A phenolic small molecule inhibitor of RNase L prevents cell death from ADAR1 deficiency

Figures

• 

  Fig. 1.. Identification of a small-molecule inhibitor of the ribonuclease activity of porcine RNase L. (A) Hit profile against the Ontario Institute for Cancer Research 500-compound protein kinase inhibitor library using an in vitro fluorescence ribonuclease assay. (B) Chemical structure of the RNase L small-molecule inhibitor ellagic acid. (C) Dose–response inhibition profile of ellagic acid against RNase L using the in vitro screening assay. Data represent mean ± SEM of four binding curves.

• 

  Fig. 2.. SAR and cross-reactivity analysis of a phenolic analog series (A–D) on the ribonuclease activities of porcine RNase L and murine IRE1: chemical structures (Left) and ribonuclease inhibition profiles (black for RNase L and blue for IRE1; Right). Data represents mean ± SEM of three binding curves. Substituent numbering scheme is shown in A for the primary hit, ellagic acid.

• 

  Fig. 3.. Enzymatic and biophysical characterization of the mechanism of action of EA and VAL on porcine RNase L. (A, i) Binding of BODIPY-ATP to RNase L as assessed by monitoring the fluorescence polarization signal of BODIPY-ATP in the presence of increasing concentrations of RNase L. Kd represents mean ± SEM of three binding curves. (A, ii) In vitro competition binding profiles of EA, VAL, and ATP to RNase L using a BODIPY-ATP fluorescent probe as assessed by fluorescence polarization. EC50 represents mean ± SEM of three displacement curves. (B) Effect of enzyme concentration on the dose–response inhibition profiles of VAL against RNase L ribonuclease activity. IC50 represents mean ± SEM of three inhibition profiles. (C) Effect of RNA substrate concentration on the dose–response inhibition profiles of VAL against RNase L ribonuclease activity. A total of 0.4 nM of RNase L was used for the reactions. IC50 values represent mean ± SEM of three inhibition profiles.

• 

  Fig. 4.. Binding of VAL to porcine RNase L assessed by NMR and SPR. (A–D) Dose-dependent CPMG-NMR spectra for VAL recorded in the presence of the indicated concentrations of RNase L, 2-5A, and ATP/MgCl2. (E–G) Steady-state binding analysis of VAL to immobilized RNase L using SPR. Representative plots are shown for one of three replicate experiments. Kd represents the average for n = 3 ± SD.

• 

  Fig. 5.. Inhibition of RNase L from different species by VAL in intact cell lines and in primary cells. (A) Human A549 cells were incubated with different concentrations (as indicated) of EA, VAL, and sunitinib (SU) for 3 h before transfection with 12 ng/mL pIC for 2 h; (B) WT and RNase L (RL) KO A549 cells were incubated with EA or VAL for 3 h and then transfected with 10 ng/mL of pIC for 2 h; and (C) mouse WT L929 cells were incubated with or without EA or VAL for 3 h and then transfected with 0.4 or 2 µg/mL of pIC for 2 h or mock-transfected without removing compounds. (D) Mouse bone marrow macrophages (BMMs) were treated with 10 µM VAL for 3 h and then transfected with 1 µg/mL of pIC for 5 h or mock-transfected without removing VAL. Two (A and D) or three (B and C) biological replicates were done. Total RNA isolated from the cells was separated on RNA chips as described previously (9).

• 

  Fig. 6.. VAL lacks cytotoxicity and prevents cell death from dsRNA or ADAR1 deficiency. A549 cells were cultured in the absence or presence of the indicated concentrations of VAL (A), EA (B), or sunitinib (SU; C). Percent cell survival was measured by real-time imaging of dye-labeled cells (Methods). Data represent the mean ± SD of three survival profiles. (D) Effect of VAL on pIC-induced cell death in A549. A549 cells were preincubated with or without 10 µM VAL for 2 h. The cells were then either mock-transfected with Lipofectamine 2000 (Invitrogen) alone (number of replicates, n = 3) or transfected with 20 ng/mL pIC (number of replicates, n = 9). Percentage of cell survival was determined by real-time imaging (Methods). Data are averages ± SD. (E) MHV NS2 expressing ADAR1 WT or ADAR KO A549 cells were treated with siRNAs against NS2 (siNS2-1, siNS2-2) or nonspecific control siGFP. At the time of transfection, cells were treated with 20 μM of VAL or mock-treated, and, at 96 h post transfection, cells were fixed and stained with crystal violet. (F) Western blots of A549 cells transfected with control siRNA or siRNA against ADAR1 for 48 h probed with anti-ADAR1 or anti–β-actin antibody as…