Novel inhibitors of poly(ADP-ribose) polymerase/PARP1 and PARP2 identifiedusing a cell-based screen in yeast

Multicellular organisms must have means of preserving their genomic integri ty or face catastrophic consequences such as uncontrolled cell proliferatio n or massive cell death. One response is a modification of nuclear proteins by the addition and removal of polymers of ADP-ribose that modulate the pr operties of DNA-binding proteins involved in DNA repair and metabolism, The se ADP-ribose units are added by poly(ADP-ribose) polymerase (PARP) and rem oved by poly(ADP-ribose) glycohydrolase, Although budding yeast Saccharomyc es cerevisiae does not possess proteins with significant sequence similarit y to the human PARP family of proteins, we identified novel small molecule inhibitors against two family members, PARP1 and PARP2, using a fell-based assay in yeast. The assay was based on the reversal of growth inhibition ca used by the heterologous expression of either PARP1 or PARP2. Validation of the assay was achieved by showing that the growth inhibition was relieved by a mutation in a single residue in the catalytic site of PARP1 or PARP2 o r exposure of yeast to a known PARP1 inhibitor, 6(5H)-phenanthridinone. In separate experiments, when a putative protein regulator of PARP activity, h uman poly(ADP-ribose) glycohydrolase, was coexpressed with PARP1 or PARP2, yeast growth was restored, Finally, the inhibitors identified by screening the yeast assay are active in a mammalian PARP biochemical assay and inhibi t PARP1 and PARP2 activity in yeast cell extracts, Thus, our data reflect t he strength of using yeast to identify small molecule inhibitors of therape utically relevant gene families, including those that are not found in yeas t, such as PARP, The resultant inhibitors have two critical uses (a) as lea ds for drug development and (b) as tools to dissect cellular function.