RESUMO
MMP-2 is a validated target for the development of anticancer agents. Herein we describe the synthesis of a new series of potent phenylalanine derived hydroxamates, with increased MMP-2/MMP-9 selectivity compared to analogous hydroxamates described previously. Docking and molecular dynamics experiments have been used to account for this selectivity, and to clarify the role of the triazole ring in the binding process.
Assuntos
Desenho de Fármacos , Gelatinases/antagonistas & inibidores , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/farmacologia , Inibidores de Metaloproteinases de Matriz/síntese química , Inibidores de Metaloproteinases de Matriz/farmacologia , Técnicas de Química Sintética , Gelatinases/química , Gelatinases/metabolismo , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/metabolismo , Inibidores de Metaloproteinases de Matriz/química , Inibidores de Metaloproteinases de Matriz/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fenilalanina/química , Especificidade por Substrato , Triazóis/químicaRESUMO
Matrix metalloproteinase 2 (MMP-2) is involved in cancer development and is overexpressed in a variety of malignant tumors. MMP-2 activity is controlled mainly by transcription, proteolytic activation, and inhibition by endogenous inhibitors. It had previously been demonstrated that MMP-2 activity is also regulated by phosphorylation at several sites by protein kinase C. Here we demonstrate, by means of bioinformatics and biochemical and cellular assays, that protein kinase CK2 also acts as a modulator of MMP-2 activity. CK2 down-regulates MMP-2 in vitro, and inhibition of CK2 in human fibrosarcoma cells results in up-regulation of MMP-2. The discovery of the crosstalk between MMP-2 and CK2 opens the possibility of new combined anticancer therapies.
Assuntos
Caseína Quinase II/metabolismo , Caseína Quinase II/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Linhagem Celular Tumoral , Biologia Computacional , Regulação para Baixo/efeitos dos fármacos , Fibrossarcoma/metabolismo , Humanos , Fosforilação , Receptor Cross-Talk/efeitos dos fármacosRESUMO
Looking for water-soluble inhibitors of matrix metalloproteinase-2 (MMP-2 or gelatinase A), we have previously reported compound 1, a potent MMP-2 inhibitor with a promising selectivity over the structurally homologous MMP-9 (gelatinase B). Here we report the results of Molecular Dynamics (MD) simulations for both gelatinases (MMP-2 and MMP-9), and for the corresponding MMP/1 complexes, in an attempt to shed light on the observed selectivity between the two enzymes. These studies indicated a higher plasticity of MMP-2 at the S1' pocket and suggested an induced-fit effect at the "back door" of this pocket. On the basis of these observations, we designed 11 a-d to aid further discrimination between MMP-2 and MMP-9. Those compounds displayed notably lower inhibitory activities against MMP-9; in particular, 11 b proved to be over 100 times more active against MMP-2 than against MMP-9. MD simulations of the MMP/11 b complexes and thermodynamic integration calculations provided structural insight and relative binding energies consistent with the experimentally observed activity data. These findings demonstrate that structural differences in the S1' pocket bottom permit an improvement in selectivity in the inhibition of MMP-2 over that of MMP-9; this is of great relevance for future structure-based drug design because MMP-2 is a validated target for cancer therapy, whereas MMP-9 plays both detrimental and protective roles in cancer. This study also supports the need to consider the dynamics of the S1' pocket in order to achieve selectivity in the inhibition of MMPs.
Assuntos
Metaloproteinase 2 da Matriz/química , Inibidores de Metaloproteinases de Matriz/química , Simulação de Dinâmica Molecular , Sulfonas/química , Sítios de Ligação , Domínio Catalítico , Desenho de Fármacos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/química , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/síntese química , Inibidores de Metaloproteinases de Matriz/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Sulfonas/síntese química , Sulfonas/metabolismo , TermodinâmicaRESUMO
Water solubility is a key aspect that needs to be addressed to obtain drug-like compounds. In an effort to improve the water solubility of our recently reported nanomolar matrix metalloproteinase type 2 (MMP-2) inhibitors based on triazole-substituted hydroxamates, we synthesized a new series of α-sulfone, α-tetrahydropyran and α-piperidine, α-sulfone clicked hydroxamates and determined their inhibitory activities against both MMP-2 and MMP-9. The best results were found for 13e, a water-soluble compound that displays a low nanomolar activity against MMP-2 and is 26-fold less active against MMP-9. This finding allowed us to pursue in vitro permeability through the Caco-2 monolayer and opened the possibility of carrying out further preclinical investigations. Docking and MD simulations have been performed in order to rationalize the biological results. The inhibitory activity of this compound against a panel of ten MMPs was determined showing an interesting MMP-2/MMP-1, -8, and -14 selectivity profile. The cytotoxicity and anti-invasive activity of the compounds on highly metastatic human fibrosarcoma tumor cells (HT1080) were determined, showing, at 10 µM concentration, a decrease in cell invasiveness up to 80%.
Assuntos
Antineoplásicos/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Triazóis/farmacologia , Água/química , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Inibidores de Metaloproteinases de Matriz/síntese química , Inibidores de Metaloproteinases de Matriz/química , Modelos Moleculares , Estrutura Molecular , Teoria Quântica , Solubilidade , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/químicaRESUMO
Based on the benzo[b]naphtho[1,2-d]furan and benzo[b]naphtho[1,2-d]thiophene frameworks, a series of ligands with different basic side chains (BSCs) has been synthesized and pharmacologically evaluated. Also, their binding modes have been modelled using docking techniques. It was found that the introduction of a BSC in these systems brings about a decrease of affinity for both estrogen receptors α and ß in an in vitro competitive binding assay. However, two full antagonists of the estrogen receptor ß (9c and 9f) have been discovered, with potency in the low micromolar concentration in a cell-based luciferase reporter assay, and completely devoid of activity against the α receptor at the same concentration range. Differences in the ERα/ERß binding modes have also been rationalized with the help of molecular modelling techniques. This interesting functional profile could be used to elucidate the physiological role of each ER subtype.
Assuntos
Antineoplásicos/farmacologia , Benzofuranos/farmacologia , Naftalenos/farmacologia , Receptores de Estrogênio/antagonistas & inibidores , Tiofenos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Benzofuranos/síntese química , Benzofuranos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligantes , Células MCF-7 , Naftalenos/síntese química , Naftalenos/química , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/químicaRESUMO
A new series of MMP2 inhibitors is described, following a fragment-based drug design approach. One fragment containing an azide group and a well known hydroxamate Zinc Binding Group in a α-sulfone, α-tetrahydropyrane scaffold, has been synthesized. Water-LOGSY, STD and competition-STD experiments indicate that this fragment binds to the active site of the enzyme. A click chemistry reaction was used to connect the azide to lipophilic alkynes selected to interact selectively with the S1' subunit of MMP2, as shown by docking and molecular dynamic experiments of the designed compounds. The most potent compounds 18 and 19 displayed an IC(50) of 1.4 and 0.3 nM against MMP2 respectively, and showed negligible activity towards MMP1 and MMP7, two metalloproteinases which have a shallow S1' subsite. Compound 18 also showed a promising selectivity profile against some antitarget metalloproteinases, such as MMP8, and considerably less activity against MMP14 (IC(50) = 65 nM), and MMP9 (IC(50) = 98 nM), other MMPs characterized by having a deep S1' pocket and, therefore, more similar to MMP2.
Assuntos
Antineoplásicos/síntese química , Química Farmacêutica , Ácidos Hidroxâmicos/síntese química , Inibidores de Metaloproteinases de Matriz , Inibidores de Proteases/síntese química , Antineoplásicos/farmacologia , Azidas/química , Sítios de Ligação , Química Click , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Ácidos Hidroxâmicos/farmacologia , Concentração Inibidora 50 , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 7 da Matriz/metabolismo , Modelos Moleculares , Neoplasias/tratamento farmacológico , Inibidores de Proteases/farmacologia , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Sulfonas/químicaRESUMO
Gallic acid and anthocyanins are abundant plant food bioactives present in many fruits and vegetables, being especially important in the composition of berries. Gallic acid has been shown to possess cytotoxic properties in several cancer cell lines and to inhibit carcinogenesis in animal models. However, its mechanism of action is not yet fully understood. The aim of this study was to elucidate whether the observed inhibitory activity of gallic acid against gelatinases corresponds to its cytotoxic activity in HT1080 cells and to determine if anthocyanins could exhibit a similar behavior. Gallic acid and delphinidin-3-glucoside have shown selective cytotoxicity towards HT1080 cells. Further analysis by a migration and invasion assay showed anti-invasive activities of gallic acid, delphinidin and pelargonidin-3-glucosides. Zymographic analysis demonstrated the inhibitory activity of gallic acid at the level of secreted and activated gelatinases. Moreover, gallic acid inhibited MMP-2 and MMP-9 proteolytic activity with very similar potency. NMR and molecular modelling experiments confirmed the interaction of gallic acid with MMP-2, and suggested that it takes place within the catalytic center. In this work we give some new experimental data supporting the role of these compounds in the inhibition of metalloproteases as the mechanism for their cytotoxic activity against fibrosarcoma.