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During the last few years, Poly(ADP-ribose)Polymerase (PARP) proteins became a very popular target for anti-cancer treatment. Many PARP inhibitors have been generated and tested by pharmacological industry. However, most of them were designed to disrupt the DNA-dependent PARP1 protein activation pathway and were based on a competition with NAD for a binding site on PARP molecule and, therefore, on disruption of PARP-mediated enzymatic reaction. This limitation resulted in a discovery of mainly nucleotide-like PARP1-inhibitors which may target not only PARP, but also other pathways involving NAD and other nucleotides. Here, we describe a strategy for the identification PARP inhibitors that target a different pathway, the histone H4 dependent PARP1 activation. Besides the identification of NAD competitors in a small molecules collection, this approach allows finding novel classes of PARP inhibitors that specifically disrupt H4 based PARP activation.
PARP1 is a constitutive component of chromatin, crucial for both DNA repair and transcription. PARP1 takes an active part in DNA repair processes by binding to DNA breaks and catalyzing polymerization of ADP-ribose into long chains on nuclear proteins, such as histones, transcription factors, and PARP1 itself (1, 2). In the past 40 years, many PARP1 inhibitors have been identified. However, confirming the effectiveness of PARP1 inhibitors and identifying their mechanism of action are fundamental to a successful treatment of aggressive and non-responsive tumors, such as triple-negative breast cancer (-BRCA, –HER, -PR) (3). So far, only one pathway of PARP1 activation is vigorously tested by pharmacological industry – DNA-dependent PARP1 activation. Here, we describe an alternative approach based on PARP1 activation by histone H4. This approach, combined with the development of a high-throughput automated PARP1 assay, affords an opportunity to screen relatively large drug libraries for potential inhibitors with different mechanisms of action.
Previously, we have developed an assay confirming that PARP1 can be activated by two distinct pathways: DNA- and histone-dependent. Most known PARP1 inhibitors are “related” to the substrate of PARP1 known as nicotinamide adenine dinucleotide (NAD+). As such, they belong to the groups of monoaryl amides and bi-, tri-, tetracyclic lactams. These inhibitors act as competitive inhibitors because they block NAD-binding domains of PARP1 (4). Our newly developed assay is based on the protein-dependent pathway of activation and can be adapted to screen potential novel inhibitors as well as to determine their mechanism of action. This method provides a basis for determining whether drugs specifically impair PARP1 via gene transcription or DNA repair, thus aiding in the discovery of new drugs with increasing specificity in future treatment protocols.
Our assay is suitable for assessing PARP1 inhibition by a subset of 50,000 drugs within the Fox Chase Cancer Center Translational Research Library. It is an ELISA-like assay where 1) a 96/384-well ELISA plate is coated with protein-activator, 2) PARP reactions are set up in each well, and 3) a product of these reactions – poly-(ADP)-ribose – is quantified (5). Absorbance at 650 or 450 nm is used as an indicator of PARP1 activity. Positive hits from these screens, as well as known PARP1 inhibitors, could then be tested in vivo using Drosophila cell culture, human cell cultures, and Drosophila as a model organism.
See Figure 1a.
See Figure 1b.
Nunc Maxisorp plate had much more sensitivity (even lowest amounts of H4 (0.125mkg/well) and PARP1 (0.625u/well) gave a good reading); therefore, all further experiments were done using this kind of plate.
See Figure 4. (See Note 8).
It can be observed that applying Stop Solution leads to higher sensitivity or better ratio between negative and positive hits at lower concentrations of H4 and PARP1.
H4 concentration at 0.0156mkg/well and PARP1 concentration at 0.312u/well were therefore chosen for further experiments.
To shorten screening time, blocking of the plate with PBST-5% non-fat milk before PARPI reaction was tried in comparison with the original conditions. Reaction was set up as follows:
See Figure 5.
Here it can be seen that blocking of a plate with PBST- 5% milk before reaction works as well as blocking after reaction. There is a slight decrease in Negative/Positive signal ratio, but this can be attributed to a longer incubation time with SureBlue HRP substrate reagent (3min vs. 6min) (See Note 8). In all further experiments, plates were blocked with 5% milk before PARP1 reaction.
See Figure 6.
From the data given in Figure 6b, it can be seen that the absorbance curve steadily goes down with the dilution for BD polyclonal antibodies, while for 10H antibodies, more dilutions can be tested.
See Figure 7.
Note that secondary antibody dilution of 1:3000 reduces reaction sensitivity, while for primary antibody even a 1:4000 dilution gives a good DMSO/3AB signal ratio. Decreasing the amount of PARP1 to 0.075 U/well also does not significantly change DMSO/3AB signal ratio.
In further experiments, 1:1500 dilution of secondary antibody was used along with 0.075 u/well of PARP1 enzyme.
See Figure 8.
As can be seen in Figure 8, DMSO/3AB signal ratio is almost 2-fold higher with a reaction time of 30 min, as opposed to a reaction time of 60 min. Therefore, a reaction time of 30 min was used in the screening experiments (See Note 9).
Originally, reaction was set up as follows: 25 mkL of 20 mkM NAD+ were distributed to H4-coated wells; then 93 nL of library were added to each well by CyBio Robotic Transfer. 2x PARP1 mix was prepared separately as follows: 5 mkL 10x PARP1 buffer and 20 mkL water, together with 0.0075 mkL PARP1, were mixed in a well with NAD/compound mixture.
To make robotic transfer more consistent, the original volume of NAD+ solution was increased to 40 mkL: 25 mkL 20 mkM NAD and 15 mkL water. Volume of PARP1 mix was reduced to 10 mkL: 5 mkL 10x PARP1 buffer, 5 mkL water and 0.0075 mkL PARP1 (See Note 10).
Z-Factor is a simple statistical parameter for high-throughput screening assays. Z-score is a useful tool for evaluating the quality of such assays and should be used for assay validation before performing a screening (12, 13) (See Note 11).
To assess whether several known inhibitors proceed in a DNA-independent pathway, we will compare the degree of inhibition of DNA and H4-dependent PARP1 activation.
See Figure 13a.
See Figure 13b.
We thank Dr. M. Einarson of the FCCC Translational Facility for her advice and assistance with small molecules collection screening experiments, Dr. I. Serebriiskii for helpful discussions, and Dr. M. Robinson for BT474 and HR6 breast cancer cell lines. The research was supported by grants from the National Institutes of Health (R01 DK082623) to A.V.T.