RESUMO
Cytosolic 5'-nucleotidase II (cN-II) regulates the intracellular nucleotide pools within the cell by catalyzing the dephosphorylation of 6-hydroxypurine nucleoside 5'-monophosphates. Beside this physiological function, high level of cN-II expression is correlated with abnormal patient outcome when treated with cytotoxic nucleoside analogues. To identify its specific role in the resistance phenomenon observed during cancer therapy, we screened a particular class of chemical compounds, namely ribonucleoside phosphonates to predict them as potential cN-II inhibitors. These compounds incorporate a chemically and enzymatically stable phosphorus-carbon linkage instead of a regular phosphoester bond. Amongst them, six compounds were predicted as better ligands than the natural substrate of cN-II, inosine 5'-monophosphate (IMP). The study of purine and pyrimidine containing analogues and the introduction of chemical modifications within the phosphonate chain has allowed us to define general rules governing the theoretical affinity of such ligands. The binding strength of these compounds was scrutinized in silico and explained by an impressive number of van der Waals contacts, highlighting the decisive role of three cN-II residues that are Phe 157, His 209 and Tyr 210. Docking predictions were confirmed by experimental measurements of the nucleotidase activity in the presence of the three best available phosphonate analogues. These compounds were shown to induce a total inhibition of the cN-II activity at 2 mM. Altogether, this study emphasizes the importance of the non-hydrolysable phosphonate bond in the design of new competitive cN-II inhibitors and the crucial hydrophobic stacking promoted by three protein residues.
Assuntos
5'-Nucleotidase/antagonistas & inibidores , 5'-Nucleotidase/química , Ribonucleotídeos/química , 5'-Nucleotidase/metabolismo , Sítios de Ligação , Hidrólise , Inosina Monofosfato/química , Inosina Monofosfato/metabolismo , Modelos Moleculares , Ribonucleotídeos/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Ovarian cancer is the most lethal gynecological malignancy among US women. Paclitaxel/carboplatin is the current drug therapy used to treat ovarian cancer, but most women develop drug resistance and recurrence of the disease, necessitating alternative strategies for treatment. A possible molecular target for cancer therapy is glycogen synthase kinase 3ß (GSK3ß), a downstream kinase in the Wnt signaling pathway that is overexpressed in serous ovarian cancer. Novel maleimide-based GSK3ß inhibitors (GSK3ßi) were synthesized, selected, and tested in vitro using SKOV3 and OVCA432 serous ovarian cancer cell lines. From a panel of 10 inhibitors, GSK3ßi 9ING41 was found to be the most effective in vitro. 9ING41 induced apoptosis as indicated by 4',6-diamidino-2-phenylindole-positive nuclear condensation, poly (ADP-ribose) polymerase cleavage, and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The mechanism for apoptosis was through caspase-3 cleavage. GSK3ßi upregulated phosphorylation of the inhibitory serine residue of GSK3ß in OVCA432 and SKOV3 cell lines and also inhibited phosphorylation of the downstream target glycogen synthase. An in-vivo xenograft study using SKOV3 cells demonstrated that tumor progression was hindered by 9ING41 in vivo. The maximum tolerated dose for 9ING41 was greater than 500 mg/kg in rats. Pharmacokinetic analysis showed 9ING41 to have a bioavailability of 4.5% and to be well distributed in tissues. Therefore, GSK3ß inhibitors alone or in combination with existing drugs may hinder the growth of serous ovarian cancers.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Animais , Disponibilidade Biológica , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/farmacocinética , Feminino , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Camundongos , Camundongos Nus , Poli(ADP-Ribose) Polimerases/metabolismo , Ratos , Ratos Sprague-Dawley , Serina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Via de Sinalização Wnt , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Nucleoside phosphonates have been designed as stable 5'-mononucleotide mimics and are nowadays considered a potent class of antiviral agents. Within cells, they must be metabolised to the corresponding diphosphate to exert their biological activity. In this process, the first phosphorylation step, catalysed by nucleoside monophosphate kinases (NMP kinases), has been proposed as a bottleneck. Herein, we report the synthesis of a series of ribonucleoside phosphonate derivatives isosteric to 5'-mononucleotides, with different degrees of flexibility within the 5',6'-C-C bond, as well as different polarities, through the introduction of hydroxy groups. The influence of these modifications on the capacity of the compounds to act as substrates for appropriate human NMP kinases, involved in nucleic acids metabolism, has been investigated. Low flexibility, as well as an absence of hydroxy groups within the ribose-phosphorus architecture, is critical for efficient phosphotransfer. Among the series of pyrimidine analogues, one derivative was shown to be phosphorylated by human UMP-CMP kinase, with rates similar to those of dUMP and even better than dCMP.
Assuntos
Núcleosídeo-Fosfato Quinase/metabolismo , Organofosfonatos/química , Organofosfonatos/farmacologia , Ribonucleosídeos/química , Ribonucleosídeos/farmacologia , Antivirais/síntese química , Antivirais/química , Antivirais/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Modelos Moleculares , Organofosfonatos/síntese química , Ligação Proteica , Ribonucleosídeos/síntese química , Especificidade por SubstratoRESUMO
Recent studies have demonstrated that glycogen synthase kinase 3beta (GSK-3beta) is overexpressed in human colon and pancreatic carcinomas, contributing to cancer cell proliferation and survival. Here, we report the design, synthesis, and biological evaluation of benzofuran-3-yl-(indol-3-yl)maleimides, potent GSK-3beta inhibitors. Some of these compounds show picomolar inhibitory activity toward GSK-3beta and an enhanced selectivity against cyclin-dependent kinase 2 (CDK-2). Selected GSK-3beta inhibitors were tested in the pancreatic cancer cell lines MiaPaCa-2, BXPC-3, and HupT3. We determined that some of these compounds, namely compounds 5, 6, 11, 20, and 26, demonstrate antiproliferative activity against some or all of the pancreatic cancer cells at low micromolar to nanomolar concentrations. We found that the treatment of pancreatic cancer cells with GSK-3beta inhibitors 5 and 26 resulted in suppression of GSK-3beta activity and a distinct decrease of the X-linked inhibitor of apoptosis (XIAP) expression, leading to significant apoptosis. The present data suggest a possible role for GSK-3beta inhibitors in cancer therapy, in addition to their more prominent applications in CNS disorders.