RESUMO
S-Trt Cys are used as precursors for the synthesis of protected NMe-Cys. N-Methylation of Alloc-Cys(Trt)-OH and Boc-Cys(Trt)-OH gives the corresponding N-methylated derivatives in good yields and purities, which can be further derivatized in solution to obtain a myriad of S-protected derivatives. To further broaden the scope of this methodology, the N (alpha)-amino protecting group of the NMe- S-protected Cys can be replaced easily either on the solid phase (from the Alloc precursor) or in solution (from the Boc precursor). Thus, this convenient route allows us to obtain many different protected NMe-Cys, which were of limited accessibility until now.
Assuntos
Técnicas de Química Combinatória , Cisteína/análogos & derivados , Cisteína/síntese química , Cisteína/química , Metilação , Conformação Molecular , SoluçõesRESUMO
The modulation of protein-protein interactions (PPIs) is emerging as a highly promising tool to fight diseases. However, whereas an increasing number of compounds are able to disrupt peptide-mediated PPIs efficiently, the inhibition of domain-domain PPIs appears to be much more challenging. Herein, we report our results related to the interaction between vascular endothelial growth factor (VEGF) and its receptor (VEGFR). The VEGF-VEGFR interaction is a typical domain-domain PPI that is highly relevant for the treatment of cancer and some retinopathies. Our final goal was to identify ligands able to bind VEGF at the region used by the growth factor to interact with its receptor. We undertook an extensive study, combining a variety of experimental approaches, including NMR-spectroscopy-based screening of small organic fragments, peptide libraries, and medicinal plant extracts. The key feature of the successful ligands that emerged from this study was their capacity to expose hydrophobic functional groups able to interact with the hydrophobic hot spots at the interacting VEGF surface patch.
Assuntos
Produtos Biológicos/farmacologia , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Sítios de Ligação/efeitos dos fármacos , Produtos Biológicos/síntese química , Produtos Biológicos/química , Relação Dose-Resposta a Droga , Humanos , Ligantes , Modelos Moleculares , Oligopeptídeos/síntese química , Oligopeptídeos/química , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Biblioteca de Peptídeos , Ligação Proteica/efeitos dos fármacos , Receptores de Fatores de Crescimento do Endotélio Vascular/química , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Relação Estrutura-Atividade , Fator A de Crescimento do Endotélio Vascular/químicaRESUMO
Antisense oligonucleotides (AONs) are a class of compounds with high therapeutic potential. One of the challenges facing this platform is the development of effective techniques to achieve cellular delivery. AON conjugates, in which traditional AONs are attached to certain biomolecules, can exhibit improved intracellular bioavailability in the absence of delivery systems. In this study, the lipophilic moieties docosahexaenoic acid, cholesterol, and docosanoic acid (DSA) were conjugated to various phosphorothioated DNA and chemically-modified 2'-fluoro-arabinonucleic acid AONs via an amino-hexanol-linker added to the 5'-end of the molecule. The gene silencing potential of these compounds was evaluated in vitro in the absence or presence of a transfecting agent (polyion complex micelle). Incubation with sub-micromolar concentration of DSA-conjugates could, in the absence of serum proteins, downregulate more than 60% of the targeted mRNA under carrier-free and carrier-loaded delivery methods. Gene silencing activity of carrier-free DSA-conjugates was, however, decreased in a dose-dependent fashion by adding albumin in the transfection medium. Supplementing the medium with free fatty acid prevented the interaction of the DSA-conjugate with albumin, and restored its silencing activity. These findings suggest that strategies aiming at preventing the association of hydrophobized AONs to serum proteins at the site of action may improve their activity.
Assuntos
Inativação Gênica , Oligonucleotídeos Antissenso/metabolismo , Albumina Sérica/metabolismo , Tensoativos/metabolismo , Bioensaio , Linhagem Celular Tumoral , Ensaio de Desvio de Mobilidade Eletroforética , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Masculino , Micelas , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
Polyion complex micelles (PICMs) arise from the spontaneous self-assembly of ionic polymers of opposite charges to form a condensate that is dispersed in aqueous media by a hydrophilic segment, usually poly(ethylene glycol) (PEG), present on at least one of the two ionic polymers. PICMs are used for many applications, especially drug delivery. This protocol paper describes the preparation by atom transfer radical polymerization (ATRP) of diblock copolymers of PEG bearing either positive or negative charges, both of which have been shown to form PICMs. Furthermore, methods of preparation and characterization of PICMs loaded with nucleic acid drugs are presented.
Assuntos
Portadores de Fármacos/química , Micelas , Ácidos Nucleicos/administração & dosagem , Polietilenoglicóis/química , Interações Hidrofóbicas e Hidrofílicas , Íons/química , PolimerizaçãoRESUMO
Bridged N-methyl amides are used as isosteres for depsi and thiodepsi bonds in thiocoraline. The introduction of NMe-amides in bridges mimics the thioester bonds without imposing steric hindrance and allows conservation of the hydrogen bonding map of the natural product. NMe-azathiocoraline was constructed by solid-phase N-methylation of the side chain of diaminopropionic acid (Dap). The three consecutive N-methyl amino acids could be coupled in good yields by using HATU/HOAt/DIEA in DMF, and the final octapeptide was also obtained on solid phase following a 4 + 4 fragment coupling approach. NMe-azathiocoraline (NMA) displayed nanomolar activity in the same order as the natural product and the same mode of action. In fact, modeling of NMe-azathiocoraline bonded to a TCGA sequence showed how the methyl groups remained further away from the DNA strand without changing the recognition pattern of thiocoraline. Moreover, NMe-azathiocoraline displayed an increased stability in human serum as compared to the parent natural product. This approach could be used in other depsipeptides and side chain to side chain cyclic peptides.
Assuntos
Amidas/química , Depsipeptídeos/química , Sulfetos/química , Neoplasias da Mama/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular , Linhagem Celular Tumoral , Colo/efeitos dos fármacos , Depsipeptídeos/síntese química , Depsipeptídeos/uso terapêutico , Estabilidade de Medicamentos , Ésteres/química , Humanos , Ligação de Hidrogênio , Masculino , Metilação , Conformação ProteicaRESUMO
Thiocoraline is a potent antitumor agent isolated from the marine organism Micromonospora sp. This symmetric bicyclic depsipeptide binds the minor groove of DNA. Here we report two solid-phase strategies for the syntheses of azathiocoraline and its analogues. The thioester linkage was replaced by an amide bond to improve the compound's pharmacokinetic properties. The first strategy is based on a convergent (4+4) approach, whilst the second is a stepwise synthesis, cyclizations in both approaches occurring on the solid support. These two strategies were designed to overcome problems caused by the presence of consecutive noncommercial N-methyl amino acids, to avoid epimerization during cyclization and/or fragment condensation, and to form the disulfide bridge under solid-phase conditions. The heterocyclic moiety was added in the last step of the synthesis to assist the preparation of libraries of new compounds with potential therapeutic applications.