Stereospecific targeting of MTH1 by (S)-crizotinib as an anticancer strategy

Abstract

SummaryActivated Ras GTPase signalling is a critical driver of oncogenic transformation and malignant disease. Cellular models of RAS-dependent cancers have been used to identify experimental small-molecules, such as SCH51344, but their molecular mechanism of action remains generally enigmatic. Here, using a chemical proteomic approach we identify the target of SCH51344 as the human mutT homologue MTH1, a nucleotide pool sanitising enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells whereas MTH1 overexpression mitigated sensitivity toward SCH51344. Searching for more drug-like inhibitors, we identified the kinase inhibitor crizotinib as a nanomolar suppressor of MTH1 activity. Surprisingly, the clinically used (R)-enantiomer of the drug was inactive, whereas the (S)-enantiomer selectively inhibited MTH1 catalytic activity. Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys, and MTH1 co-crystal structures of both enantiomers provided a rationale for this remarkable stereospecificity. Disruption of nucleotide pool homeostasis via MTH1 inhibition by (S)-crizotinib induced an increase in DNA single strand breaks, activated DNA repair in human colon carcinoma cells, and effectively suppressed tumour growth in animal models. Our results propose (S)-crizotinib as an attractive chemical entity for further pre-clinical evaluation and small molecule inhibitors of MTH1 in general as a promising novel class of anti-cancer agents.

Proteomic profiling of small-molecule inhibitors reveals dispensability of MTH1 for cancer cell survival

“…Since recent publications suggest that MTH1 is essential for cancer cell survival 8,9 , we analyzed the cytotoxic effects of our MTH1 inhibitors against cancer cells. Contrary to our expectations, NPD7155 and NPD9948 exhibited only weak cytotoxicity against HeLa cells (IC 50 = 65 μ M and 35 μ M, respectively; Fig.…”

“…To analyze the interaction between small-molecule ligands and their target proteins in intact cells, a cellular thermal shift assay (CETSA) was performed as described previously with minor modifications 9,34 . Briefly, HeLa cells cultured in a 100-mm dish to 80% confluency, were treated with NPD7155 (300 μ M), NPD9948 (300 μ M), or (S)-crizotinib (30 μ M) for 1 h. After treatment, cells were collected with trypsin and resuspended in Tris-buffered saline (TBS).…”

“…Rai et al reported that overexpression of MTH1 protected primary fibroblasts from RAS-induced senescence by suppressing DNA damage caused by ROS 7 . In addition, it was reported that cancer cells frequently overexpress MTH1, and that inhibition of MTH1 by genetic and pharmacologic methods suppressed xenograft tumors derived from a number of different tumor cell lines [8][9][10] . Four types of first-in-class small-molecule inhibitors of MTH1 were recently reported-TH287/TH588, (S)-crizotinib, SCH51344, and organometallic complexesamong which TH287/TH588, (S)-crizotinib, and SCH51344 preferentially kill cancer cells or RAS-transformed fibroblast cells 8,9,11 .…”

Replication stress and cancer: It takes two to tango

“…The need of cancer cells to maintain a healthy pool of dNTPs is highlighted by the recent discovery of inhibitors of the MTH1 enzyme, responsible for sanitizing oxidized nucleotides in the cells. MTH1 inhibition leads to an increase in DNA damage, and is preferentially toxic for cancer cells due to the increased levels of oxidation stress [103,104]. Interestingly, studies in yeast suggest that ATR might suppress RS by modulating nucleotide metabolic pathways [105,106], further linking the connection between the RS-targeting strategies.…”

Cancer therapy

“…Finally, there is evidence that malignant cells exhibit elevated basal levels of autophagy, raising the possibility that inhibiting this process could lead to cancer-specific toxicity. 7 Two recent reports 8,9 highlight another potential generic Achilles heel in cancer; namely the possibility of converting endogenous oxidized nucleotide precursors, generally more abundant in tumor cells, into toxic DNA damage lesions. A study by Gad et al.…”