RESUMO
DNA-encoded libraries (DELs) have been broadly applied to identify chemical probes for target validation and lead discovery. To date, the main application of the DEL platform has been the identification of reversible ligands using multiple rounds of affinity selection. Irreversible (covalent) inhibition offers a unique mechanism of action for drug discovery research. In this study, we report a developing method of identifying irreversible (covalent) ligands from DELs. The new method was validated by using 3C protease (3CP) and on-DNA irreversible tool compounds (rupintrivir derivatives) spiked into a library at the same concentration as individual members of that library. After affinity selections against 3CP, the irreversible tool compounds were specifically enriched compared with the library members. In addition, we compared two immobilization methods and concluded that microscale columns packed with the appropriate affinity resin gave higher tool compound recovery than magnetic beads.
Assuntos
Descoberta de Drogas/métodos , Biblioteca Gênica , Proteases Virais 3C , Cromatografia de Afinidade , Cisteína Endopeptidases/metabolismo , Humanos , Microesferas , Proteínas Virais/metabolismoRESUMO
A DNA-encoded macrocyclic peptide library was designed and synthesized with 2.4 × 1012 members composed of 4-20 natural and non-natural amino acids. Affinity-based selection was performed against two therapeutic targets, VHL and RSV N protein. On the basis of selection data, some peptides were selected for resynthesis without a DNA tag, and their activity was confirmed.
Assuntos
Biblioteca de Peptídeos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Proteínas Virais/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Aminoácidos/química , DNA/química , Avaliação Pré-Clínica de Medicamentos/métodos , Terapia de Alvo Molecular , Peptídeos Cíclicos/genética , Reação em Cadeia da Polimerase , Vírus Sinciciais Respiratórios , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Proteína Supressora de Tumor Von Hippel-Lindau/químicaRESUMO
The sirtuins SIRT1, SIRT2, and SIRT3 are NAD(+) dependent deacetylases that are considered potential targets for metabolic, inflammatory, oncologic, and neurodegenerative disorders. Encoded library technology (ELT) was used to affinity screen a 1.2 million heterocycle enriched library of DNA encoded small molecules, which identified pan-inhibitors of SIRT1/2/3 with nanomolar potency (e.g., 11c: IC50 = 3.6, 2.7, and 4.0 nM for SIRT1, SIRT2, and SIRT3, respectively). Subsequent SAR studies to improve physiochemical properties identified the potent drug like analogues 28 and 31. Crystallographic studies of 11c, 28, and 31 bound in the SIRT3 active site revealed that the common carboxamide binds in the nicotinamide C-pocket and the aliphatic portions of the inhibitors extend through the substrate channel, explaining the observable SAR. These pan SIRT1/2/3 inhibitors, representing a novel chemotype, are significantly more potent than currently available inhibitors, which makes them valuable tools for sirtuin research.
Assuntos
Descoberta de Drogas , Pirimidinas/química , Pirimidinas/farmacologia , Sirtuínas/antagonistas & inibidores , Humanos , Modelos Moleculares , Conformação Proteica , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/química , Sirtuína 2/antagonistas & inibidores , Sirtuína 2/química , Sirtuína 3/antagonistas & inibidores , Sirtuína 3/química , Sirtuínas/químicaRESUMO
A method for the synthesis of polypeptides modified with a tetrazole ring at the N-terminus is described. Reaction of the N-terminal amino group of solid-supported peptides with arylisothiocyanates generates thiourea intermediates, which upon treatment with Mukaiyama's reagent (2-chloro-1-methylpyridinium iodide) generate electrophilic carbodiimide functionality. Trapping by the azide anion and electrocyclization of the intermediate imidoylazide generates an aryl-substituted 5-aminotetrazole at the N-terminus of the peptide. To prevent competitive cyclization of a neighboring amide N-H into the carbodiimide, there should not be a free N-H at the [X-1] position relative to the activated carbodiimide. Protection of the N-H group at this position or incorporation of a secondary amino acid is thus required for optimal tetrazole formation. Cleavage from the resin releases the hybrid molecules incorporating a 5-aminotetrazole ring conjugated onto a peptidic fragment.
Assuntos
Azidas/química , Elétrons , Compostos Heterocíclicos/química , Imidas/química , Peptídeos/síntese química , Tetrazóis/química , Ciclização , Bases de Dados de Proteínas , Compostos Heterocíclicos/síntese química , Hidantoínas/química , Estrutura Molecular , Peptídeos/química , Tioureia/químicaRESUMO
A method for the heterocyclic modification of the N-terminus of a peptide is described. Reaction of the N-terminal amino group of solid-supported peptides with arylisothiocyanates generates a thiourea intermediate, as in the first step of Edman degradation. Treatment of the resin-supported peptide-thioureas with Mukaiyama's reagent (2-chloro-1-methylpyridinium iodide) generates an electrophilic carbodiimide functionality, which undergoes rapid intramolecular trapping by the adjacent amide group to give a 2-iminohydantoin ring at the N-terminus of the peptide. The dehydrothiolation step in the presence of Mukaiyama's reagent prevents Edman degradation from occurring, in essence leading to a "diversion" of the Edman degradation. Cleavage from the resin then releases the hybrid molecules incorporating a 2-iminohydantoin ring conjugated onto a peptidic fragment. A 400-member library of the iminohydantoin-peptide hybrids was synthesized using this approach, starting from a chlorotrityl resin-supported tripeptides.