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1.
Bioorg Med Chem Lett ; 87: 129261, 2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-36990245

RESUMO

RAD51 is a pivotal protein of the homologous recombination DNA repair pathway, and is overexpressed in some cancer cells, disrupting then the efficiency of cancer-treatments. The development of RAD51 inhibitors appears as a promising solution to restore these cancer cells sensitization to radio- or chemotherapy. From a small molecule identified as a modulator of RAD51, the 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), two series of analogues with small or bulky substituents on the aromatic parts of the stilbene moiety were prepared for a structure-activity relationship study. Three compounds, the cyano analogue (12), and benzamide (23) or phenylcarbamate (29) analogues of DIDS were characterized as novel potent RAD51 inhibitors with HR inhibition in the micromolar range.


Assuntos
Recombinação Homóloga , Rad51 Recombinase , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Ligação Proteica , Relação Estrutura-Atividade
2.
Int J Mol Sci ; 24(5)2023 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-36901779

RESUMO

High-throughput protein assays are crucial for modern diagnostics, drug discovery, proteomics, and other fields of biology and medicine. It allows simultaneous detection of hundreds of analytes and miniaturization of both fabrication and analytical procedures. Photonic crystal surface mode (PC SM) imaging is an effective alternative to surface plasmon resonance (SPR) imaging used in conventional gold-coated, label-free biosensors. PC SM imaging is advantageous as a quick, label-free, and reproducible technique for multiplexed analysis of biomolecular interactions. PC SM sensors are characterized by a longer signal propagation at the cost of a lower spatial resolution, which makes them more sensitive than classical SPR imaging sensors. We describe an approach for designing label-free protein biosensing assays employing PC SM imaging in the microfluidic mode. Label-free, real-time detection of PC SM imaging biosensors using two-dimensional imaging of binding events has been designed to study arrays of model proteins (antibodies, immunoglobulin G-binding proteins, serum proteins, and DNA repair proteins) at 96 points prepared by automated spotting. The data prove feasibility of simultaneous PC SM imaging of multiple protein interactions. The results pave the way to further develop PC SM imaging as an advanced label-free microfluidic assay for the multiplexed detection of protein interactions.


Assuntos
Técnicas Biossensoriais , Técnicas Analíticas Microfluídicas , Técnicas Biossensoriais/métodos , Ressonância de Plasmônio de Superfície/métodos , Anticorpos , Proteínas , Técnicas Analíticas Microfluídicas/métodos
3.
Mar Drugs ; 19(7)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34210084

RESUMO

Very little is known about chemical interactions between fungi and their mollusc host within marine environments. Here, we investigated the metabolome of a Penicillium restrictum MMS417 strain isolated from the blue mussel Mytilus edulis collected on the Loire estuary, France. Following the OSMAC approach with the use of 14 culture media, the effect of salinity and of a mussel-derived medium on the metabolic expression were analysed using HPLC-UV/DAD-HRMS/MS. An untargeted metabolomics study was performed using principal component analysis (PCA), orthogonal projection to latent structure discriminant analysis (O-PLSDA) and molecular networking (MN). It highlighted some compounds belonging to sterols, macrolides and pyran-2-ones, which were specifically induced in marine conditions. In particular, a high chemical diversity of pyran-2-ones was found to be related to the presence of mussel extract in the culture medium. Mass spectrometry (MS)- and UV-guided purification resulted in the isolation of five new natural fungal pyran-2-one derivatives-5,6-dihydro-6S-hydroxymethyl-4-methoxy-2H-pyran-2-one (1), (6S, 1'R, 2'S)-LL-P880ß (3), 5,6-dihydro-4-methoxy-6S-(1'S, 2'S-dihydroxy pent-3'(E)-enyl)-2H-pyran-2-one (4), 4-methoxy-6-(1'R, 2'S-dihydroxy pent-3'(E)-enyl)-2H-pyran-2-one (6) and 4-methoxy-2H-pyran-2-one (7)-together with the known (6S, 1'S, 2'S)-LL-P880ß (2), (1'R, 2'S)-LL-P880γ (5), 5,6-dihydro-4-methoxy-2H-pyran-2-one (8), (6S, 1'S, 2'R)-LL-P880ß (9), (6S, 1'S)-pestalotin (10), 1'R-dehydropestalotin (11) and 6-pentyl-4-methoxy-2H-pyran-2-one (12) from the mussel-derived culture medium extract. The structures of 1-12 were determined by 1D- and 2D-MMR experiments as well as high-resolution tandem MS, ECD and DP4 calculations. Some of these compounds were evaluated for their cytotoxic, antibacterial, antileishmanial and in-silico PTP1B inhibitory activities. These results illustrate the utility in using host-derived media for the discovery of new natural products.


Assuntos
Bivalves , Penicillium/metabolismo , Piranos/metabolismo , Animais , Organismos Aquáticos , França , Metabolômica , Penicillium/química , Piranos/química , Relação Estrutura-Atividade
4.
Molecules ; 26(18)2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34576930

RESUMO

RAD51 is the central protein in DNA repair by homologous recombination (HR), involved in several steps of this process. It is shown that overexpression of the RAD51 protein is correlated with increased survival of cancer cells to cancer treatments. For the past decade, RAD51 overexpression-mediated resistance has justified the development of targeted inhibitors. One of the first molecules described to inhibit RAD51 was the 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) molecule. This small molecule is effective in inhibiting different functions of RAD51, however its mode of action and the chemical functions involved in this inhibition have not been identified. In this work, we used several commercial molecules derived from DIDS to characterize the structural determinants involved in modulating the activity of RAD51. By combining biochemical and biophysical approaches, we have shown that DIDS and two analogs were able to inhibit the binding of RAD51 to ssDNA and prevent the formation of D-loop by RAD51. Both isothiocyanate substituents of DIDS appear to be essential in the inhibition of RAD51. These results open the way to the synthesis of new molecules derived from DIDS that should be greater modulators of RAD51 and more efficient for HR inhibition.


Assuntos
Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/análogos & derivados , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/administração & dosagem , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/administração & dosagem , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , DNA de Cadeia Simples/metabolismo , Relação Dose-Resposta a Droga , Rad51 Recombinase/antagonistas & inibidores
5.
Chemistry ; 26(21): 4734-4751, 2020 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-31774931

RESUMO

The catalyst H3+x PMo12-x +6 Mox +5 O40 supported on SiO2 was developed for peroxidation of 1,3- and 1,5-diketones with hydrogen peroxide with the formation of bridged 1,2,4,5-tetraoxanes and bridged 1,2,4-trioxolanes (ozonides) with high yield based on isolated products (up to 86 and 90 %, respectively) under heterogeneous conditions. Synthesis of peroxides under heterogeneous conditions is a rare process and represents a challenge for this field of chemistry, because peroxides tend to decompose on the surface of a catalyst . A new class of antifungal agents for crop protection, that is, cyclic peroxides: bridged 1,2,4,5-tetraoxanes and bridged ozonides, was discovered. Some ozonides and tetraoxanes exhibit a very high antifungal activity and are superior to commercial fungicides, such as Triadimefon and Kresoxim-methyl. It is important to note that none of the fungicides used in agricultural chemistry contains a peroxide fragment.


Assuntos
Fungicidas Industriais/química , Compostos Heterocíclicos/química , Peróxido de Hidrogênio/química , Cetonas/química , Peróxidos/síntese química , Dióxido de Silício/química , Tetraoxanos/síntese química , Catálise , Fungicidas Industriais/síntese química , Peróxidos/química , Tetraoxanos/química
6.
Int J Mol Sci ; 21(23)2020 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-33291743

RESUMO

Fucoxanthin is a well-known carotenoid of the xanthophyll family, mainly produced by marine organisms such as the macroalgae of the fucus genus or microalgae such as Phaeodactylum tricornutum. Fucoxanthin has antioxidant and anti-inflammatory properties but also several anticancer effects. Fucoxanthin induces cell growth arrest, apoptosis, and/or autophagy in several cancer cell lines as well as in animal models of cancer. Fucoxanthin treatment leads to the inhibition of metastasis-related migration, invasion, epithelial-mesenchymal transition, and angiogenesis. Fucoxanthin also affects the DNA repair pathways, which could be involved in the resistance phenotype of tumor cells. Moreover, combined treatments of fucoxanthin, or its metabolite fucoxanthinol, with usual anticancer treatments can support conventional therapeutic strategies by reducing drug resistance. This review focuses on the current knowledge of fucoxanthin with its potential anticancer properties, showing that fucoxanthin could be a promising compound for cancer therapy by acting on most of the classical hallmarks of tumor cells.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Organismos Aquáticos/química , Microalgas/química , Alga Marinha/química , Xantofilas/química , Xantofilas/farmacologia , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaios Clínicos como Assunto , Dano ao DNA , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Estrutura Molecular , Resultado do Tratamento , Xantofilas/uso terapêutico
7.
Phys Chem Chem Phys ; 20(26): 18020-18030, 2018 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-29931001

RESUMO

Disulfonic stilbene (DS) derivatives are a member of the large family of compounds widely employed in medicine and biology as modulators for membrane transporters or inhibitors of a protein involved in DNA repair. They constitute interesting compounds that have not yet been investigated within the bioavailability framework. No crystallographic structures exist involving such compounds embedded in the most common drug carrier, human serum albumin (HSA). The present work studies, for the first time, the physico-chemical features driving the inclusion of three DS derivatives (amino, nitro and acetamido, named DADS, DNDS and DATDS, respectively) within the four common HSA binding sites using combined molecular docking and molecular dynamics simulations. A careful analysis of each ligand within each of the studied binding sites is carried out, highlighting specific interactions and key residues playing a role in stabilizing the ligand within each pocket. The comparison between DADS, DNDS and DATDS reveals that depending on the binding site, the conclusions are rather different. For instance, the IB binding site shows a specificity to DADS compounds while IIIA is the most favorable site for DNDS and DATDS.


Assuntos
Simulação por Computador , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Albumina Sérica Humana/química , Estilbenos/química , Humanos , Ligantes , Ligação Proteica , Conformação Proteica
8.
Chemphyschem ; 17(15): 2434-45, 2016 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-27062098

RESUMO

4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) is a well-known ion-exchange inhibitor targeting cardiac functions and indirectly impeding both radio- and chemo-resistance. A joint computational and experimental study is presented to provide deeper insights into DIDS and other members of this family of compounds. To this end, we applied state-of-the-art density functional theory (DFT) and time-dependent DFT methods, in addition to measuring the optical properties. The experimental data show that such compounds are highly sensitive to their environment and that the optical properties change within as little time as 7 h. However, the optical properties of DIDS are similar in various acidic/basic environments, which were confirmed by pKa computations on both cis and trans isomers. The protonation analysis also highlights that the singly protonated form of DIDS behaves like a proton sponge compound. The experimentally observed redshift that can be seen when going from water to DMSO was reproduced solely by using the solvation model based on density, although the polarization continuum model and implicit/explicit hybrid schemes were also tested. The characteristic broadening of the absorption peak in water and the vibronic fine structure in DMSO were also reproduced thanks to vibronic coupling simulations associated with the solvent reorganization energy. For other stilbene derivatives, a correlation is found between the maximum absorption wavelength and the Hammett parameters.


Assuntos
Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/química , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/análogos & derivados , Isomerismo , Modelos Moleculares , Prótons , Teoria Quântica , Espectrofotometria , Termodinâmica
9.
Int J Mol Sci ; 17(5)2016 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-27187356

RESUMO

The expression and activity of DNA-dependent protein kinase (DNA-PK) is related to DNA repair status in the response of cells to exogenous and endogenous factors. Recent studies indicate that Epidermal Growth Factor Receptor (EGFR) is involved in modulating DNA-PK. It has been shown that a compound 4-nitro-7-[(1-oxidopyridin-2-yl)sulfanyl]-2,1,3-benzoxadiazole (NSC), bearing a nitro-benzoxadiazole (NBD) scaffold, enhances tyrosine phosphorylation of EGFR and triggers downstream signaling pathways. Here, we studied the behavior of DNA-PK and other DNA repair proteins in prostate cancer cells exposed to compound NSC. We showed that both the expression and activity of DNA-PKcs (catalytic subunit of DNA-PK) rapidly decreased upon exposure of cells to the compound. The decline in DNA-PKcs was associated with enhanced protein ubiquitination, indicating the activation of cellular proteasome. However, pretreatment of cells with thioglycerol abolished the action of compound NSC and restored the level of DNA-PKcs. Moreover, the decreased level of DNA-PKcs was associated with the production of intracellular hydrogen peroxide by stable dimeric forms of Cu/Zn SOD1 induced by NSC. Our findings indicate that reactive oxygen species and electrophilic intermediates, generated and accumulated during the redox transformation of NBD compounds, are primarily responsible for the rapid modulation of DNA-PKcs functions in cancer cells.


Assuntos
Proteína Quinase Ativada por DNA/metabolismo , Regulação para Baixo/efeitos dos fármacos , Oxidiazóis/farmacologia , Neoplasias da Próstata/metabolismo , Linhagem Celular Tumoral , Reparo do DNA/efeitos dos fármacos , Proteína Quinase Ativada por DNA/genética , Glicerol/análogos & derivados , Glicerol/farmacologia , Humanos , Peróxido de Hidrogênio/metabolismo , Masculino , Superóxido Dismutase/metabolismo , Ubiquitinação
10.
Genes Cells ; 19(10): 755-65, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25200281

RESUMO

The homologous recombination factor RAD51 is highly conserved. This criterion enabled us to identify a RAD51 ortholog in Physarum polycephalum. We found that the Physarum protein presents a high homology to the human protein and cross-reacted with antibodies directed against the human RAD51. Taking advantage of the natural synchrony of millions of nuclei within a single cell of Physarum, we investigated the fluctuation of the amount of the PpRAD51 throughout the cell cycle. Our results showed that in the late G2-phase, RAD51 was transiently expressed in a large quantity. Furthermore, knocking-down RAD51 in the G2-phase abolished this transient expression before mitosis and affected cell cycle progression. These results support the idea that RAD51 plays a role in the progression of the cell cycle in the late G2-phase.


Assuntos
Fase G2 , Physarum/metabolismo , Rad51 Recombinase/metabolismo , Humanos , Physarum/citologia , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/genética
11.
Bioconjug Chem ; 26(4): 609-24, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25710410

RESUMO

Quantum dots (QDs) are highly fluorescent nanoscale crystals with size-dependent emission spectra. Due to their excellent photophysical properties, QDs are a promising alternative to organic fluorescent dyes and fluorescent proteins for cell targeting, imaging, and drug delivery. For biomedical applications, QDs should be chemically modified to be stable in aqueous solutions and tagged with the recognition molecules or drugs. Here, we review surface modification approaches to, and strategies for, conjugation of bioactive molecules with QDs. There are a variety of methods of QD surface modification and QD incorporation into larger delivery systems that yield fluorescent nanocarriers from 10 nm to several micrometers. Conjugates of QDs with peptides, proteins, antibodies, oligonucleotides, and small molecules have been used for fluorescent targeting, tracking, and imaging both in vitro and in vivo. Due to an extremely high stability to photobleaching, QDs were used for long-term visualization. QD applications pave the way for new generations of ultrasensitive detection, diagnostic systems, as well as drug delivery approaches, combining accurate targeting, delivery, and imaging in a single assay.


Assuntos
Portadores de Fármacos/química , Sondas Moleculares/química , Nanopartículas/química , Pontos Quânticos/química , Animais , Anticorpos/química , Anticorpos/metabolismo , Rastreamento de Células/métodos , Portadores de Fármacos/síntese química , Células Eucarióticas/efeitos dos fármacos , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Humanos , Imunoconjugados/química , Imunoconjugados/metabolismo , Imagem Molecular/métodos , Sondas Moleculares/síntese química , Sondas Moleculares/farmacologia , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Tamanho da Partícula , Peptídeos/química , Peptídeos/metabolismo , Coloração e Rotulagem/métodos , Propriedades de Superfície
12.
Chemistry ; 20(32): 10160-9, 2014 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-24989116

RESUMO

The reaction of ß,δ-triketones with an ethereal solution of H2O2 catalyzed by heteropoly acids in the presence of a polar aprotic co-solvent proceeds via three pathways to form three classes of peroxides: tricyclic monoperoxides, bridged tetraoxanes, and a pair of stereoisomeric ozonides. The reaction is unusual in that produces bridged tetraoxanes and ozonides with one of the three carbonyl groups remaining intact. In the synthesis of bridged tetraoxanes, the peroxide ring is formed by the reaction of hydrogen peroxide with two carbonyl groups at the ß positions. The synthesis of ozonides from ketones and hydrogen peroxide is a unique process in which the ozonide ring is formed with the participation of two carbonyl groups at the δ positions. Rearrangements of ozonides were found for the first time after more than one century of their active investigation. Ozonides are interconverted with each other and rearranged into tricyclic monoperoxides, whereas ozonides and tricyclic monoperoxides are transformed into bridged tetraoxanes. The individual reaction products were isolated by column chromatography and characterized by NMR spectroscopy, mass spectrometry, and elemental analysis. One representative of each class of peroxides was characterized by X-ray diffraction.


Assuntos
Compostos Heterocíclicos/síntese química , Peróxido de Hidrogênio/química , Cetonas/química , Peróxidos/síntese química , Ciclização , Compostos Heterocíclicos/química , Peróxidos/química , Tetraoxanos/síntese química , Tetraoxanos/química , Difração de Raios X
13.
Chemphyschem ; 15(17): 3753-60, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25208912

RESUMO

The interaction of human Rad51 protein (HsRad51) with single-stranded deoxyribonucleic acid (ssDNA) was investigated by using quartz crystal microbalance (QCM) monitoring and atomic force microscopy (AFM) visualization. Gold surfaces for QCM and AFM were modified by electrografting of the in situ generated aryldiazonium salt from the sulfanilic acid to obtain the organic layer Au-ArSO3 H. The Au-ArSO3 H layer was activated by using a solution of PCl5 in CH2 Cl2 to give a Au-ArSO2 Cl layer. The modified surface was then used to immobilize long ssDNA molecules. The results obtained showed that the presence of adenosine diphosphate promotes the protein autoassociation rather than nucleation around DNA. In addition, when the BRC4-28 peptide inhibitor was used, both QCM and AFM confirmed the inhibitory effect of BRC4-28 toward HsRad51 autoassociation. Altogether these results show the suitability of this modified surface to investigate the kinetics and structure of DNA-protein interactions and for the screening of inhibitors.


Assuntos
Difosfato de Adenosina/farmacologia , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/ultraestrutura , Microscopia de Força Atômica , Peptídeos/farmacologia , Técnicas de Microbalança de Cristal de Quartzo , Rad51 Recombinase/metabolismo , Rad51 Recombinase/ultraestrutura , DNA de Cadeia Simples/química , Humanos , Cinética , Compostos Organoáuricos/química , Ligação Proteica/efeitos dos fármacos , Rad51 Recombinase/química , Relação Estrutura-Atividade , Propriedades de Superfície
14.
Biosensors (Basel) ; 14(1)2024 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-38248420

RESUMO

Photonic crystals (PCs) are promising tools for label-free sensing in drug discovery screening, diagnostics, and analysis of ligand-receptor interactions. Imaging of PC surface modes has emerged as a novel approach to the detection of multiple binding events at the sensor surface. PC surface modification and decoration with recognition units yield an interface providing the highly sensitive detection of cancer biomarkers, antibodies, and oligonucleotides. The RAD51 protein plays a central role in DNA repair via the homologous recombination pathway. This recombinase is essential for the genome stability and its overexpression is often correlated with aggressive cancer. RAD51 is therefore a potential target in the therapeutic strategy for cancer. Here, we report the designing of a PC-based array sensor for real-time monitoring of oligonucleotide-RAD51 recruitment by means of surface mode imaging and validation of the concept of this approach. Our data demonstrate that the designed biosensor ensures the highly sensitive multiplexed analysis of association-dissociation events and detection of the biomarker of DNA damage using a microfluidic PC array. The obtained results highlight the potential of the developed technique for testing the functionality of candidate drugs, discovering new molecular targets and drug entities. This paves the way to further adaption and bioanalytical use of the biosensor for high-content screening to identify new DNA repair inhibitor drugs targeting the RAD51 nucleoprotein filament or to discover new molecular targets.


Assuntos
Anticorpos , Neoplasias , Humanos , Diagnóstico por Imagem , Biomarcadores Tumorais , Reparo do DNA , DNA de Cadeia Simples , Oligonucleotídeos , Rad51 Recombinase
15.
Blood ; 118(4): 1062-8, 2011 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-21653319

RESUMO

Chronic myeloid leukemia chronic phase (CML-CP) CD34(+) cells contain numerous DNA double-strand breaks whose unfaithful repair may contribute to chromosomal instability and disease progression to blast phase (CML-BP). These phenomena are often associated with the appearance of imatinib-resistant BCR-ABL1 kinase mutants (eg, T315I) and overexpression of BCR-ABL1. Here we show that BCR-ABL1 (nonmutated and T315I mutant) promoted RAD51 recombinase-mediated unfaithful homeologous recombination repair (HomeoRR) in a dosage-dependent manner. BCR-ABL1 SH3 domain interacts with RAD51 proline-rich regions, resulting in direct phosphorylation of RAD51 on Y315 (pY315). RAD51(pY315) facilitates dissociation from the complex with BCR-ABL1 kinase, migrates to the nucleus, and enhances formation of the nuclear foci indicative of recombination sites. HomeoRR and RAD51 nuclear foci were strongly reduced by RAD51(Y315F) phosphorylation-less mutant. In addition, peptide aptamer mimicking RAD51(pY315) fragment, but not that with Y315F phosphorylation-less substitution, diminished RAD51 foci formation and inhibited HomeoRR in leukemia cells. In conclusion, we postulate that BCR-ABL1 kinase-mediated RAD51(pY315) promotes unfaithful HomeoRR in leukemia cells, which may contribute to accumulation of secondary chromosomal aberrations responsible for CML relapse and progression.


Assuntos
Reparo do DNA/fisiologia , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Rad51 Recombinase/genética , Animais , Western Blotting , Linhagem Celular Tumoral , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Camundongos , Reação em Cadeia da Polimerase , Rad51 Recombinase/metabolismo , Transfecção , Tirosina/metabolismo
16.
Biochim Biophys Acta Rev Cancer ; 1875(1): 188495, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33346130

RESUMO

Receptor Tyrosine Kinases (RTK) are an important family involved in numerous signaling pathways essential for proliferation, cell survival, transcription or cell-cycle regulation. Their role and involvement in cancer cell survival have been widely described in the literature, and are generally associated with overexpression and/or excessive activity in the cancer pathology. Because of these characteristics, RTKs are relevant targets in the fight against cancer. In the last decade, increasingly numerous works describe the role of RTK signaling in the modulation of DNA repair, thus providing evidence of the relationship between RTKs and the protein actors in the repair pathways. In this review, we propose a summary of RTKs described as potential modulators of double-stranded DNA repair pathways in order to put forward new lines of research aimed at the implementation of new therapeutic strategies targeting both DNA repair pathways and RTK-mediated signaling pathways.


Assuntos
Reparo do DNA/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Receptores Proteína Tirosina Quinases/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias/genética , Neoplasias/patologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos
17.
Biochim Biophys Acta Gen Subj ; 1864(12): 129705, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32805318

RESUMO

BACKGROUND: DNA dependent Protein Kinase (DNA-PK) is an heterotrimeric complex regulating the Non Homologous End Joining (NHEJ) double strand break (DSB) repair pathway. The activity of its catalytic subunit (DNA-PKcs) is regulated by multiple phosphorylations, like the Ser2056 one that impacts DSB end processing and telomeres integrity. O-GlcNAcylation is a post translational modification (PTM) closely related to phosphorylation and its implication in the modulation of DNA-PKcs activity during the DNA Damage Response (DDR) is unknown. METHODS: Using IP techniques, and HeLa cell line, we evaluated the effect of pharmacological or siOGT mediated O-GlcNAc level modulation on DNA-PKcs O-GlcNAcylation. We used the RPA32 phosphorylation as a DNA-PKcs activity reporter substrate to evaluate the effect of O-GlcNAc modulators. RESULTS: We show here that human DNA-PKcs is an O-GlcNAc modified protein and that this new PTM is responsive to the cell O-GlcNAcylation level modulation. Our findings reveal that DNA-PKcs hypo O-GlcNAcylation affects its kinase activity and that the bleomycin-induced Ser2056 phosphorylation, is modulated by DNA-PKcs O-GlcNAcylation. CONCLUSIONS: DNA-PKcs Ser2056 phosphorylation is antagonistically linked to DNA-PKcs O-GlcNAcylation level modulation. GENERAL SIGNIFICANCE: Given the essential role of DNA-PKcs Ser2056 phosphorylation in the DDR, this study brings data about the role of cell O-GlcNAc level on genome integrity maintenance.


Assuntos
Acetilglucosamina/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Acilação , Células HeLa , Humanos , Fosforilação , Processamento de Proteína Pós-Traducional
18.
Methods Mol Biol ; 2135: 259-273, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32246341

RESUMO

Antibody microarrays have become a powerful tool in multiplexed immunoassay technologies. The advantage of microarray technology is the possibility of rapid analysis of multiple targets in a single sample with a high sensitivity, which makes them ideal for high throughput screening. Usually these microarrays contain biological recognition molecules, such as full-size antibodies, antigen-binding fragments, and single-domain antibodies, and a label for detection. Organic fluorophores are the most popular labels, but they suffer from low sensitivity and instability due to their photodegradation. Here, we describe a protocol for fabricating an antibody microarray with highly fluorescent semiconductor nanocrystals or quantum dots (QDs) as the source of fluorescent signals, which may significantly improve the properties of microarrays, including their sensitivity and specificity. Our approach to analyte detection is based on the use of sandwich approach with streptavidin-biotin to assess and monitor the fluorescence signal instead of direct labeling of samples, which helps improve the reproducibility of results and sensitivity of the microarrays. The antibody microarray developed has been tested for its capacity of detecting DNA-PKcs in glial cell lines and measuring cell protein phosphorylation changes caused by camptothecin-induced DNA damage with different protein kinase inhibitors in HeLa cells.


Assuntos
Análise Serial de Proteínas/métodos , Pontos Quânticos/química , Anticorpos/imunologia , Biotina/química , Corantes Fluorescentes/química , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Imunoensaio/métodos , Análise em Microsséries/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estreptavidina/química
19.
ChemMedChem ; 15(13): 1118-1127, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32154637

RESUMO

This article discloses a new horizon for the application of peroxides in medical chemistry. Stable cyclic peroxides are demonstrated to have cytotoxic activity against cancer cells; in addition a mechanism of cytotoxic action is proposed. Synthetic bridged 1,2,4,5-tetraoxanes and ozonides were effective against HepG2 cancer cells and some ozonides selectively targeted liver cancer cells (the selectivity indexes for compounds 11 b and 12 a are 8 and 5, respectively). In some cases, tetraoxanes and ozonides were more selective than paclitaxel, artemisinin, and artesunic acid. Annexin V flow-cytometry analysis revealed that the active ozonides 22 a and 23 a induced cell death of HepG2 by apoptosis. Further study showed that compounds 22 a and 23 a exhibited a strong inhibitory effect on P-glycoprotein (P-gp/ABCB5)-overexpressing HepG2 cancer cells. ABCB5 is a key player in the multidrug-resistant phenotype of liver cancer. Peroxides failed to demonstrate a direct correlation between oxidative potential and their biological activity. To our knowledge this is the first time that peroxide diastereoisomers have been found to show stereospecific antimalarial action against the chloroquine-sensitive 3D7 strain of Plasmodium falciparum. Stereoisomeric ozonide 12 b is 11 times more active than stereoisomeric ozonide 12 a (IC50 =5.81 vs 65.18 µm). Current findings mean that ozonides merit further investigation as potential therapeutic agents for drug-resistant hepatocellular carcinoma.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Antimaláricos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Peróxidos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Antimaláricos/síntese química , Antimaláricos/química , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Testes de Sensibilidade Parasitária , Peróxidos/síntese química , Peróxidos/química , Plasmodium falciparum/crescimento & desenvolvimento , Relação Estrutura-Atividade
20.
Biochem Biophys Res Commun ; 382(2): 286-91, 2009 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-19285032

RESUMO

The assembly of RAD51 recombinase on DNA substrates at sites of breakage is essential for their repair by homologous recombination repair (HRR). The signaling pathway that triggers RAD51 assembly at damage sites to form subnuclear foci is unclear. Here, we provide evidence that c-ABL, a tyrosine kinase activated by DNA damage which phosphorylates RAD51 on Tyr-315, works at a previously unrecognized, proximal step to initiate RAD51 assembly. We first show that c-ABL associates with chromatin after DNA damage in a manner dependent on its kinase activity. Using RAD51 mutants that are unable to oligomerize to form a nucleoprotein filament, we separate RAD51 assembly on DNA to form foci into two steps: stable chromatin association followed by oligomerization. We show that phosphorylation on Tyr-315 by c-ABL is required for chromatin association of oligomerization-defective RAD51 mutants, but is insufficient to restore oligomerization. Our findings suggest a new model for the regulation of early steps of HRR.


Assuntos
Cromatina/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Rad51 Recombinase/metabolismo , Linhagem Celular , Dano ao DNA , Humanos , Fosforilação , Rad51 Recombinase/genética , Tirosina/genética , Tirosina/metabolismo
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