Having a diverse network of substrates, NUDIX hydrolases emerged like a key group of nucleotide-metabolizing enzymes. NUDT5 (also known as NUDIX5) continues to be implicated in ADP-ribose and eight-oxo-guanine metabolic process and it was lately recognized as a rheostat of hormone-dependent gene regulation and proliferation in cancer of the breast cells. Here, we further elucidate the physiological relevance of known NUDT5 substrates and underscore the biological requirement of NUDT5 in gene regulation and proliferation of cancer of the breast cells. We read the participation of NUDT5 in ADP-ribose metabolic process and dissociate rapport to oxidized nucleotide sanitation. In addition, we identify potent NUDT5 inhibitors, that are enhanced to advertise maximal NUDT5 cellular target engagement by CETSA. Lead compound, TH5427, blocks progestin-dependent, Componen-derived nuclear ATP synthesis and subsequent chromatin remodeling, gene regulation and proliferation in cancer of the breast cells. We herein present TH5427 like a promising, targeted inhibitor you can use to help study NUDT5 activity and ADP-ribose metabolic process.