A-966492

A-966492 is one of the most potent PARP inhibitors. This compound displays excellent potency against the PARP-1 enzyme with a K_iof 1 nM and an EC50 of 1 nM in a whole cell assay. It significantly enhances the efficacy of TMZ in a dose-dependent manner. In addition, this chemical is orally bioavailable across multiple species, crosses the blood−brain barrier, and appears to distribute into tumor tissue. It represents a promising, structurally diverse benzimidazole analogue and is being further characterized preclinically. [1]

A-966492 also demonstrates good in vivo efficacy in a B16F10 subcutaneous murine melanoma model in combination and in an MX-1 breast cancer xenograft model both as a single agent and in combination. In addition, this compound has excellent pharmaceutical properties and has demonstrated in vivo efficacy in preclinical mouse tumor models in combination with TMZ, as well as single agent activity in a BRCA1-deficient MX-1 tumor model. It is further characterized in Sprague−Dawley rats, beagle dogs, and cynomolgus monkeys, with this chemical demonstrating oral bioavailabilities of 34−72% and half-lives of 1.7−1.9 hours. In vivo, it demonstrates significant enhancement of the efficacy of TMZ in a murine B16F10 syngeneic melanoma model, with the combination groups showing superior efficacy. [1]

• Essai enzymatique PARP

  L'essai enzymatique est réalisé dans un tampon contenant 50 mM Tris, pH 8,0, 1 mM dithiothréitol (DTT) et 4 mM MgCl₂. Les réactions PARP contiennent 1,5 μM [³H]-NAD⁺ (1,6 μCi/mmol), 200 nM d'histone H1 biotinylée, 200 nM d'ADNdb, et 1 nM d'enzyme PARP-1 ou 4 nM d'enzyme PARP-2. Les auto-réactions utilisant une détection basée sur des billes SPA sont réalisées dans des volumes de 100 μL dans des plaques à 96 puits blanches. Les réactions sont initiées par l'ajout de 50 μL de mélange de substrat NAD⁺ 2X à 50 μL de mélange enzymatique 2× contenant PARP et ADN. Ces réactions sont terminées par l'ajout de 150 μL de 1,5 mM de benzamide (environ 1 × 10³ fois son IC50). Une quantité de 170 μL des mélanges réactionnels arrêtés est transférée sur des plaques Flash Plates recouvertes de streptavidine, incubée pendant 1 heure et comptée à l'aide d'un compteur à scintillation de microplaques TopCount. Les données K_i sont déterminées à partir des courbes d'inhibition à diverses concentrations de substrat.

• Lignées cellulaires

  C41 cell

• Concentrations

  ~10 nM

• Temps d'incubation

  30 minutes

• Méthode

  C41 cells are treated with A-966492 for 30 minutes in a 96-well plate. PARP are activated by damaging DNA with 1 mM H₂O₂ for 10 minutes. Cells are washed with ice-cold phosphate-buffered saline (PBS) once and fixed with prechilled methanol/acetone (7:3) at -20 °C for 10 minutes. After they are air-dried, plates are rehydrated with PBS and blocked using 5% nonfat dry milk in PBS-Tween(0.05%) (blocking solution) for 30 minutes at room temperature. Cells are incubated with anti-PAR antibody 10H (1:50) in blocking solution at room temperature for 60 minutes followed by washing with PBS-Tween20 five times, and incubation with goat antimouse 5(6)-isothiocyanate (FITC)-coupled antibody (1:50) and 1 μg/mL 40,6-diamidino-2-phenylindole (DAPI) in blocking solution at room temperature for 60 minutes. After washing with PBS-Tween20 5 times, analysis is performed using an fmax Fluorescence Microplate Reader set at the excitation and emission wavelength for FITC or the excitation and emission wavelength for DAPI. PARP activity (FITC signal) is normalized with cell numbers (DAPI).

• Modèles animaux

  A 0.2 cc amount of a 1:10 dilution of tumor brei in 45% Matrigel and 45% Spinner MEM is injected subcutaneously into the flank of female SCID mice.

• Posologies

  12.5, 25, and 50 mg/kg/day, for 14 days

• Administration

  Orally administrated