RESUMO
The purpose of this study was to design a polyamidoamine (PAMAM)-based nanovector for the efficient delivery of methotrexate to U87 glioma cells. To this end, 0-100% acetylated PAMAM dendrimers of the fourth generation were synthesized and evaluated using drug encapsulation measurements, molecular dynamics simulations, neurotoxicity assays and neuronal internalization experiments. The best system was tested as a nanovector for methotrexate delivery to U87 glioma cells. The authors found that 25% acetylated PAMAM dendrimers of the fourth-generation combine low intrinsic toxicity, large drug complexation capacity and efficient internalization into hippocampal neurons. Nanovector complexation enhances the cytotoxic response of methotrexate against U87 glioma cells compared with free drug solutions. In conclusion, 25% acetylated PAMAM dendrimers of the fourth-generation increase drug uptake by glioma cells and thereby act as efficient nanovectors for methotrexate delivery.
Assuntos
Dendrímeros , Glioma , Dendrímeros/uso terapêutico , Sistemas de Liberação de Medicamentos , Glioma/tratamento farmacológico , Humanos , Metotrexato/uso terapêutico , PoliaminasRESUMO
Microtubules (MT) are cytoskeletal polymers of αß-tubulin dimers that play a critical role in many cellular functions. Diverse antimitotic drugs bind to MT and disrupt their dynamics acting as MT stabilizing or destabilizing agents. The occurrence of undesired side effects and drug resistance encourages the search for novel MT binding agents with chemically diverse structures and different interaction profiles compared to known active compounds. This work reports the rational discovery of seven novel MT stabilizers using a combination of molecular modeling methods and in vitro experimental assays. Virtual screening, similarity filtering, and molecular mechanics generalized Born surface area (MM/GBSA) binding free energy refinement were employed to select seven potential candidates with high predicted affinity toward the non-taxoid site for MT stabilizers on ß-tubulin. MD simulations of 150 ns on reduced MT models suggest that candidate compounds strengthen the longitudinal interactions between tubulin dimers across protofilaments, which is a primary molecular mechanism of action for known MT stabilizers. In vitro MT polymerization assays confirmed that all candidates promote MT assembly at concentrations of >50 mM and exhibit noncompetitive MT polymerization profiles when cotreating with Taxol. Preliminary HeLa cell viability assays revealed a moderate cytotoxic effect for the compounds under study at 100 µM concentration. These results support the validity of our rational discovery strategy and the use of molecular modeling methods to pursue the search and optimization of new MT targeting agents.
Assuntos
Excipientes , Paclitaxel , Células HeLa , Humanos , Microtúbulos , Paclitaxel/farmacologia , Tubulina (Proteína)RESUMO
Glycine receptors (GlyRs) are members of the pentameric ligand-gated ionic channel family (pLGICs) and mediate fast inhibitory neurotransmission in the brain stem and spinal cord. The function of GlyRs can be modulated by positive allosteric modulators (PAMs). So far, it is largely accepted that both the extracellular (ECD) and transmembrane (TMD) domains constitute the primary target for many of these PAMs. On the other hand, the contribution of the intracellular domain (ICD) to the PAM effects on GlyRs remains poorly understood. To gain insight about the role of the ICD in the pharmacology of GlyRs, we examined the contribution of each domain using a chimeric receptor. Two chimeras were generated, one consisting of the ECD of the prokaryotic homologue Gloeobacter violaceus ligand-gated ion channel (GLIC) fused to the TMD of the human α1GlyR lacking the ICD (Lily) and a second with the ICD (Lily-ICD). The sensitivity to PAMs of both chimeric receptors was studied using electrophysiological techniques. The Lily receptor showed a significant decrease in the sensitivity to four recognized PAMs. Remarkably, the incorporation of the ICD into the Lily background was sufficient to restore the wild-type α1GlyR sensitivity to these PAMs. Based on these data, we can suggest that the ICD is necessary to form a pLGIC having full sensitivity to positive allosteric modulators.