TH1760

This is a top-quality chemical probe for in vitro and cellular studies of NUDT15. Nanomolar affinity to NUDT15 is shown, and direct engagement with the protein has clearly been demonstrated (TSA, X-ray). Also, cellular target engagement has been shown (CETSA, DARTS). The compound is selective to NUDT15 over closely related protein homologues (TH1760 here tested at 100 µM), and is only little active against two broad panels of counter-targets (TH1760 tested at 10 µM in a safety screen and a range of kinases). TH1760 has been shown to induce the expected biochemical outcome in various cell lines (10 µM). A close analogue (TH7285) has been identified, which can serve an excellent negative control compound. This is the first bona fide small-molecule NUDT15 inhibitor. Currently, there is no information about in vivo activity of TH1760 available in literature.

TH1760 chemical probe for NUDT15 was extensively characterized in vitro and in cells. It is highly potent in vitro and shows a remarkable selectivity within the Nudix enzyme family. The cellular characterization of this probe includes CETSA and DARTS assays. The biological sensitization to thiopurines via accumulation of 6-thio-dGTP was shown as mechanism for the compound and NUDT15 knockdown sensitization; thus, providing additional evidence of compound activity in cells.

Sufficient efforts have been made to characterize the lead compound and compare it with its negative control, which is structurally similar to the parental by the difference of a methyl group at a key interacting site. The chemical probe has demonstrated target engagement and modes of interaction with multiple biochemical assays and in multiple cellular contexts and the on-target selectivity/minimal off-target effects. The biological outcomes of the chemical probe are consistent with the underlying mechanism and modes of action. Therefore, this chemical probe, paired with its negative control, is well justified for its use for cellular assays. The reviewer recommends this chemical probe to suppress the enzymatic activity of NUDT15 for biochemical and cellular assays. Given that the recommended dose of 10 uM is modest, the reviewer suggests pairing this chemical probe with its negative control to further avoid unexpected off-target effects. It is worth noting that this chemical probe is not ready for use in model organisms because its on/off-target effect has only been evaluated against human NUDT15 with human cell lines.