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

Huber KV, Salah E, Radic B, Gridling M, Elkins JM, Stukalov A, Jemth AS, Göktürk C, Sanjiv K, Strömberg K, Pham T, Berglund UW, Colinge J, Bennett KL, Loizou JI, Helleday T, Knapp S, Superti-Furga G - Nature (2014)

Bottom Line: Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys and MTH1 co-crystal structures of both enantiomers provide 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 anticancer agents.

View Article: PubMed Central - PubMed

Affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria.

ABSTRACT
Activated 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 unknown. Here, using a chemical proteomic approach, we identify the target of SCH51344 as the human mutT homologue MTH1 (also known as NUDT1), a nucleotide pool sanitizing enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells, whereas MTH1 overexpression mitigated sensitivity towards 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 provide 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 anticancer agents.

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(S)-Crizotinib is a nanomolar MTH1 inhibitora, MTH1 catalytic assay. Data are shown for both crizotinib enantiomers and the racemic mixture at 100 μM dGTP. Results indicate two technical replicates ± SEM representative for at least duplicate experiments (n ≥ 2). b, ITC for MTH1 with (R)- and (S)-crizotinib (n = 1). c, (S)-Crizotinib inhibits colony formation of PANC1 and SW480 cells. Images are representative for three independent experiments (n = 3). d, Comparison of antiproliferative efficacy of (S)-crizotinib versus SCH51344 against SW480 cells. Data shown as mean ± SEM for three independent experiments (n = 3). e, Cellular thermal shift assay MTH1 target engagement by (S)-crizotinib in intact KRASV12-expressing BJ cells. Images are representative of two independent experiments (n = 2). f, The (S)-crizotinibaffinity probe selectively binds MTH1, but not ALK, in SW480 lysates, whereas the (R)-enantiomer exerts inverse properties.
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Figure 2: (S)-Crizotinib is a nanomolar MTH1 inhibitora, MTH1 catalytic assay. Data are shown for both crizotinib enantiomers and the racemic mixture at 100 μM dGTP. Results indicate two technical replicates ± SEM representative for at least duplicate experiments (n ≥ 2). b, ITC for MTH1 with (R)- and (S)-crizotinib (n = 1). c, (S)-Crizotinib inhibits colony formation of PANC1 and SW480 cells. Images are representative for three independent experiments (n = 3). d, Comparison of antiproliferative efficacy of (S)-crizotinib versus SCH51344 against SW480 cells. Data shown as mean ± SEM for three independent experiments (n = 3). e, Cellular thermal shift assay MTH1 target engagement by (S)-crizotinib in intact KRASV12-expressing BJ cells. Images are representative of two independent experiments (n = 2). f, The (S)-crizotinibaffinity probe selectively binds MTH1, but not ALK, in SW480 lysates, whereas the (R)-enantiomer exerts inverse properties.

Mentions: Since SCH51344 has not been evaluated in a clinical setting, we decided to screen for other, more potent MTH1 inhibitors with favourable pharmacokinetic and pharmacodynamic properties. Based on substrates and active site architecture we hypothesised that kinase inhibitors may target MTH1. Screening a kinase inhibitor collection in a thermal shift stability asay17 we found that the dual Met/ALK inhibitor crizotinib18,19 exhibited high affinity toward MTH1 (data not shown). Crizotinib recently received approval for the treatment of EML4-ALK-positive non-small cell lung cancer (NSCLC) and is in several other clinical trials20-23. However, using the catalytic MTH1 assay, we found that crizotinib batches obtained from different vendors resulted in varying IC50 values. This could not be explained by impurities or degradation products as analytical data were in accordance with literature18. Since crizotinib bears a chiral centre, we speculated that variable amounts of crizotinib stereoisomers may occur in different batches of inhibitor. We prepared and tested both the pure, clinically used (R)-as well as the so far unexplored (S)-enantiomer of crizotinib in the MTH1 catalytic assay, which suggested that the screening hit batch contained a racemic mixture. We found that pure (S)-crizotinib was a low nanomolar MTH1 inhibitor whereas the (R)-enantiomer gave IC50 values in the micromolar range (Fig. 2a). These data were confirmed by direct binding assays (ITC) indicating a 16-fold higher affinity of the (S)-enantiomer toward MTH1 (Fig. 2b, Extended Data Fig. 2a). Using Km concentrations of substrates,12,15 we determined average IC50 values of 330 nM and 408 nM for (S)-crizotinib and the MTH1 substrates 8-oxo-dGTP and 2-OH-dATP, respectively (n = 2). Consistent with these data, (S)-crizotinib efficiently inhibited colony formation of SW480 and KRAS mutated PANC1 cells, similar to SCH51344 (Fig. 2c, d). In vitro Kd measurements indicated that (S)-crizotinib was considerably less potent than the (R)-enantiomer against the established targets ALK, MET and ROS1 (Extended Data Fig. 2b).Treatment of SW480 cells with a specific c-MET inhibitor, a potential off-target for (S)-crizotinib,18 did not lead to the detection of any significant effects on proliferation (Extended Data Fig. 2c). However, investigating whether MTH1 overexpression could rescue SW480 cells from cell death induced by (S)-crizotinib in a similar manner as for SCH51344, we failed to observe any significant shift in IC50 values (Extended Data Fig. 2d), raising the question if other targets contributed to the cell killing effect. We started investigating whether MTH1 was indeed targeted by (S)-crizotinib in intact cells. If a cellular protein is bound by a chemical agent, it is stabilized by the physical engagement as compared to the non-engaged counterpart24. In a cellular thermal shift assay using BJ-KRASV12 cells, (S)-crizotinib, in contrast to (R)-crizotinib, efficiently stabilized MTH1 validating the differential targeting within cells (Fig. 2e).


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

Huber KV, Salah E, Radic B, Gridling M, Elkins JM, Stukalov A, Jemth AS, Göktürk C, Sanjiv K, Strömberg K, Pham T, Berglund UW, Colinge J, Bennett KL, Loizou JI, Helleday T, Knapp S, Superti-Furga G - Nature (2014)

(S)-Crizotinib is a nanomolar MTH1 inhibitora, MTH1 catalytic assay. Data are shown for both crizotinib enantiomers and the racemic mixture at 100 μM dGTP. Results indicate two technical replicates ± SEM representative for at least duplicate experiments (n ≥ 2). b, ITC for MTH1 with (R)- and (S)-crizotinib (n = 1). c, (S)-Crizotinib inhibits colony formation of PANC1 and SW480 cells. Images are representative for three independent experiments (n = 3). d, Comparison of antiproliferative efficacy of (S)-crizotinib versus SCH51344 against SW480 cells. Data shown as mean ± SEM for three independent experiments (n = 3). e, Cellular thermal shift assay MTH1 target engagement by (S)-crizotinib in intact KRASV12-expressing BJ cells. Images are representative of two independent experiments (n = 2). f, The (S)-crizotinibaffinity probe selectively binds MTH1, but not ALK, in SW480 lysates, whereas the (R)-enantiomer exerts inverse properties.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4150021&req=5

Figure 2: (S)-Crizotinib is a nanomolar MTH1 inhibitora, MTH1 catalytic assay. Data are shown for both crizotinib enantiomers and the racemic mixture at 100 μM dGTP. Results indicate two technical replicates ± SEM representative for at least duplicate experiments (n ≥ 2). b, ITC for MTH1 with (R)- and (S)-crizotinib (n = 1). c, (S)-Crizotinib inhibits colony formation of PANC1 and SW480 cells. Images are representative for three independent experiments (n = 3). d, Comparison of antiproliferative efficacy of (S)-crizotinib versus SCH51344 against SW480 cells. Data shown as mean ± SEM for three independent experiments (n = 3). e, Cellular thermal shift assay MTH1 target engagement by (S)-crizotinib in intact KRASV12-expressing BJ cells. Images are representative of two independent experiments (n = 2). f, The (S)-crizotinibaffinity probe selectively binds MTH1, but not ALK, in SW480 lysates, whereas the (R)-enantiomer exerts inverse properties.
Mentions: Since SCH51344 has not been evaluated in a clinical setting, we decided to screen for other, more potent MTH1 inhibitors with favourable pharmacokinetic and pharmacodynamic properties. Based on substrates and active site architecture we hypothesised that kinase inhibitors may target MTH1. Screening a kinase inhibitor collection in a thermal shift stability asay17 we found that the dual Met/ALK inhibitor crizotinib18,19 exhibited high affinity toward MTH1 (data not shown). Crizotinib recently received approval for the treatment of EML4-ALK-positive non-small cell lung cancer (NSCLC) and is in several other clinical trials20-23. However, using the catalytic MTH1 assay, we found that crizotinib batches obtained from different vendors resulted in varying IC50 values. This could not be explained by impurities or degradation products as analytical data were in accordance with literature18. Since crizotinib bears a chiral centre, we speculated that variable amounts of crizotinib stereoisomers may occur in different batches of inhibitor. We prepared and tested both the pure, clinically used (R)-as well as the so far unexplored (S)-enantiomer of crizotinib in the MTH1 catalytic assay, which suggested that the screening hit batch contained a racemic mixture. We found that pure (S)-crizotinib was a low nanomolar MTH1 inhibitor whereas the (R)-enantiomer gave IC50 values in the micromolar range (Fig. 2a). These data were confirmed by direct binding assays (ITC) indicating a 16-fold higher affinity of the (S)-enantiomer toward MTH1 (Fig. 2b, Extended Data Fig. 2a). Using Km concentrations of substrates,12,15 we determined average IC50 values of 330 nM and 408 nM for (S)-crizotinib and the MTH1 substrates 8-oxo-dGTP and 2-OH-dATP, respectively (n = 2). Consistent with these data, (S)-crizotinib efficiently inhibited colony formation of SW480 and KRAS mutated PANC1 cells, similar to SCH51344 (Fig. 2c, d). In vitro Kd measurements indicated that (S)-crizotinib was considerably less potent than the (R)-enantiomer against the established targets ALK, MET and ROS1 (Extended Data Fig. 2b).Treatment of SW480 cells with a specific c-MET inhibitor, a potential off-target for (S)-crizotinib,18 did not lead to the detection of any significant effects on proliferation (Extended Data Fig. 2c). However, investigating whether MTH1 overexpression could rescue SW480 cells from cell death induced by (S)-crizotinib in a similar manner as for SCH51344, we failed to observe any significant shift in IC50 values (Extended Data Fig. 2d), raising the question if other targets contributed to the cell killing effect. We started investigating whether MTH1 was indeed targeted by (S)-crizotinib in intact cells. If a cellular protein is bound by a chemical agent, it is stabilized by the physical engagement as compared to the non-engaged counterpart24. In a cellular thermal shift assay using BJ-KRASV12 cells, (S)-crizotinib, in contrast to (R)-crizotinib, efficiently stabilized MTH1 validating the differential targeting within cells (Fig. 2e).

Bottom Line: Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys and MTH1 co-crystal structures of both enantiomers provide 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 anticancer agents.

View Article: PubMed Central - PubMed

Affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria.

ABSTRACT
Activated 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 unknown. Here, using a chemical proteomic approach, we identify the target of SCH51344 as the human mutT homologue MTH1 (also known as NUDT1), a nucleotide pool sanitizing enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells, whereas MTH1 overexpression mitigated sensitivity towards 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 provide 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 anticancer agents.

Show MeSH
Related in: MedlinePlus