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 & inibidoresRESUMO
Rad51 is a key protein in DNA repair by homologous recombination and an important target for development of drugs in cancer therapy. 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) has been used in clinic during the past 30 years as an inhibitor of anion transporters and channels. Recently DIDS has been demonstrated to affect Rad51-mediated homologous pairing and strand exchange, key processes in homologous recombination. Consequently, DIDS has been considered as a potential revertant of radio- and chemo-resistance of cancer cells, the major causes of therapy failure. Here, we have investigated the behavior of DIDS towards serum albumins. The effects of environmental factors, primarily, solvent polarity, on DIDS stability were evaluated, and the mechanisms of interaction of DIDS with human or bovine serum albumin were analyzed using isothermal calorimetry, circular dichroism and fluorescence spectroscopies. DIDS interaction with both serum albumins have been demonstrated, and the interaction characteristics have been determined. By comparing these characteristics for several DIDS derivatives, we have identified the DIDS moiety essential for the interaction. Furthermore, site competition data indicate that human albumin has two DIDS-binding sites: a high-affinity site in the IIIA subdomain and a low-affinity one in the IB subdomain. Molecular docking has revealed the key molecular moieties of DIDS responsible for its interactions in each site and shown that the IB site can bind two ligands. These findings show that binding of DIDS to serum albumin may change the balance between the free and bound DIDS forms, thereby affecting its bioavailability and efficacy against Rad51.
Assuntos
Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/análogos & derivados , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Soroalbumina Bovina/metabolismo , Albumina Sérica Humana/metabolismo , Animais , Sítios de Ligação , Bovinos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Rad51 Recombinase/metabolismoRESUMO
A family of bi- and tetrametallic gold(I) phosphine dithiocarbamate complexes were synthesized, starting from cyclam and dimethylcyclam polyazamacrocycles, respectively, along with their monometallic gold(I) chloridophosphine precursors. Their antiproliferative properties were evaluated on two cancer cell lines (A549 and NSCLC-N6-L16). Most of the mono- and bimetallic complexes displayed strong activities and, in particular, one bimetallic derivative showed antiproliferative properties in the low micromolar range. Insights into the structure-activity relationships are given, along with determination of the thioredoxin reductase inhibition potential, two-photon imaging of the fluorescent derivatives, and evaluation of gold uptake.
Assuntos
Antineoplásicos/síntese química , Ouro/farmacocinética , Fosfinas , Tiocarbamatos/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Complexos de Coordenação/síntese química , Complexos de Coordenação/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imagem Óptica , Relação Estrutura-Atividade , Tiocarbamatos/síntese química , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidoresRESUMO
2-Ethylphenyl 4-(3-ethylureido)benzenesulfonate (SFOM-0046) is a novel anticancer agent that arrests cell cycle in S-phase and causes DNA replication stress leading to the phosphorylation of H2AX into γ-H2AX. First, using the M21, HT29, HT-1080 and HeLa cell lines, we confirmed that S-phase cell cycle arrest and γ-H2AX foci induction by SFOM-0046 is a general mechanism occurring in diverse cancer cell lines. In addition to γ-H2AX, SFOM-0046 activates preferentially ATR-Chk1 in M21 and HT29 cells while both ATR-Chk1 and ATM-Chk2 pathways are activated in HCT116 cells. Co-localization of SFOM-0046-induced 53BP1 foci with γ-H2AX foci validates that the DNA damage generated corresponds to double-strand-breaks (DSBs). Consistent with an S-phase arrest, SFOM-0046 treatment induces RAD51 foci formation but not DNA-PKcs foci, confirming that homologous recombination is the major DSB repair pathway targeted by the drug. Furthermore, using isogenic HCT116 p53+/+ and HCT116 p53-/- cells, we showed that p53 plays a key role in the survival mechanism to SFOM-0046. Finally, SFOM-0046 exhibits a dose-dependent antitumor activity on human fibrosarcoma HT-1080 tumours grafted onto chick chorioallantoic membranes without showing embryo toxicity even at high doses. Altogether, our results highlight SFOM-0046 as a very promising drug that induces a replication stress response.
Assuntos
Benzenossulfonatos/farmacologia , Dano ao DNA , Reparo do DNA , Compostos de Fenilureia/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Replicação do DNA , HumanosRESUMO
For decades, radiotherapy and chemotherapy were the two only approaches exploiting DNA repair processes to fight against cancer. Nowadays, cancer therapeutics can be a major challenge when it comes to seeking personalized targeted medicine that is both effective and selective to the malignancy. Over the last decade, the discovery of new targeted therapies against DNA damage signalling and repair has offered the possibility of therapeutic improvements in oncology. In this review, we summarize the current knowledge of DNA damage signalling and repair inhibitors, their molecular and cellular effects, and future therapeutic use.