Linking off-target kinase pharmacology to the differential cellular effects observed among PARP inhibitors.

PARP inhibitors hold promise as a novel class of targeted anticancer drugs. However, their true mechanism of action is still not well understood following recent reports that show marked differences in cellular effects. Here, we demonstrate that three PARP drug candidates, namely, rucaparib, veliparib, and olaparib, have a clearly different in vitro affinity profile across a panel of diverse kinases selected using a computational approach that relates proteins by ligand similarity. In this respect, rucaparib inhibits nine kinases with micromolar affinity, including PIM1, PIM2, PRKD2, DYRK1A, CDK1, CDK9, HIPK2, CK2, and ALK. In contrast, olaparib does not inhibit any of the sixteen kinases tested. In between, veliparib inhibits only two, namely, PIM1 and CDK9. The differential kinase pharmacology observed among PARP inhibitors provides a plausible explanation to their different cellular effects and offers unexplored opportunities for this drug class, but alerts also on the risk associated to transferring directly both preclinical and clinical outcomes from one PARP drug candidate to another.

3 software developed at Cerep (Hill software) and validated by comparison with data generated by the commercial software SigmaPlot® 4.0 for Windows® (© 1997 by SPSS Inc.).

Human PRKD2 kinase in vitro inhibition assay
All details for the PRKD2 in vitro assay can be consulted in the Cerep Catalogue available online under references 1729 (742-pkd2): http://www.cerep.fr/cerep/users/pages/catalog/Affiche_CondExp_Test.asp?test=1729 Evaluation of the effects of compounds on the activity of the human PKD2 quantified by measuring the phosphorylation of the substrate biotinyl-βAβAβAKKKVSRSGLYRSPSMPENLNRPR using a human recombinant enzyme expressed in insect cells and the HTRF detection method.
The test compound, reference compound or water (control) are preincubated for 5 min at room temperature with the enzyme (3 ng) in a buffer containing 50 mM Hepes/NaOH (pH 7.4), 2 mM MgCl 2 , 1 mM DTT, 40 µM Na 3 VO 4 and 0.005% Tween 20. Thereafter, the reaction is initiated by adding 25 nM of the substrate biotinyl-βAβAβAKKKVSRSGLYRSPSMPENLNRPR and 30 µM ATP, and the mixture is incubated for 30 min at room temperature. For control basal measurements, the enzyme is omitted from the reaction mixture. Following incubation, the reaction is stopped by adding 33 mM EDTA. The fluorescence acceptor (XL665labeled streptavidine) and the fluorescence donor (anti-phospho-Ser-K antibody labeled with europium cryptate) are then added. After 60 min, the fluorescence transfer is measured at λex=337 nm, λem=620 and λem=665 nm using a microplate reader (Rubystar, BMG). The enzyme activity is determined by dividing the signal measured at λem=665 nm by that measured at λem=620 nm (ratio). The results are expressed as a percent inhibition of the control enzyme activity. The standard inhibitory reference compound is staurosporine, which is tested in each experiment at several concentrations to obtain an inhibition curve from which its IC 50 value is calculated.  ARTKQTARKSTGGKAPRKQLAGCG (histone H3) and 10 µM ATP, and the mixture is incubated for 120 min at room temperature. For control basal measurements, the enzyme is omitted from the reaction mixture. Following incubation, the reaction is stopped by adding 13 mM EDTA. After 5 min, the anti-phopho-histone H3 antibody labeled with europium chelate is added. After 60 more min, the fluorescence transfer is measured at λex=337 nm, λem=620 nm and λem=665 nm using a microplate reader (Envision, Perkin Elmer). The enzyme activity is determined by dividing the signal measured at λem=665 nm by that measured at λem=620 nm (ratio). The results are expressed as a percent inhibition of the control enzyme activity. The standard inhibitory reference compound is staurosporine, which is tested in each experiment at several concentrations to obtain an inhibition curve from which its IC 50 value is calculated. the enzyme is omitted from the reaction mixture. Following incubation, the reaction is stopped by adding 13 mM EDTA. After 5 min, the anti-phospho-MBP antibody labeled with europium chelate is added. After 60 more min, the fluorescence transfer is measured at λex=337 nm, λem=620 nm and λem=665 nm using a microplate reader (Envision, Perkin Elmer). The enzyme activity is determined by dividing the signal measured at λem=665 nm by that measured at λem=620 nm (ratio). The results are expressed as a percent inhibition of the control enzyme activity. The standard inhibitory reference compound is staurosporine, which is tested in each experiment at several concentrations to obtain an inhibition curve from which its IC 50 value is calculated.

Human CDK1 kinase in vitro inhibition assay
The IC 50 values (concentration causing a half-maximal inhibition of control specific activity) and Hill coefficients (

Human DYRK1A kinase in vitro inhibition assay
All details for the DYRK1A in vitro assay can be consulted in the Cerep the enzyme is omitted from the reaction mixture. Following incubation, the reaction is stopped by adding 13 mM EDTA. After 5 min, the anti-phospho-MBP antibody labeled with europium chelate is added. After 60 more min, the fluorescence transfer is measured at λex=337 nm, λem=620 nm and λem=665 nm using a microplate reader (Envision, Perkin Elmer). The enzyme activity is determined by dividing the signal measured at λem=665 nm by that measured at λem=620 nm (ratio). The results are expressed as a percent inhibition of the control enzyme activity. The standard inhibitory reference compound is staurosporine, which is tested in each experiment at several concentrations to obtain an inhibition curve from which its IC 50 value is calculated.
The IC 50 values (concentration causing a half-maximal inhibition of control specific activity) and Hill coefficients (  the enzyme is omitted from the reaction mixture. Following incubation, the reaction is stopped by adding 13 mM EDTA. After 5 min, the anti-phospho-MBP antibody labeled with europium chelate is added. After 60 more min, the fluorescence transfer is measured at λex=337 nm, λem=620 nm and λem=665 nm using a microplate reader (Envision, Perkin Elmer). The enzyme activity is determined by dividing the signal measured at λem=665 nm by that measured at λem=620 nm (ratio). The results are expressed as a percent inhibition of the control enzyme activity. The standard inhibitory reference compound is staurosporine, which is tested in each experiment at several concentrations to obtain an inhibition curve from which its IC 50 value is calculated.

Human CDK9 kinase in vitro inhibition assay
The IC 50 values (concentration causing a half-maximal inhibition of control specific activity) and Hill coefficients (  the enzyme is omitted from the reaction mixture. Following incubation, the reaction is stopped by adding 13 mM EDTA. After 5 min, the anti-phospho-MBP antibody labeled with europium chelate is added. After 60 more min, the fluorescence transfer is measured at λex=337 nm, λem=620 nm and λem=665 nm using a microplate reader (Envision, Perkin Elmer). The enzyme activity is determined by dividing the signal measured at λem=665 nm by that measured at λem=620 nm (ratio). The results are expressed as a percent inhibition of the control enzyme activity. The standard inhibitory reference compound is staurosporine, which is tested in each experiment at several concentrations to obtain an inhibition curve from which its IC 50 value is calculated.

Human HIPK2 kinase in vitro inhibition assay
The IC 50 values (concentration causing a half-maximal inhibition of control specific activity) and Hill coefficients (

Human CK2 kinase in vitro inhibition assay
All details for the CK2 (casein kinase 2) in vitro assay can be consulted in the Tween 20. Thereafter, the reaction is initiated by adding 100 nM of the substrate Ulight-IkappaB-alpha and 500 nM ATP, and the mixture is incubated for 60 min at room temperature. For control basal measurements, the enzyme is omitted from the reaction mixture. Following incubation, the reaction is stopped by adding 13 mM EDTA. After 5 min, the anti-phopho-IkappaB-alpha antibody labeled with europium chelate is added. After 60 more min, the fluorescence transfer is measured at λex=337 nm, λem=620 nm and λem=665 nm using a microplate reader (Envision, Perkin Elmer). The enzyme activity is determined by dividing the signal measured at λem=665 nm by that measured at λem=620 nm (ratio). The results are expressed as a percent inhibition of the control enzyme activity. The standard inhibitory reference compound is heparin, which is tested in each experiment at several concentrations to obtain an inhibition curve from which its IC 50 value is calculated.  Tween 20. Thereafter, the reaction is initiated by adding 50 nM of the substrate Ulight-ARTKQTARKSTGGKAPRKQLAGCG (histone H3) and 10 µM ATP, and the mixture is incubated for 60 min at room temperature. For control basal measurements, the enzyme is omitted from the reaction mixture. Following incubation, the reaction is stopped by adding 13 mM EDTA. After 5 min, the anti-phopho-histone H3 antibody labeled with europium chelate is added. After 60 more min, the fluorescence transfer is measured at λex=337 nm, λem=620 nm and λem=665 nm using a microplate reader (Envision, Perkin Elmer). The enzyme activity is determined by dividing the signal measured at λem=665 nm by that measured at λem=620 nm (ratio). The results are expressed as a percent inhibition of the control enzyme activity. The standard inhibitory reference compound is staurosporine, which is tested in each experiment at several concentrations to obtain an inhibition curve from which its IC 50 value is calculated. Tween 20. Thereafter, the reaction is initiated by adding 150 nM of the substrate Ulight-CKKSRGDYMTMQIG (IRS-1) and 10 µM ATP, and the mixture is incubated for 60 min at room temperature. For control basal measurements, the enzyme is omitted from the reaction mixture. Following incubation, the reaction is stopped by adding 13 mM EDTA. After 5 min, the anti-phopho-PT66 antibody labeled with europium chelate is added. After 60 more min, the fluorescence transfer is measured at λex=337 nm, λem=620 nm and λem=665 nm using a microplate reader (Envision, Perkin Elmer). The enzyme activity is determined by dividing the signal measured at λem=665 nm by that measured at λem=620 nm (ratio). The results are expressed as a percent inhibition of the control enzyme activity. The standard inhibitory reference compound is staurosporine, which is tested in each experiment at several concentrations to obtain an inhibition curve from which its IC 50 value is calculated.

Human ABL kinase in vitro inhibition assay
All details for the ABL in vitro assay can be consulted in the Cerep Catalogue available online under references 3056 (781-ablh): http://www.cerep.fr/cerep/users/pages/catalog/Affiche_CondExp_Test.asp?test=3056 Evaluation of the effects of compounds on the activity of the human Abl kinase quantified by measuring the phosphorylation of the substrate Ulight-TK peptide using a human recombinant enzyme expressed in insect cells and the LANCE ® detection method.
Thereafter, the reaction is initiated by adding 100 nM of the substrate Ulight-TK peptide and 10 µM ATP, and the mixture is incubated for 60 min at room temperature. For control basal measurements, the enzyme is omitted from the reaction mixture. Following incubation, the reaction is stopped by adding 13 mM EDTA. After 5 min, the anti-phospho-PT66 antibody labeled with europium chelate is added. After 60 more min, the fluorescence transfer is measured at λex=337 nm, λem=620 nm and λem=665 nm using a microplate reader (Envision, Perkin Elmer). The enzyme activity is determined by dividing the signal measured at λem=665 nm by that measured at λem=620 nm (ratio). The results are expressed as a percent inhibition of the control enzyme activity. The standard inhibitory reference compound is staurosporine, which is tested in each experiment at several concentrations to obtain an inhibition curve from which its IC 50 value is calculated.

Human AKT3 kinase in vitro inhibition assay
All details for the AKT3 in vitro assay can be consulted in the Cerep Catalogue min at room temperature. For control basal measurements, the enzyme is omitted from the reaction mixture. Following incubation, the reaction is stopped by adding 13 mM EDTA. After 5 min, the anti-phospho-PLK antibody labeled with europium chelate is added. After 60 more min, the fluorescence transfer is measured at λex=337 nm, λem=620 nm and λem=665 nm using a microplate reader (Envision, 13 Perkin Elmer). The enzyme activity is determined by dividing the signal measured at λem=665 nm by that measured at λem=620 nm (ratio). The results are expressed as a percent inhibition of the control enzyme activity. The standard inhibitory reference compound is staurosporine, which is tested in each experiment at several concentrations to obtain an inhibition curve from which its IC 50 value is calculated.

Human PRKCG kinase in vitro inhibition assay
All details for the PRKCG (PKCγ) in vitro assay can be consulted in the Cerep 0.014% phosphatidyl-serine. Thereafter, the reaction is initiated by adding 500 nM of the substrate biotinyl-βAβAβAKIQASFRGHMARKK and 1 µM ATP, and the mixture is incubated for 30 min at room temperature. For control basal measurements, the enzyme is omitted from the reaction mixture. Following incubation, the reaction is stopped by adding 33 mM EDTA. The fluorescence acceptor (XL665-labeled streptavidine) and the fluorescence donor (anti-phospho-Creb-K antibody labeled with europium cryptate) are then added. After 60 min, the fluorescence transfer is measured at λex=337 nm, λem=620 and λem=665 nm using a microplate reader (Rubystar, BMG). The enzyme activity is determined by dividing the signal measured at λem=665 nm by that measured at λem=620 nm (ratio). The results are expressed as a percent inhibition of the control enzyme activity. The standard inhibitory reference compound is Bis 10, which is tested in each experiment at several concentrations to obtain an inhibition curve from which its IC 50 value is calculated. measurements, the enzyme is omitted from the reaction mixture. Following incubation, the reaction is stopped by adding 33 mM EDTA. The fluorescence acceptor (XL665labeled streptavidine) and the fluorescence donor (anti-phospho-Creb K antibody labeled with europium cryptate) are then added. After 60 min, the fluorescence transfer is measured at λex=337 nm, λem=620 and λem=665 nm using a microplate reader (Rubystar, BMG). The enzyme activity is determined by dividing the signal measured at λem=665 nm by that measured at λem=620 nm (ratio).The results are expressed as a percent inhibition of the control enzyme activity. The standard inhibitory reference compound is Bis 10, which is tested in each experiment at several concentrations to obtain an inhibition curve from which its IC 50 value is calculated. substrate Ulight-RRRSLLE (PLK) and 1 µM ATP, and the mixture is incubated for 10 min at room temperature. For control basal measurements, the enzyme is omitted from the reaction mixture. Following incubation, the reaction is stopped by adding 13 mM EDTA. After 5 min, the anti-phospho-PLK antibody labeled with europium chelate is added. After 60 more min, the fluorescence transfer is measured at λex=337 nm, λem=620 nm and λem=665 nm using a microplate reader (Envision, Perkin Elmer). The enzyme activity is determined by dividing the signal measured at λem=665 nm by that measured at λem=620 nm (ratio). The results are expressed as a percent inhibition of the control enzyme activity. The standard inhibitory reference compound is staurosporine, which is tested in each experiment at several concentrations to obtain an inhibition curve from which its IC 50 value is calculated.

Kinase in vitro screening at 10µM
Supplementary Table 1 provides the raw data, in duplicate, for the interaction of olaparib, veliparib and rucaparib with the 16 kinases analyzed. Values are reported as percent inhibition of the control enzyme activity at 10µM drug concentration.

Dose-response curves and IC50s from the in vitro assays
Supplementary Figure 1 provides the raw data, in duplicate, and the doseresponse curve for the interaction of veliparib with human PIM1 kinase, as provided by the CRO (Cerep, http://www.cerep.fr/). Values are reported as percent inhibition of the control enzyme activity (see Supplementary Methods above). The IC 50 value calculated by non-linear regression analysis of the dose-response curve generated with mean replicate values using Hill equation curve fitting is also included as provided by Cerep.