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]

• Ensayo enzimático de PARP

  El ensayo enzimático se lleva a cabo en un tampón que contiene 50 mM Tris, pH 8,0, 1 mM ditiotreitol (DTT) y 4 mM MgCl₂. Las reacciones de PARP contienen 1,5 μM [³H]-NAD⁺ (1,6 μCi/mmol), 200 nM de histona H1 biotinilada, 200 nM de ADN de cadena sencilla, y 1 nM de la enzima PARP-1 o 4 nM de la enzima PARP-2. Las autorreacciones que utilizan detección basada en perlas de SPA se realizan en volúmenes de 100 μL en placas blancas de 96 pocillos. Las reacciones se inician añadiendo 50 μL de una mezcla de sustrato NAD⁺ 2X a 50 μL de una mezcla enzimática 2× que contiene PARP y ADN. Estas reacciones se terminan mediante la adición de 150 μL de 1,5 mM de benzamida (aproximadamente 1 × 10³ veces su IC50). Una cantidad de 170 μL de las mezclas de reacción detenidas se transfiere a placas Flash recubiertas de estreptavidina, se incuba durante 1 hora y se cuenta utilizando un contador de centelleo de microplacas TopCount. Los datos de K_i se determinan a partir de curvas de inhibición a varias concentraciones de sustrato.

• Líneas celulares

  C41 cell

• Concentraciones

  ~10 nM

• Tiempo de incubación

  30 minutes

• Método

  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).

• Modelos animales

  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.

• Dosificaciones

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

• Administración

  Orally administrated