanopartículas Metálicas/química , Animais , Metais/química , Metais/metabolismo , Compostos Inorgânicos/química
3.
Inorg Chem ; 62(28): 10940-10954, 2023 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-37405779

RESUMO

While cancer cells rely heavily upon glycolysis to meet their energetic needs, reducing the importance of mitochondrial oxidative respiration processes, more recent studies have shown that their mitochondria still play an active role in the bioenergetics of metastases. This feature, in combination with the regulatory role of mitochondria in cell death, has made this organelle an attractive anticancer target. Here, we report the synthesis and biological characterization of triarylphosphine-containing bipyridyl ruthenium (Ru(II)) compounds and found distinct differences as a function of the substituents on the bipyridine and phosphine ligands. 4,4'-Dimethylbipyridyl-substituted compound 3 exhibited especially high depolarizing capabilities, and this depolarization was selective for the mitochondrial membrane and occurred within minutes of treatment in cancer cells. The Ru(II) complex 3 exhibited an 8-fold increase in depolarized mitochondrial membranes, as determined by flow cytometry, which compares favorably to the 2-fold increase observed by carbonyl cyanide chlorophenylhydrazone (CCCP), a proton ionophore that shuttles protons across membranes, depositing them into the mitochondrial matrix. Fluorination of the triphenylphosphine ligand provided a scaffold that maintained potency against a range of cancer cells but avoided inducing toxicity in zebrafish embryos at higher concentrations, displaying the potential of these Ru(II) compounds for anticancer applications. This study provides essential information regarding the role of ancillary ligands for the anticancer activity of Ru(II) coordination compounds that induce mitochondrial dysfunction.


Assuntos
Antineoplásicos , Complexos de Coordenação , Rutênio , Animais , 2,2'-Dipiridil , Ligantes , Peixe-Zebra , Mitocôndrias , Rutênio/farmacologia , Rutênio/metabolismo
4.
RSC Chem Biol ; 4(5): 344-353, 2023 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-37181632

RESUMO

Ruthenium complexes are often investigated as potential replacements for platinum-based chemotherapeutics in hopes of identifying systems with improved tolerability in vivo and reduced susceptibility to cellular resistance mechanisms. Inspired by phenanthriplatin, a non-traditional platinum agent that contains only one labile ligand, monofunctional ruthenium polypyridyl agents have been developed, but until now, few demonstrated promising anticancer activity. Here we introduce a potent new scaffold, based on [Ru(tpy)(dip)Cl]Cl (tpy = 2,2':6',2''-terpyridine and dip = 4,7-diphenyl-1,10-phenanthroline) in pursuit of effective Ru(ii)-based monofunctional agents. Notably, the extension of the terpyridine at the 4' position with an aromatic ring resulted in a molecule that was cytotoxic in several cancer cell lines with sub-micromolar IC50 values, induced ribosome biogenesis stress, and exhibited minimal zebrafish embryo toxicity. This study demonstrates the successful design of a Ru(ii) agent that mimics many of the biological effects and phenotypes seen with phenanthriplatin, despite numerous differences in both the ligands and metal center structure.

5.
J Med Chem ; 66(1): 398-412, 2023 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-36520541

RESUMO

Cytochrome P450 1B1 (CYP1B1) is a potential drug target in cancer research that is overexpressed in several solid tumors but is present only at low levels in healthy tissues. Its expression is associated with resistance to common chemotherapeutics, while inhibitors restore efficacy to these drugs in model systems. The majority of CYP1B1 inhibitors are derived from a limited number of scaffolds, and few have achieved outstanding selectivity against other human CYPs, which could impede clinical development. This study explores a new chemical space for CYP1B1 inhibitors using a scaffold-hopping approach and establishes 2,4-diarylthiazoles as a promising framework for further development. From a small library, compound 15 emerged as the lead, with picomolar CYP1B1 inhibition, and over 19,000-fold selectivity against its relative, CYP1A1. To investigate the activity of 15, molecular dynamics, optical spectroscopy, point mutations, and traditional structure-activity relationships were employed and revealed key interactions important for the development of CYP1B1 inhibitors.


Assuntos
Inibidores das Enzimas do Citocromo P-450 , Neoplasias , Humanos , Inibidores das Enzimas do Citocromo P-450/farmacologia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Relação Estrutura-Atividade
6.
J Inorg Biochem ; 238: 112031, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36327501

RESUMO

Photoreactive Ru(II) complexes capable of ejecting ligands have been used extensively for photocaging applications and for the creation of "photocisplatin" reagents. The incorporation of distortion into the structure of the coordination complex lowers the energy of dissociative excited states, increasing the yield of the photosubstitution reaction. While steric clash between ligands induced by adding substituents at the coordinating face of the ligand has been extensively utilized, a lesser known, more subtle approach is to distort the coordination sphere by altering the chelate ring size. Here a systematic study was performed to alter metal-ligand bond lengths, angles, and to cause intraligand distortion by introducing a "linker" atom or group between two pyridine rings. The synthesis, photochemistry, and photobiology of five Ru(II) complexes containing CH2, NH, O, and S-linked dipyridine ligands was investigated. All systems where stable in the dark, and three of the five were photochemically active in buffer. While a clear periodic trend was not observed, this study lays the foundation for the creation of photoactive systems utilizing an alternative type of distortion to facilitate photosubstitution reactions.


Assuntos
Rutênio , Rutênio/química , Ligantes , Fotobiologia , Fotoquímica
7.
Nat Commun ; 13(1): 3636, 2022 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-35752630

RESUMO

The cytochrome P450 family of enzymes (CYPs) are important targets for medicinal chemistry. Recently, CYP1B1 has emerged as a key player in chemotherapy resistance in the treatment of cancer. This enzyme is overexpressed in a variety of tumors, and is correlated with poor treatment outcomes; thus, it is desirable to develop CYP1B1 inhibitors to restore chemotherapy efficacy. However, possible off-target effects, such as inhibition of liver CYPs responsible for first pass metabolism, make selective inhibition a high priority to avoid possible drug-drug interactions and toxicity. Here we describe the creation of light-triggered CYP1B1 inhibitors as "prodrugs", and achieve >6000-fold improvement in potency upon activation with low energy (660 nm) light. These systems provide a selectivity index of 4,000-100,000 over other off-target CYPs. One key to the design was the development of coordinating CYP1B1 inhibitors, which suppress enzyme activity at pM concentrations in live cells. The metal binding group enforces inhibitor orientation in the active site by anchoring to the iron. The second essential component was the biologically compatible Ru(II) scaffold that cages the inhibitors before photochemical release. These Ru(II) photocages are anticipated to provide similar selectivity and control for any coordinating CYP inhibitors.


Assuntos
Sistema Enzimático do Citocromo P-450 , Neoplasias , Citocromo P-450 CYP1B1/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Rutênio
8.
Eur J Inorg Chem ; 2021(35): 3611-3621, 2021 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-34539235

RESUMO

The ß-diketone scaffold is a commonly used synthetic intermediate, and is a functional group found in natural products such as curcuminoids. This core structure can also act as a chelating ligand for a variety of metals. In order to assess the potential of this scaffold for medicinal inorganic chemistry, seven different κ2-O,O'-chelating ligands were used to construct Ru(II) complexes with polypyridyl co-ligands, and their biological activity was evaluated. The complexes demonstrated promising structure-dependent cytotoxicity. Three complexes maintained high activity in a tumor spheroid model, and all complexes demonstrated low in vivo toxicity in a zebrafish model. From this series, the best compound exhibited a ~ 30-fold window between cytotoxicity in a 3-D tumor spheroid model and potential in vivo toxicity. These results suggest that κ2-O,O'-ligands can be incorporated into Ru(II)-polypyridyl complexes to create favorable candidates for future drug development.

9.
ChemMedChem ; 16(18): 2845-2850, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34224206

RESUMO

Inhibition of estrogen synthesis is an integral component of the frontline pharmacologic therapy for the treatment of estrogen receptor positive cancers. However, there is currently no direct, high-throughput-ready assay for aromatase (also known as CYP19A1) that can be performed in live cells. Herein we present a cell-based assay that allows for multiplexed assessment of enzyme activity, protein half-life, cell viability, and identification of inhibitors with slow off-rates.


Assuntos
Inibidores da Aromatase/farmacologia , Aromatase/metabolismo , Corantes Fluorescentes/farmacologia , Fluorometria , Inibidores da Aromatase/química , Relação Dose-Resposta a Droga , Corantes Fluorescentes/química , Células HEK293 , Humanos , Estrutura Molecular , Relação Estrutura-Atividade
10.
Inorg Chem ; 60(4): 2178-2187, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33502194

RESUMO

The specific recognition of AT-rich DNA sequences opens up the door to promising diagnostic and/or therapeutic strategies against gene-related diseases. Here, we demonstrate that amphiphilic PtII complexes of the type [Pt(dmba)(N∧N)]NO3 (dmba = N,N-dimethylbenzylamine-κN, κC; N∧N = dpq (3), dppz (4), and dppn (5)) recognize AT-rich oligonucleotides over other types of DNA, RNA, and model proteins. The crystal structure of 4 shows the presence of significant π-stacking interactions and a distorted coordination sphere of the d8 PtII atom. Complex 5, containing the largest π-conjugated ligand, forms supramolecular assemblies at high concentrations under aqueous environment. However, its aggregation can be promoted in the presence of DNA at concentrations as low as 10 µM in a process that "turns on" its excimer emission around 600 nm. Viscometry, gel electrophoresis, and theoretical calculations demonstrate that 5 binds to minor groove when self-assembled, while the monomers of 3 and 4 intercalate into the DNA. The complexes also inhibit cancer cell growth with low-micromolar IC50 values in 2D tissue culture and suppress tumor growth in 3D tumor spheroids with a multicellular resistance (MCR) index comparable to that of cisplatin.


Assuntos
Complexos de Coordenação/química , DNA/química , Compostos Organoplatínicos/química , Células A549 , Cristalografia por Raios X , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Substâncias Intercalantes/química , Ligantes , Estrutura Molecular , Análise Espectral/métodos , Estereoisomerismo
11.
Dalton Trans ; 49(35): 12161-12167, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32845256

RESUMO

Four structurally distinct classes of polypyridyl ruthenium complexes containing avobenzone exhibited low micromolar and submicromolar potencies in cancer cells, and were up to 273-fold more active than the parent ligand. Visible light irradiation enhanced the cytotoxicity of some complexes, making them promising candidates for combined chemo-photodynamic therapy.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Propiofenonas/química , Rutênio/química , Linhagem Celular Tumoral , Humanos , Ligantes , Fotoquimioterapia , Relação Estrutura-Atividade
12.
ACS Omega ; 5(30): 18894-18906, 2020 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-32775891

RESUMO

The discovery of new light-triggered prodrugs based on ruthenium (II) complexes is a promising approach for photoactivated chemotherapy (PACT). The light-mediated activation of "strained" Ru(II) polypyridyl complexes resulted in ligand release and produced a ligand-deficient metal center capable of forming covalent adducts with biomolecules such as DNA. Based on the strategy of exploiting structural distortion to activate photochemistry, biologically active small molecules were coordinated to a Ru(II) scaffold to create light-triggered dual-action agents. Thirteen new Ru(II) complexes with pyridyl-pyrazol(in)e ligands were synthesized, and their photochemical reactivity and anticancer properties were investigated. Isomeric bidentate ligands were investigated, where "regular" ligands (where the coordinated nitrogens in the heterocycles are linked by C-C atoms) were compared to "inverse" isomers (where the coordinated nitrogens in the heterocycles are linked by C-N atoms). Coordination of the regular 3-(pyrid-2-yl)-pyrazol(in)es to a Ru(II) bis-dimethylphenanthroline scaffold yielded photoresponsive compounds with promising photochemical and biological properties, in contrast to the inverse 1-(pyrid-2-yl)-pyrazolines. The introduction of a phenyl ring to the 1N-pyrazoline cycle increased the distortion in complexes and improved ligand release upon light irradiation (470 nm) up to 5-fold in aqueous media. Compounds 1-8, containing pyridyl-pyrazol(in)e ligands, were at least 20-80-fold more potent than the parent pyridyl-pyrazol(in)es, and exhibited biological activity in the dark, with half-maximal inhibitory concentration (IC50) values ranging from 0.2 to 7.6 µM in the HL60 cell line, with complete growth inhibition upon light irradiation. The diversification of coligands and introduction of a carboxylic acid into the Ru(II) complex resulted in compounds 9-12, with up to 146-fold improved phototoxicity indices compared with complexes 1-8.

13.
Inorg Chem ; 59(13): 8882-8892, 2020 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-32530274

RESUMO

Ruthenium(II) complexes developed for photodynamic therapy (PDT) are almost exclusively tris-bidentate systems with C2 or D3 symmetry. This is due to the fact that this structural framework commonly produces long-lived excited states, which, in turn, allow for the generation of large amounts of singlet oxygen (1O2) and other reactive oxygen species. Complexes containing tridentate ligands would be advantageous for biological applications as they are generally achiral (D2d or C2v symmetry), which eliminates the possibility of multiple isomers which could exhibit potentially different interactions with chiral biological entities. However, Ru(II) complexes containing tridentate ligands are rarely studied as candidates for photobiological applications, such as PDT, since they almost exclusively exhibit low quantum yields and very short excited-state lifetimes and, thus, are not capable of generating sufficient 1O2 or engaging in electron transfer reactions. Here, we report a proof-of-concept approach to make bis-tridentate Ru(II) complexes useful for PDT applications by altering their photophysical properties through the inclusion of N-heterocyclic carbene (NHC) ligands. Three NHC and two terpyridine ligands were studied to evaluate the effects of structural and photophysical modulations of bis-substituted Ru(II) complexes. The NHC complexes were found to have superior excited-state lifetimes, 1O2 production, and photocytotoxicity. To the best of our knowledge, these complexes are the most potent light-activated bis-tridentate complexes reported.


Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/efeitos da radiação , Linhagem Celular Tumoral , Complexos de Coordenação/síntese química , Complexos de Coordenação/efeitos da radiação , DNA/metabolismo , Quebras de DNA de Cadeia Simples/efeitos da radiação , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligantes , Luz , Estudo de Prova de Conceito , Rutênio/química , Oxigênio Singlete/metabolismo
14.
Biophys J ; 115(7): 1251-1263, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30224054

RESUMO

Cytochrome P450BM3 catalyzes the hydroxylation and/or epoxidation of fatty acids, fatty amides, and alcohols. Protein engineering has produced P450BM3 variants capable of accepting drug molecules normally metabolized by human P450 enzymes. The enhanced substrate promiscuity has been attributed to the greater flexibility of the lid of the substrate channel. However, it is not well understood how structurally different and highly polar drug molecules can stably bind in the active site nor how the activity and coupling efficiency of the enzyme may be affected by the lack of enzyme-substrate complementarity. To address these important aspects of non-native small molecule binding, this study investigated the binding of drug molecules with different size, charge, polar surface area, and human P450 affinity on the promiscuous R47L/F87V/L188Q/E267V/F81I pentuple mutant of P450BM3. Binding free energy data and energy decomposition analysis showed that pentuple mutant P450BM3 stably binds (i.e., negative ΔGb°) a broad range of substrate and inhibitor types because dispersion interactions with active site residues overcome unfavorable repulsive and electrostatic effects. Molecular dynamics simulations revealed that 1) acidic substrates tend to disrupt the heme propionate A-K69 salt bridge, which may reduce heme oxidizing ability, and 2) the lack of complementarity leads to high substrate mobility and water density in the active site, which may lead to uncoupling. These factors must be considered in future developments of P450BM3 as a biocatalyst in the large-scale production of drug metabolites.


Assuntos
Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Simulação de Dinâmica Molecular , Mutação , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Heme/metabolismo , Mutagênese Sítio-Dirigida , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/genética , Ligação Proteica , Termodinâmica
15.
Eur J Med Chem ; 156: 790-799, 2018 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-30055464

RESUMO

8-Hydroxyquinolines (HQ), including clioquinol, possess cytotoxic properties and are widely used as ligands for metal-based anticancer drug research. The number and identity of substituents on the HQ can have a profound effect on activity for a variety of inorganic compounds. Ruthenium complexes of HQ exhibit radically improved potencies, and operate by a new, currently unknown, mechanism of action. To define structure-activity relationships (SAR), a family of 22 Ru(II) coordination complexes containing mono-, di- and tri-substituted hydroxyquinoline ligands were synthesized and their biological activity evaluated. The complexes exhibited promising cytotoxic activity against a cancer cell line, and the SAR data revealed the 2- and 7-positions as key sites for the incorporation of halogens to improve potency. The Ru(II) complexes potently inhibited translation, as demonstrated by an in-cell translation assay. The effects were seen at 2-15-fold higher concentrations than those required to observe cytotoxicity, suggesting that prevention of protein synthesis may be a primary, but not the exclusive mechanism for the observed cytotoxic activity.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Hidroxiquinolinas/química , Hidroxiquinolinas/farmacologia , Rutênio/química , Rutênio/farmacologia , Linhagem Celular Tumoral , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Humanos , Ligantes , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/genética , Biossíntese de Proteínas/efeitos dos fármacos , Relação Estrutura-Atividade , Transcrição Gênica/efeitos dos fármacos
16.
Mol Pharm ; 15(8): 3404-3416, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-29865789

RESUMO

Target identification and mechanistic studies of cytotoxic agents are challenging processes that are both time-consuming and costly. Here we describe an approach to mechanism of action studies for potential anticancer compounds by utilizing the simple prokaryotic system, E. coli, and we demonstrate its utility with the characterization of a ruthenium polypyridyl complex [Ru(bpy)2dmbpy2+]. Expression of the photoconvertible fluorescent protein Dendra2 facilitated both high throughput studies and single-cell imaging. This allowed for simultaneous ratiometric analysis of inhibition of protein production and phenotypic investigations. The profile of protein production, filament size and population, and nucleoid morphology revealed important differences between inorganic agents that damage DNA vs more selective inhibitors of transcription and translation. Trace metal analysis demonstrated that DNA is the preferred nucleic acid target of the ruthenium complex, but further studies in human cancer cells revealed altered cell signaling pathways compared to the commonly administrated anticancer agent cisplatin. This study demonstrates E. coli can be used to rapidly distinguish between compounds with disparate mechanisms of action and also for more subtle distinctions within in studies in mammalian cells.


Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Rutênio/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Complexos de Coordenação/química , Dano ao DNA/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Humanos , Biossíntese de Proteínas/efeitos dos fármacos , Rutênio/química , Transcrição Gênica/efeitos dos fármacos
17.
J Biol Methods ; 5(4): e105, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-31453255

RESUMO

We present a new approach to quantify the half-life of membrane proteins on the cell surface, through tagging the protein with the photoconvertible fluorescent protein, Dendra2. Upon exposure to 405 nm light, Dendra2 is photoconverted from green to red emission. Total internal reflection fluorescence microscopy (TIRF) is applied to limit visualization of fluorescence to proteins located on the plasma membrane. Conversion of Dendra2 works as a pulse chase experiment through monitoring only the population of protein that has been photoconverted. As the protein is endocytosed the red emission decreases due to the protein leaving the TIRF field of view. This method is not impacted by the insertion of new protein into the plasma membrane as newly synthesized protein only exhibits green emission. We used this approach to determine the half-life of ENaC on the plasma membrane illustrating the high temporal resolution capability of this technique compared to current methods.

18.
Eur J Inorg Chem ; 2017(12): 1687-1694, 2017 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-29200939

RESUMO

Ruthenium complexes capable of light-triggered cytotoxicity are appealing potential prodrugs for photodynamic therapy (PDT) and photoactivated chemotherapy (PACT). Two groups of Ru(II) polypyridyl complexes with 2-(2-pyridyl)-benzazole ligands were synthesized and investigated for their photochemical properties and anticancer activity to compare strained and unstrained systems that are likely to have different biological mechanisms of action. The structure-activity relationship was focused on the benzazole core bioisosterism and replacement of coligands in Ru(II) complexes. Strained compounds rapidly ejected the 2-(2-pyridyl)-benzazole ligand after light irradiation, and possessed strong toxicity in the HL-60 cell line both under dark and light conditions. In contrast, unstrained Ru(II) complexes were non-toxic in the absence of light, induced cytotoxicity at nanomolar concentrations after light irradiation, and are capable of light-induced DNA damage. The 90-220-fold difference in light and dark IC50 values provides a large potential therapeutic window to allow for selective targeting of cells by exposure to light.

19.
Inorg Chem ; 56(20): 12214-12223, 2017 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-28949518

RESUMO

Light-activated compounds are powerful tools and potential agents for medical applications, as biological effects can be controlled in space and time. Ruthenium polypyridyl complexes can induce cytotoxic effects through multiple mechanisms, including acting as photosensitizers for singlet oxygen (1O2) production, generating other reactive oxygen species (ROS), releasing biologically active ligands, and creating reactive intermediates that form covalent bonds to biological molecules. A structure-activity relationship (SAR) study was performed on a series of Ru(II) complexes containing isomeric tetramethyl-substituted bipyridyl-type ligands. Three of the ligand systems studied contained strain-inducing methyl groups and created photolabile metal complexes, which can form covalent bonds to biomolecules upon light activation, while the fourth was unstrained and resulted in photostable complexes, which can generate 1O2. The compounds studied included both bis-heteroleptic complexes containing two bipyridine ligands and a third, substituted ligand and tris-homoleptic complexes containing only the substituted ligand. The photophysics, electrochemistry, photochemistry, and photobiology were assessed. Strained heteroleptic complexes were found to be more photoactive and cytotoxic then tris-homoleptic complexes, and bipyridine ligands were superior to bipyrimidine. However, the homoleptic complexes exhibited an enhanced ability to inhibit protein production in live cells. Specific methylation patterns were associated with improved activation with red light, and photolabile complexes were generally more potent cytotoxic agents than the photostable 1O2-generating compounds.


Assuntos
2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/efeitos da radiação , Complexos de Coordenação/efeitos da radiação , Rutênio/química , 2,2'-Dipiridil/síntese química , 2,2'-Dipiridil/farmacologia , Quelantes/química , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Adutos de DNA/efeitos dos fármacos , Quebras de DNA , Replicação do DNA/efeitos dos fármacos , Células HL-60 , Humanos , Ligantes , Luz , Metilação , Biossíntese de Proteínas , Pirimidinas/síntese química , Pirimidinas/química , Pirimidinas/farmacologia , Pirimidinas/efeitos da radiação , Oxigênio Singlete/química , Relação Estrutura-Atividade
20.
SLAS Discov ; 22(4): 399-407, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28328316

RESUMO

Dysregulated transcription, translation, and protein degradation are common features of cancer cells, regardless of specific genetic profiles. Several clinical anticancer agents take advantage of this characteristic vulnerability and interfere with the processes of transcription and translation or inhibit protein degradation. However, traditional assays that follow the process of protein production and removal require multistep processing and are not easily amenable to high-throughput screening. The use of recombinant fluorescent proteins provides a convenient solution to this problem, and moreover, photoconvertable fluorescent proteins allow for ratiometric detection of both new protein production and removal of existing proteins. Here, the photoconvertable protein Dendra2 is used in the development of in-cell assays of protein production and degradation that are optimized and validated for high-throughput screening. Conversion from the green to red emissive form can be achieved using a high-intensity light-emitting diode array, producing a stable pool of the red fluorescent form of Dendra2. This allows for rates of protein production or removal to be quantified in a plate reader or by fluorescence microscopy, providing a means to measure the potencies of inhibitors that affect these key processes.


Assuntos
Ensaios de Triagem em Larga Escala , Proteínas Luminescentes/genética , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Bortezomib/farmacologia , Linhagem Celular , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/antagonistas & inibidores , Proteínas Luminescentes/metabolismo , Inibidor de NF-kappaB alfa/antagonistas & inibidores , Inibidor de NF-kappaB alfa/genética , Inibidor de NF-kappaB alfa/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
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