Development of a chemical probe against NUDT15

Abstract

The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides but was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we sought to develop the first small-molecule NUDT15 inhibitors to elucidate its biological functions, and potentially for improving NUDT15-dependent chemotherapeutics. Lead compound TH1760, demonstrated low-nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic pocket and engaged cellular NUDT15 in the low-micromolar range. We further employed thiopurine potentiation as a proxy functional read-out and demonstrated that TH1760 sensitized cells to 6-thioguanine through enhanced accumulation of 6-thio-(d)GTP in nucleic acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased thiopurine toxicity is via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.

NUDT15-mediated hydrolysis limits the efficacy of anti-HCMV drug ganciclovir

“…A promising hit compound NSC56456 was identified, with its activity further confirmed using an in-house resynthesized and purified compound (referred to hereafter as TH7410; IC 50 in MG enzyme-coupled assay = 620 nM) (see supplemental experimental procedure). Subsequent medicinal chemistry efforts (inhibitor development and structuralactivity relationship study will be detailed in a separate paper, manuscript under preparation) further led to approximately 20fold potency improvement from TH7410, resulting in the lead compound TH8321 (MG IC 50 = 35 nM), which demonstrated potency comparable with that of the previously identified non-thiopurine derivative inhibitor, TH1760 (MG IC 50 = 34 nM) (Zhang et al, 2020) (Figure 3A). When assayed at 100 mM, TH8321 further demonstrated satisfactory selectivity among a panel of related proteins with sequential or functional resemblance to NUDT15, i.e., NUDT22, NUDT18, NUDT12, NUDT9, NUDT5, NUDT2, MTH1, ITPase, dCTPase, and dUTPase (Figures 3B and S3B).…”

“…The binding modality between TH8321 and NUDT15 was further elucidated by a NUDT15/TH8321 co-crystal structure at a resolution of 2.35 Å (Figures 3E and S3C). Comparing the binding interactions of the ligands in the different NUDT15 structures -in complex with TH8321, GCV-TP, 6-thio-GMP (PDB: 5LPG), or TH1760 (PDB: 6T5J) (Zhang et al, 2020), we see that TH8321 binds in a manner most similar to that of 6-thio-GMP (Figures S3D-S3F). Notably, the guanine moiety of TH8321 is situated the same as that of 6-thio-GMP, with the same hydrogen bonding to Leu45 and Gly137.…”

“…We next investigated whether GCV potentiation could also be achieved through pharmacological inhibition of NUDT15. To this end, in addition to the inhibitor series recently reported (Zhang et al, 2020), we reasoned that derivatization of thiopurine analogs would create potent NUDT15 inhibitors due to the high affinity of thiopurine moieties for the active site (Valerie et al, 2016). As a first step, we screened a curated library of 62 thiopurine analogs from the National Cancer Institute Developmental Therapeutics Program (http://dtp.cancer.gov/) for NUDT15 inhibition, via the previously established enzyme-coupled MG assay (Zhang et al, 2020) (Figure S3A and Table S1).…”

“…Article of thiopurine drugs, a validated system previously used to develop the first NUDT15 inhibitor TH1760 (Zhang et al, 2020). We co-applied TH8321 and thioguanine (6-TG) to an acute myeloid leukemia (AML) cell line, HL-60, and assessed viability by resazurin reduction.…”

“…Clinically, patients harboring inactivating NUDT15 missense mutations experience thiopurine hypersensitivity (Moriyama et al, 2016;Yang et al, 2014). Depletion and/or inhibition of NUDT15 leads to enhanced thiopurine responsiveness in vitro (Valerie et al, 2016;Zhang et al, 2020) and in vivo (Nishii et al, 2018), suggesting NUDT15 as a potential avenue to modulate existing nucleoside analog therapeutics.…”