Updated project metadata. Previously, we showed cancer cells rely on the MTH1 protein to prevent incorporation of otherwise deadly oxidative nucleotides into DNA and we developed MTH1 inhibitors which selectively kill cancer cells. Recently, several new and potent inhibitors of MTH1 were demonstrated to be non-toxic to cancer cells, challenging the utility of MTH1 inhibition as a target for cancer treatment. Here, we demonstrate that these recently described MTH1 inhibitors, which fail to kill cancer cells, also fail to introduce the toxic oxidized nucleotides into DNA. We further describe a new MTH1 inhibitor TH1579, an analogue of TH588, which is a potent, selective MTH1 inhibitor with good oral availability and demonstrates excellent pharmacokinetic and anti-cancer properties in vivo. Furthermore, we describe TH1579 as a best-in-class MTH1 inhibitor, which we expect to be useful in order to test the MTH1 inhibitor concept. Cellular thermal shift assay (CETSA) combined with basic reversed phase (bRP) separation and LC-MS was used to study drug binding in BJ Ras Sv40 cells treated with the MTH1 inhibitor TH1579.