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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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KINOMEscan results for both crizotinib enantiomersScreening of both (R)- and (S)-crizotinib against a panel of 456 recombinant human protein kinases indicates a dramatic difference in the ability of the two enantiomers to bind kinases. (R)-crizotinib has high affinity toward a large number of kinases including its cognate targets MET, ALK, and ROS1. Selectivity Score or S-score is a quantitative measure of compound selectivity. It is calculated by dividing the number of kinases that compounds bind to by the total number of distinct kinases tested, excluding mutant variants. S(35) = (number of non-mutant kinases with %Ctrl <35)/(number of non-mutant kinases tested).
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Figure 7: KINOMEscan results for both crizotinib enantiomersScreening of both (R)- and (S)-crizotinib against a panel of 456 recombinant human protein kinases indicates a dramatic difference in the ability of the two enantiomers to bind kinases. (R)-crizotinib has high affinity toward a large number of kinases including its cognate targets MET, ALK, and ROS1. Selectivity Score or S-score is a quantitative measure of compound selectivity. It is calculated by dividing the number of kinases that compounds bind to by the total number of distinct kinases tested, excluding mutant variants. S(35) = (number of non-mutant kinases with %Ctrl <35)/(number of non-mutant kinases tested).

Mentions: To further investigate the ability of the two crizotinib enantiomers to engage cellular proteins we derived chemical probes suitable for drug pull-downs (Supplementary Information). We tested the two derivatized compounds for their ability to target ALK and MTH1 in SW480 cell extracts. The two enantiomers were remarkably specific for their cognate targets (Fig. 2f). If MTH1 was indeed the key target of (S)-crizotinib in other RAS-transformed cells, it should rank at the top of the specific interactors in an unbiased chemical proteomic experiment as done before with SCH51344. MTH1 was by far the most specific and prominent interactor of (S)-crizotinib (Extended Data Fig. 2e). Plotting the chemical proteomic results of SCH51344 and (S)-crizotinib against each other singled out MTH1 as the only common high-significance interactor (Fig. 3a). We also performed the reciprocal analysis with (R)-crizotinib which identified a plethora of protein kinases, all efficiently competed by free drug, but not MTH1 (Extended Data Fig. 2f). Notably, comparison of both profiles did not reveal any proteins that were significantly bound by both enantiomers (Fig. 3b). To exclude that either crizotinib enantiomer may target kinases of low abundance we interrogated a panel of 456 different recombinant kinases (KINOMEscan, Extended Data Fig. 3)25. In line with the chemoproteomic results the two enantiomers displayed a remarkable stereoselectivity with very distinct profiles. The few kinases to which (S)-crizotinib showed some affinity were not calculated to be significantly inhibited. (R)-Crizotinib not only bound to ten times more kinases, but also was predicted to efficiently inhibit at least ten of them, including the well characterized cognate targets ALK, MET, but also LCK, IRAK1, JAK3, LOK and SLK. To understand the differences in MTH1 binding, we co-crystallised both (R)- and (S)-crizotinib with recombinant protein. The structure revealed an unfavourable eclipsed conformation of the methyl group at the chiral centre and the halogen substituents on the benzyl ring is likely to reduce the energetic favourability of (R)-crizotinib binding (Fig. 3c,d, Extended Data Fig. 4, 5). ITC data confirmed that the difference in binding between (R)- and (S)-crizotinib was entirely entropic and therefore not due to different binding interactions with the protein (Fig. 2b).


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)

KINOMEscan results for both crizotinib enantiomersScreening of both (R)- and (S)-crizotinib against a panel of 456 recombinant human protein kinases indicates a dramatic difference in the ability of the two enantiomers to bind kinases. (R)-crizotinib has high affinity toward a large number of kinases including its cognate targets MET, ALK, and ROS1. Selectivity Score or S-score is a quantitative measure of compound selectivity. It is calculated by dividing the number of kinases that compounds bind to by the total number of distinct kinases tested, excluding mutant variants. S(35) = (number of non-mutant kinases with %Ctrl <35)/(number of non-mutant kinases tested).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4150021&req=5

Figure 7: KINOMEscan results for both crizotinib enantiomersScreening of both (R)- and (S)-crizotinib against a panel of 456 recombinant human protein kinases indicates a dramatic difference in the ability of the two enantiomers to bind kinases. (R)-crizotinib has high affinity toward a large number of kinases including its cognate targets MET, ALK, and ROS1. Selectivity Score or S-score is a quantitative measure of compound selectivity. It is calculated by dividing the number of kinases that compounds bind to by the total number of distinct kinases tested, excluding mutant variants. S(35) = (number of non-mutant kinases with %Ctrl <35)/(number of non-mutant kinases tested).
Mentions: To further investigate the ability of the two crizotinib enantiomers to engage cellular proteins we derived chemical probes suitable for drug pull-downs (Supplementary Information). We tested the two derivatized compounds for their ability to target ALK and MTH1 in SW480 cell extracts. The two enantiomers were remarkably specific for their cognate targets (Fig. 2f). If MTH1 was indeed the key target of (S)-crizotinib in other RAS-transformed cells, it should rank at the top of the specific interactors in an unbiased chemical proteomic experiment as done before with SCH51344. MTH1 was by far the most specific and prominent interactor of (S)-crizotinib (Extended Data Fig. 2e). Plotting the chemical proteomic results of SCH51344 and (S)-crizotinib against each other singled out MTH1 as the only common high-significance interactor (Fig. 3a). We also performed the reciprocal analysis with (R)-crizotinib which identified a plethora of protein kinases, all efficiently competed by free drug, but not MTH1 (Extended Data Fig. 2f). Notably, comparison of both profiles did not reveal any proteins that were significantly bound by both enantiomers (Fig. 3b). To exclude that either crizotinib enantiomer may target kinases of low abundance we interrogated a panel of 456 different recombinant kinases (KINOMEscan, Extended Data Fig. 3)25. In line with the chemoproteomic results the two enantiomers displayed a remarkable stereoselectivity with very distinct profiles. The few kinases to which (S)-crizotinib showed some affinity were not calculated to be significantly inhibited. (R)-Crizotinib not only bound to ten times more kinases, but also was predicted to efficiently inhibit at least ten of them, including the well characterized cognate targets ALK, MET, but also LCK, IRAK1, JAK3, LOK and SLK. To understand the differences in MTH1 binding, we co-crystallised both (R)- and (S)-crizotinib with recombinant protein. The structure revealed an unfavourable eclipsed conformation of the methyl group at the chiral centre and the halogen substituents on the benzyl ring is likely to reduce the energetic favourability of (R)-crizotinib binding (Fig. 3c,d, Extended Data Fig. 4, 5). ITC data confirmed that the difference in binding between (R)- and (S)-crizotinib was entirely entropic and therefore not due to different binding interactions with the protein (Fig. 2b).

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