RESUMEN
3-(ß-d-Glucopyranosyl)-5-substituted-1,2,4-triazoles have been revealed as an effective scaffold for the development of potent glycogen phosphorylase (GP) inhibitors but with the potency very sensitive to the nature of the alkyl/aryl 5-substituent (Kun et al., Eur. J. Med. Chem. 2014, 76, 567). For a training set of these ligands, quantum mechanics-polarized ligand docking (QM-PLD) demonstrated good potential to identify larger differences in potencies (predictive index PIâ¯=â¯0.82) and potent inhibitors with Ki'sâ¯<â¯10⯵M (AU-ROCâ¯=â¯0.86). Accordingly, in silico screening of 2335 new analogues exploiting the ZINC docking database was performed and nine predicted candidates selected for synthesis. The compounds were prepared in O-perbenzoylated forms by either ring transformation of 5-ß-d-glucopyranosyl tetrazole by N-benzyl-arenecarboximidoyl chlorides, ring closure of C-(ß-d-glucopyranosyl)formamidrazone with aroyl chlorides, or that of N-(ß-d-glucopyranosylcarbonyl)arenethiocarboxamides by hydrazine, followed by deprotections. Kinetics experiments against rabbit muscle GPb (rmGPb) and human liver GPa (hlGPa) revealed five compounds as potent low µM inhibitors with three of these on the submicromolar range for rmGPa. X-ray crystallographic analysis sourced the potency to a combination of favorable interactions from the 1,2,4-triazole and suitable aryl substituents in the GP catalytic site. The compounds also revealed promising calculated pharmacokinetic profiles.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Glucógeno Fosforilasa/antagonistas & inhibidores , Teoría Cuántica , Triazoles/farmacología , Células CACO-2 , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Glucógeno Fosforilasa/metabolismo , Humanos , Cinética , Ligandos , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/químicaRESUMEN
Phosphorylase kinase (PhK) has been linked with a number of conditions such as glycogen storage diseases, psoriasis, type 2 diabetes and more recently, cancer (Camus et al., 2012 [6]). However, with few reported structural studies on PhK inhibitors, this hinders a structure based drug design approach. In this study, the inhibitory potential of 38 indirubin analogues have been investigated. 11 of these ligands had IC50 values in the range 0.170-0.360µM, with indirubin-3'-acetoxime (1c) the most potent. 7-Bromoindirubin-3'-oxime (13b), an antitumor compound which induces caspase-independent cell-death (Ribas et al., 2006 [20]) is revealed as a specific inhibitor of PhK (IC50=1.8µM). Binding assay experiments performed using both PhK-holo and PhK-γtrnc confirmed the inhibitory effects to arise from binding at the kinase domain (γ subunit). High level computations using QM/MM-PBSA binding free energy calculations were in good agreement with experimental binding data, as determined using statistical analysis, and support binding at the ATP-binding site. The value of a QM description for the binding of halogenated ligands exhibiting σ-hole effects is highlighted. A new statistical metric, the 'sum of the modified logarithm of ranks' (SMLR), has been defined which measures performance of a model for both the "early recognition" (ranking earlier/higher) of active compounds and their relative ordering by potency. Through a detailed structure activity relationship analysis considering other kinases (CDK2, CDK5 and GSK-3α/ß), 6'(Z) and 7(L) indirubin substitutions have been identified to achieve selective PhK inhibition. The key PhK binding site residues involved can also be targeted using other ligand scaffolds in future work.
Asunto(s)
Hipoglucemiantes/química , Indoles/química , Oximas/química , Fosforilasa Quinasa/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Secuencias de Aminoácidos , Sitios de Unión , Quinasa 2 Dependiente de la Ciclina/química , Quinasa 5 Dependiente de la Ciclina/química , Glucógeno Sintasa Quinasa 3/química , Glucógeno Sintasa Quinasa 3 beta , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Fosforilasa Quinasa/química , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Termodinámica , Interfaz Usuario-ComputadorRESUMEN
All possible isomers of N-ß-D-glucopyranosyl aryl-substituted oxadiazolecarboxamides were synthesised. O-Peracetylated N-cyanocarbonyl-ß-D-glucopyranosylamine was transformed into the corresponding N-glucosyl tetrazole-5-carboxamide, which upon acylation gave N-glucosyl 5-aryl-1,3,4-oxadiazole-2-carboxamides. The nitrile group of the N-cyanocarbonyl derivative was converted to amidoxime which was ring closed by acylation to N-glucosyl 5-aryl-1,2,4-oxadiazole-3-carboxamides. A one-pot reaction of protected ß-D-glucopyranosylamine with oxalyl chloride and then with arenecarboxamidoximes furnished N-glucosyl 3-aryl-1,2,4-oxadiazole-5-carboxamides. Removal of the O-acetyl protecting groups by the Zemplén method produced test compounds which were evaluated as inhibitors of glycogen phosphorylase. Best inhibitors of these series were N-(ß-D-glucopyranosyl) 5-(naphth-1-yl)-1,2,4-oxadiazol-3-carboxamide (Ki = 30 µM), N-(ß-D-glucopyranosyl) 5-(naphth-2-yl)-1,3,4-oxadiazol-2-carboxamide (Ki =33 µM), and N-(ß-D-glucopyranosyl) 3-phenyl-1,2,4-oxadiazol-5-carboxamide (Ki = 104 µM). ADMET property predictions revealed these compounds to have promising oral drug-like properties without any toxicity.