Sirtuins are Unaffected by PARP Inhibitors Containing Planar Nicotinamide Bioisosteres
Torun Ekblad and Herwig Schuler
Introduction
PARP-family ADP-ribosyltransferases (PARPs) and sirtuin deacetylases all use NAD+ as cosubstrate for ADP-ribosyl transfer. PARP inhibitors are important research tools and several are being evaluated in cancer treatment. With the exception of a few tankyrase inhibitors, all current PARP inhibitors mimic the nicotinamide moiety in NAD+ and block the nicotinamide binding pocket. We report here that while the activities of the four human sirtuin isoforms SIRT1, SIRT2, SIRT3 and SIRT6 are blocked by sirtuin inhibitor Ex527 in vitro, they are unaffected by the seven clinical and commonly used PARP inhibitors niraparib, olaparib, rucaparib, talazoparib, veliparib, PJ34, and XAV939. These findings indicate that PARP inhibitors containing planar nicotinamide mimetics do not bind to sirtuin cofactor sites. In conclusion, a simple commercially available assay can be used to rule out interference of novel PARP inhibitors with sirtuin NAD+ binding.
Key words: chemical biology, structure-based drug design, therapeutic target
Summary
Inhibitors of ADP-ribosyltransferases (ARTs) of the poly (ADP-ribose) polymerase (PARP) family, in particular of PARP1 and PARP2, are promising candidates for treatment of cancer. Several PARP inhibitors have advanced to clinical trials (1,2), and olaparib was recently approved for treatment of BRCA-positive ovarian cancer (3). Many PARP inhibitors are also used as chemical tools to study signaling pathways and DNA repair mechanisms (4). Meanwhile, these compounds are still poorly characterized with respect to their off-target actions both within and outside of the PARP family (5).
The main therapeutic targets for PARP inhibitors, PARP1 and PARP2, and their prokaryotic ancestors are structurally unique; but they share functional similarity with a family of deacetylases: the sirtuins (silent information regulator-2; SIR2). Both PARPs and sirtuins cleave NAD+ into nicotinamide and ADP-ribose. Sirtuins couple this reaction to a number of different activities, the most prominent being protein deacetylation (6). Thus, in the deacetylation reaction pathway, sirtuins ADP-ribosylate acetyl-lysine residues prior to elimination of O-acetylADP-ribose (7–9). Sirtuin-mediated ADP-ribosylation of other amino acid side chains has also been reported (10– 14). Regardless of whether the latter are side reactions or significant in a physiological context, this highlights important similarities between sirtuins and other ARTs. Furthermore, different forms of coregulation and functional interplay between sirtuin and PARP enzymes have been reported (15). Therefore, from a pharmacological point of view, it was important to systematically investigate whether PARP inhibitors affect sirtuin activities.
We measured the deacetylase activities of recombinant human SIRT1, SIRT2, SIRT3, and SIRT6 using a fluorimetric assay. First, we conducted control experiments to verify that we could reproduce previously published IC50 values for the sirtuin inhibitor, Ex527 (16) (Figure 1A). We compared initial enzymatic rates of sirtuin activities in the presence of Ex527 over a range of inhibitor concentrations (Figure 2A), and calculated an IC50 value of 102 nM for SIRT1 inhibition from the data, which is similar to two previous estimates of 98 nM and 90 nM (16,17). IC50 values for Ex527 inhibition of the four human sirtuins are reported in Figure 2B. Incidentally, Ex527 did not inhibit PARP1, PARP10, or TNKS1 (data not shown).
Next, we tested the effects of seven PARP inhibitors on sirtuin activities. We focused on the clinical inhibitors rucaparib, veliparib, olaparib, niraparib and talazoparib as well as the commonly used research tools, PJ34 (a general PARP inhibitor) and XAV939 (a tankyrase inhibitor) (18–24). All of these compounds were designed to anchor in the nicotinamide pocket and then extend into the active site of PARP enzymes (Figure 1B) (25). At 100 lM compound concentration, no major effects were seen on any of the four sirtuin activities tested, with two exceptions: niraparib and rucaparib reduced the initial enzymatic rate of SIRT6 (Figure 2C). We then carried out concentration–response experiments on SIRT6 activity, with niraparib and rucaparib concentrations ranging from 3 to 200 lM. No significant inhibition could be established below 100 lM compound concentration (Figure 2D).
We conclude that at concentrations sufficient to inhibit PARP activities, the seven PARP inhibitors tested here are unlikely to affect sirtuin activities in cellular or in vivo experiments. Our results indicate that (i) sirtuin cofactor sites are inaccessible for PARP inhibitors that are based on planar nicotinamide bioisosteres, and (ii) sirtuin active sites discriminate between NAD+ and the most important PARP inhibitors.
A rational explanation for the first conclusion is thus the planarity of these PARP inhibitors in their nicotinamide mimicking moieties (Figure 1): Ex527 is characterized by an asymmetric carbon; only the S-isomer inhibits sirtuins efficiently which is illustrated by the crystal complexes of sirtuins with this and similar compounds (16,17,26). Ex527 requires presence of NAD+ and substrate for sirtuin binding (16) and the crystal structures show Ex527 binding in the so-called C-pocket that is otherwise occupied by the nicotinamide moiety of NAD+ (17,26). Accordingly, full occupancy of Ex527 in the C-pocket was achieved in the postcatalytic state, in a co-complex with O-acetyl-ADPribose (17; Figure 1C).
The molecular basis for the second observation likely lies in the fundamentally different structures of the NAD+-binding pockets of sirtuins and PARP enzymes (27,28). Notably in this context, Ex527 is the only well-characterized sirtuin inhibitor containing a nicotinamide bioisostere. Nevertheless, the above considerations cannot dismiss the need for experimental validation of novel PARP inhibitors with respect to pharmacologically relevant off-target effects via sirtuins.
Methods and Materials
Figure 2: Fluorimetric analysis of sirtuin activities and the effects of the sirtuin inhibitor Ex527 as well as five clinical PARP inhibitors and two commonly used research tools. (A) Concentration dependent inhibition of recombinant human SIRT1, SIRT2, SIRT3, and SIRT6 by Ex527. Initial rates were normalized against rates detected in the absence of inhibitor and presented as % inhibition. (B) Ex527 potencies determined from the data in panel A, calculated by non-linear regression. LogIC50 values are presented as means SD; IC50 values as means and, in parentheses, 95% confidence intervals. (C) Effect of PARP inhibitors (100 lM) on the activities of recombinant human SIRT-1, -2, -3, and -6. (D) Concentration–response experiments for the effects of niraparib and rucaparib on SIRT6 activity. The effects of several PARP inhibitors on SIRT3 activity could not be assessed due to compound autofluorescence (see Methods and Materials).
Materials
Sirtuin inhibitor Ex527 was purchased from SigmaAldrich, and PARP inhibitors niraparib, olaparib, PJ34, rucaparib, talazoparib, veliparib, and XAV939 were purchased from Selleck or SigmaAldrich. Enzyme containing SIRT1/Sirt2, SIRT2, SIRT3, and SIRT6 fluorometric assay kits were obtained from CycLex (cat. nos. CY-1151, CY-1152, CY-1153, and CY-1156, respectively).
Enzyme inhibition assays
Deacetylase activity of recombinant sirtuins was studied using the CycLex assay kits according to manufacturer’s protocols, with the following exceptions. Absorbance and emission spectra of PARP inhibitors indicated that niraparib, PJ34, rucaparib and talazoparib autofluorescence interfered with the SIRT1 and SIRT3 assays (Figure S1). To circumvent this problem, the reaction rates of SIRT1 and SIRT3 were also determined using the fluorescent substrate provided with the SIRT2 assay kit, which has a longer excitation wavelength. SIRT1 had similar enzymatic rates using the two substrates and therefore the effects of the PARP inhibitors on SIRT1 could be measured using the SIRT2-substrate. SIRT3 inhibition data were only determined for Ex527, olaparib, veliparib, and XAV939.
PARP inhibitors were used at final assay concentrations of 100 lM (1% DMSO). Concentration–response experiments were performed with PARP or sirtuin inhibitors at 0 – 200 lM (1% DMSO). Fluorescence intensities were recorded over time in a CLARIOstar (BMG Labtech) microplate reader with excitation at 350 10 nm (SIRT3) or 490 10 nm (SIRT1, SIRT2 and SIRT6) and emission at 450 10 nm (SIRT3) or 530 10 nm (SIRT1, SIRT2,
SIRT6). Initial rates were determined by linear regression of fluorescence intensity over time using PRISM (GraphPad Software, La Jolla, CA, USA). Reported data represent the means of two replicates. Rates are presented as % of the rate observed in the absence of inhibitor. IC50 values were calculated by non-linear regression using a four parameter fit and no further constrains, using PRISM.
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