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]

• PARP 효소 분석

  효소 분석은 50 mM Tris, pH 8.0, 1 mM dithiothreitol(DTT) 및 4 mM MgCl₂를 포함하는 완충액에서 수행됩니다. PARP 반응물은 1.5 μM [³H]-NAD⁺(1.6 μCi/mmol), 200 nM 비오틴화 히스톤 H1, 200 nM slDNA 및 1 nM PARP-1 또는 4 nM PARP-2 효소를 포함합니다. SPA 비드 기반 검출을 활용한 자동 반응은 흰색 96-웰 플레이트에서 100 μL 용량으로 수행됩니다. 반응은 50 μL의 2X NAD⁺ 기질 혼합물을 PARP와 DNA를 포함하는 50 μL의 2× 효소 혼합물에 첨가하여 시작됩니다. 이러한 반응은 150 μL의 1.5 mM 벤즈아미드(IC50 대비 약 1 × 10³배)를 첨가하여 종료됩니다. 중단된 반응 혼합물 170 μL를 스트렙타비딘 코팅된 Flash Plate로 옮기고 1시간 동안 배양한 후, TopCount 마이크로플레이트 섬광 계수기를 사용하여 계수합니다. K_i 데이터는 다양한 기질 농도에서의 억제 곡선으로부터 결정됩니다.

• 세포주

  C41 cell

• 농도

  ~10 nM

• 배양 시간

  30 minutes

• 방법

  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).

• 동물 모델

  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.

• 용량

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

• 투여

  Orally administrated