| Poly(ADP-ribose) polymerases (PARPs) play a major role in cellular survival and maintenance of energy stores after genotoxic insult. By synthesizing ADP-ribose polymers from beta-NAD+, PARPs are able to drastically alter the overall size and charge of the modified protein and thus initiate the DNA repair response. Not surprisingly, small molecule inhibitors of PARP isozymes have been described as possible therapies for neurodegeneration, ischemia, and as potentiators of anticancer agents. In the past, there was no continuous, high-throughput method to measure PARP activity and no direct comparison of PARP isozyme activities were offered in the literature. Described herein is the development of a colorimetric PARP substrate, ADP-ribose pNP, that can be used to kinetically monitor PARP activity. PARP-1 is able to utilize this substrate to synthesize ADP-ribose polymers, and by monitoring production of p-nitrophenolate, the kinetics of PARP-1, tankyrase, and VPARP have been studied.;While several promising inhibitors of PARP have been developed, no crystal structure of the complementary enzyme poly(ADP-ribose)glycohydrolase (PARG) is available, and therefore it has been difficult to intelligently design compounds to inhibit this enzyme. To date, the known inhibitors of PARG can be predominantly grouped into the three categories of DNA intercalators, tannins, and ADP-ribose analogues. While these compounds have proven valuable in the in vitro analysis of PARG, due to toxicity (intercalators), cell permeability issues (ADP-HPD), and nonspecific activity (tannins), few if any of these compounds have been useful for evaluating the effects of PARG inhibition in cell culture and in vivo. Thus, efforts to synthesize and assess cell ADP-HPD based PARG inhibitors will be discussed in the second half of this document. Furthermore, as compounds based on rhodanine scaffolds have been identified as inhibitors of enzymes utilizing pyrophosphate-containing substrates, we have screened a collection of rhodanine-containing small molecules for their ability to inhibit PARG. Through this screen several novel PARG inhibitors were identified, and subsequent derivative synthesis has elucidated structural features important for PARG inhibition. Several of the compounds are cell permeable and induce the build-up of poly(ADP-ribose) in the cell. Details of the TLC based screen, synthesis, and evaluation of hit compounds in cell culture will be discussed. |
