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Molecular basis of human ATM kinase inhibition

Nature Structural & Molecular Biology volume 28, pages 789–798 (2021)Cite this article

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Abstract

Human checkpoint kinase ataxia telangiectasia-mutated (ATM) plays a key role in initiation of the DNA damage response following DNA double-strand breaks. ATM inhibition is a promising approach in cancer therapy, but, so far, detailed insights into the binding modes of known ATM inhibitors have been hampered due to the lack of high-resolution ATM structures. Using cryo-EM, we have determined the structure of human ATM to an overall resolution sufficient to build a near-complete atomic model and identify two hitherto unknown zinc-binding motifs. We determined the structure of the kinase domain bound to ATPγS and to the ATM inhibitors KU-55933 and M4076 at 2.8 Å, 2.8 Å and 3.0 Å resolution, respectively. The mode of action and selectivity of the ATM inhibitors can be explained by structural comparison and provide a framework for structure-based drug design.

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Fig. 1: Cryo-EM structure and activity of dimeric human ATM.
Fig. 2: Human ATM active site conformation in the KU-55933 bound state.
Fig. 3: Human ATM active site conformation in the M4076-bound state.
Fig. 4: Specificity of the inhibitor M4076 towards human ATM kinase over other PIKKs.

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Data availability

The 3D cryo-EM density maps reported in this Article have been deposited in the EMD under accession nos. EMD-12352 (ATM-ATPyS), EMD-12350 (ATM-M4076), EMD-12343 (ATM-KU-55933, Kinase), EMD-12347 (ATM-KU-55933, Spiral), EMD-12345 (ATM-KU-55933, Pincer) and EMD-12346 (ATM-KU-55933, Spiral–Pincer). The corresponding models have been deposited in the Protein Data Bank under IDs 7NI6 (ATM-ATPyS), 7NI4 (ATM-M4076) and 7NI5 (ATM-KU-55933). Raw micrographs are archived at the LRZ of the Bavarian Academy of Science and Humanities and can be accessed for legitimate research purposes upon reasonable request to K.P.H. (hopfner@genzentrum.lmu.de). Any requests for additional data by qualified scientific and medical researchers for legitimate research purposes will be subject to the Merck KGaA, Darmstadt, Germany Data Sharing Policy. All requests should be submitted in writing to the Merck KGaA, Darmstadt, Germany data-sharing portal (https://www.emdgroup.com/en/research/our-approach-to-research-and-development/healthcare/clinical-trials/commitment-responsible-data-sharing.html). When Merck KGaA, Darmstadt, Germany has a co-research, co-development, or co-marketing or co-promotion agreement, or when the product has been out-licensed, the responsibility for disclosure might be dependent on the agreement between parties. Under these circumstances, Merck KGaA, Darmstadt, Germany, will endeavor to gain agreement to share data in response to requests. Source data are provided with this paper.

References

  1. Hoeijmakers, J. H. Genome maintenance mechanisms for preventing cancer. Nature 411, 366–374 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Matsuoka, S. et al. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science 316, 1160–1166 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Bartek, J. & Lukas, J. Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer Cell 3, 421–429 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Burma, S., Chen, B. P., Murphy, M., Kurimasa, A. & Chen, D. J. ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J. Biol. Chem. 276, 42462–42467 (2001).

    Article  CAS  PubMed  Google Scholar 

  5. Roos, W. P. & Kaina, B. DNA damage-induced cell death: from specific DNA lesions to the DNA damage response and apoptosis. Cancer Lett. 332, 237–248 (2013).

    Article  CAS  PubMed  Google Scholar 

  6. Mills, K. D., Ferguson, D. O. & Alt, F. W. The role of DNA breaks in genomic instability and tumorigenesis. Immunol. Rev. 194, 77–95 (2003).

    Article  CAS  PubMed  Google Scholar 

  7. Kastan, M. B. & Bartek, J. Cell-cycle checkpoints and cancer. Nature 432, 316–323 (2004).

    Article  CAS  PubMed  Google Scholar 

  8. Lovejoy, C. A. & Cortez, D. Common mechanisms of PIKK regulation. DNA Repair (Amst.) 8, 1004–1008 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Keith, C. T. & Schreiber, S. L. PIK-related kinases: DNA repair, recombination and cell cycle checkpoints. Science 270, 50–51 (1995).

    Article  CAS  PubMed  Google Scholar 

  10. Shiloh, Y. & Ziv, Y. The ATM protein kinase: regulating the cellular response to genotoxic stress, and more. Nat. Rev. Mol. Cell Biol. 14, 197–210 (2013).

    Article  CAS  PubMed  Google Scholar 

  11. Zou, L. & Elledge, S. J. Sensing DNA damage through ATRIP recognition of RPA–ssDNA complexes. Science 300, 1542–1548 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Yamashita, A., Ohnishi, T., Kashima, I., Taya, Y. & Ohno, S. Human SMG-1, a novel phosphatidylinositol 3-kinase-related protein kinase, associates with components of the mRNA surveillance complex and is involved in the regulation of nonsense-mediated mRNA decay. Genes Dev. 15, 2215–2228 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. McMahon, S. B., Van Buskirk, H. A., Dugan, K. A., Copeland, T. D. & Cole, M. D. The novel ATM-related protein TRRAP is an essential cofactor for the c-Myc and E2F oncoproteins. Cell 94, 363–374 (1998).

    Article  CAS  PubMed  Google Scholar 

  14. Yang, H. et al. mTOR kinase structure, mechanism and regulation. Nature 497, 217–223 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Imseng, S., Aylett, C. H. & Maier, T. Architecture and activation of phosphatidylinositol 3-kinase related kinases. Curr. Opin. Struct. Biol. 49, 177–189 (2018).

    Article  CAS  PubMed  Google Scholar 

  16. Baretic, D. et al. Structures of closed and open conformations of dimeric human ATM. Sci. Adv. 3, e1700933 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Baretic, D. & Williams, R. L. PIKKs—the solenoid nest where partners and kinases meet. Curr. Opin. Struct. Biol. 29, 134–142 (2014).

    Article  CAS  PubMed  Google Scholar 

  18. Sawicka, M. et al. The dimeric architecture of checkpoint kinases Mec1ATR and Tel1ATM reveal a common structural organization. J. Biol. Chem. 291, 13436–13447 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Jansma, M. et al. Near-complete structure and model of Tel1ATM from Chaetomium thermophilum reveals a robust autoinhibited ATP state. Structure 28, 83–95 (2020).

    Article  CAS  PubMed  Google Scholar 

  20. Xin, J. et al. Structural basis of allosteric regulation of Tel1/ATM kinase. Cell Res. 29, 655–665 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Yates, L. A. et al. Cryo-EM structure of nucleotide-bound Tel1(ATM) unravels the molecular basis of inhibition and structural rationale for disease-associated mutations. Structure 28, 96–104 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Xiao, J. et al. Structural insights into the activation of ATM kinase. Cell Res. 29, 683–685 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bakkenist, C. J. & Kastan, M. B. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421, 499–506 (2003).

    Article  CAS  PubMed  Google Scholar 

  24. Guo, Z., Kozlov, S., Lavin, M. F., Person, M. D. & Paull, T. T. ATM activation by oxidative stress. Science 330, 517–521 (2010).

    Article  CAS  PubMed  Google Scholar 

  25. Lavin, M. F. & Yeo, A. J. Clinical potential of ATM inhibitors. Mutat. Res. 821, 111695 (2020).

    Article  CAS  PubMed  Google Scholar 

  26. Jin, M. H. & Oh, D. Y. ATM in DNA repair in cancer. Pharmacol. Ther. 203, 107391 (2019).

    Article  CAS  PubMed  Google Scholar 

  27. Weber, A. M. & Ryan, A. J. ATM and ATR as therapeutic targets in cancer. Pharmacol. Ther. 149, 124–138 (2015).

    Article  CAS  PubMed  Google Scholar 

  28. Rainey, M. D., Charlton, M. E., Stanton, R. V. & Kastan, M. B. Transient inhibition of ATM kinase is sufficient to enhance cellular sensitivity to ionizing radiation. Cancer Res. 68, 7466–7474 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Batey, M. A. et al. Preclinical evaluation of a novel ATM inhibitor, KU59403, in vitro and in vivo in p53 functional and dysfunctional models of human cancer. Mol. Cancer Ther. 12, 959–967 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Riches, L. C. et al. Pharmacology of the ATM inhibitor AZD0156: potentiation of irradiation and olaparib responses preclinically. Mol. Cancer Ther. 19, 13–25 (2020).

    Article  CAS  PubMed  Google Scholar 

  31. Fuchss, T. et al. Abstract 3500: highly potent and selective ATM kinase inhibitor M4076: a clinical candidate drug with strong anti-tumor activity in combination therapies. Cancer Res. 79, 3500 (2019).

    Google Scholar 

  32. Choi, M., Kipps, T. & Kurzrock, R. ATM mutations in cancer: therapeutic implications. Mol. Cancer Ther. 15, 1781–1791 (2016).

    Article  CAS  PubMed  Google Scholar 

  33. Dohmen, A. J. C. et al. Identification of a novel ATM inhibitor with cancer cell specific radiosensitization activity. Oncotarget 8, 73925–73937 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Toledo-Sherman, L. et al. Optimization of potent and selective ataxia telangiectasia-mutated inhibitors suitable for a proof-of-concept study in Huntington’s disease models. J. Med. Chem. 62, 2988–3008 (2019).

    Article  CAS  PubMed  Google Scholar 

  35. Min, A. et al. AZD6738, a novel oral inhibitor of ATR, induces synthetic lethality with ATM deficiency in gastric cancer cells. Mol. Cancer Ther. 16, 566–577 (2017).

    Article  CAS  PubMed  Google Scholar 

  36. Perkhofer, L. et al. ATM deficiency generating genomic instability sensitizes pancreatic ductal adenocarcinoma cells to therapy-induced DNA damage. Cancer Res. 77, 5576–5590 (2017).

    Article  CAS  PubMed  Google Scholar 

  37. Schmitt, A. et al. ATM deficiency is associated with sensitivity to PARP1- and ATR inhibitors in lung adenocarcinoma. Cancer Res. 77, 3040–3056 (2017).

    Article  CAS  PubMed  Google Scholar 

  38. Weston, V. J. et al. The PARP inhibitor olaparib induces significant killing of ATM-deficient lymphoid tumor cells in vitro and in vivo. Blood 116, 4578–4587 (2010).

    Article  CAS  PubMed  Google Scholar 

  39. Kantidze, O. L., Velichko, A. K., Luzhin, A. V., Petrova, N. V. & Razin, S. V. Synthetically lethal interactions of ATM, ATR and DNA-PKcs. Trends Cancer 4, 755–768 (2018).

    Article  CAS  PubMed  Google Scholar 

  40. Hickson, I. et al. Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM. Cancer Res. 64, 9152–9159 (2004).

    Article  CAS  PubMed  Google Scholar 

  41. Golding, S. E. et al. Improved ATM kinase inhibitor KU-60019 radiosensitizes glioma cells, compromises insulin, AKT and ERK prosurvival signaling, and inhibits migration and invasion. Mol. Cancer Ther. 8, 2894–2902 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Karlin, J. et al. Orally bioavailable and blood–brain barrier-penetrating ATM inhibitor (AZ32) radiosensitizes intracranial gliomas in mice. Mol. Cancer Ther. 17, 1637–1647 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Pike, K. G. et al. The identification of potent, selective and orally available inhibitors of ataxiatelangiectasia mutated (ATM) kinase: the discovery of AZD0156(8-{6-[3-(dimethylamino)propoxy]pyridin-3-yl}-3-methyl-1-(tetrahydro-2H-pyran-4-yl)-1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one). J. Med. Chem. 61, 3823–3841 (2018).

    Article  CAS  PubMed  Google Scholar 

  44. Durant, S. T. et al. The brain-penetrant clinical ATM inhibitor AZD1390 radiosensitizes and improves survival of preclinical brain tumor models. Sci. Adv. 4, eaat1719 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Morgado-Palacin, I. et al. Targeting the kinase activities of ATR and ATM exhibits antitumoral activity in mouse models of MLL-rearranged AML. Sci. Signal. 9, ra91 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Fuchss, T., Becker, A., Kubas, H. & Graedler, U. Imidazolonylquinoline compounds and therapeutic uses thereof. WO patent 2020/193660 (2020).

  47. Boland, A., Chang, L. & Barford, D. The potential of cryo-electron microscopy for structure-based drug design. Essays Biochem. 61, 543–560 (2017).

    Article  PubMed  Google Scholar 

  48. Seidel, J. J., Anderson, C. M. & Blackburn, E. H. A novel Tel1/ATM N-terminal motif, TAN, is essential for telomere length maintenance and a DNA damage response. Mol. Cell. Biol. 28, 5736–5746 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Fernandes, N. et al. DNA damage-induced association of ATM with its target proteins requires a protein interaction domain in the N terminus of ATM. J. Biol. Chem. 280, 15158–15164 (2005).

    Article  CAS  PubMed  Google Scholar 

  50. Falck, J., Coates, J. & Jackson, S. P. Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage. Nature 434, 605–611 (2005).

    Article  CAS  PubMed  Google Scholar 

  51. Tate, J. G. et al. COSMIC: the Catalogue Of Somatic Mutations In Cancer. Nucleic Acids Res. 47, D941–D947 (2019).

    Article  CAS  PubMed  Google Scholar 

  52. Weber, A. M. et al. Phenotypic consequences of somatic mutations in the ataxia-telangiectasia mutated gene in non-small cell lung cancer. Oncotarget 7, 60807–60822 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Milanovic, M. et al. The cancer-associated ATM R3008H mutation reveals the link between ATM activation and its exchange. Cancer Res. 81, 426–437 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  54. Vivek, M., & Dunbrack, R. L. Defining a new nomenclature for the structures of active and inactive kinases. Proc. Natl Acad. Sci. USA 116, 6818–6827 (2019).

    Article  CAS  Google Scholar 

  55. Tannous, E. A., Yates, L. A., Zhang, X. & Burgers, P. M. Mechanism of auto-inhibition and activation of Mec1(ATR) checkpoint kinase. Nat. Struct. Mol. Biol. 28, 50–61 (2021).

    Article  CAS  PubMed  Google Scholar 

  56. Langer, L. M., Gat, Y., Bonneau, F. & Conti, E. Structure of substrate-bound SMG1-8-9 kinase complex reveals molecular basis for phosphorylation specificity. eLife 9, e57127 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Kuhn, B., Fuchs, J. E., Reutlinger, M., Stahl, M. & Taylor, N. R. Rationalizing tight ligand binding through cooperative interaction networks. J. Chem. Inf. Model. 51, 3180–3198 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Chaplin, A. K. et al. Dimers of DNA-PK create a stage for DNA double-strand break repair. Nat. Struct. Mol. Biol. 28, 13–19 (2021).

    Article  CAS  PubMed  Google Scholar 

  59. Armstrong, S. A., Schultz, C. W., Azimi-Sadjadi, A., Brody, J. R. & Pishvaian, M. J. ATM dysfunction in pancreatic adenocarcinoma and associated therapeutic implications. Mol. Cancer Ther. 18, 1899–1908 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  63. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  64. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  65. Bepler, T. et al. Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs. Nat. Methods 16, 1153–1160 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Ramlaul, K., Palmer, C. M. & Aylett, C. H. S. A local agreement filtering algorithm for transmission EM reconstructions. J. Struct. Biol. 205, 30–40 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  67. Wood, C. et al. Collaborative computational project for electron cryo-microscopy. Acta Crystallogr. D Biol. Crystallogr. 71, 123–126 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D Struct. Biol. 75, 861–877 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  CAS  Google Scholar 

  70. Drozdetskiy, A., Cole, C., Procter, J. & Barton, G. J. JPred4: a protein secondary structure prediction server. Nucleic Acids Res. 43, W389–W394 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D Struct. Biol. 74, 531–544 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators and developers. Protein Sci. 30, 70–82 (2020).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  73. Kashishian, A. et al. DNA-dependent protein kinase inhibitors as drug candidates for the treatment of cancer. Mol. Cancer Ther. 2, 1257–1264 (2003).

    CAS  PubMed  Google Scholar 

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Acknowledgements

EM data were collected at the Cryo-EM Core Facility of the Gene Center, Department of Biochemistry, LMU, Munich. B. Kessler, a technician at the Gene Center, LMU, provided support with cloning and protein purification. We are grateful for support from B. Blume and H. Dahmen, both employees of Merck KGaA, Darmstadt, Germany, for providing cellular pCHK2 results. Funding was provided by the Deutsche Forschungsgemeinschaft (CRC1054 to K.L.; CRC1361, CRC1064, Gottfried Wilhelm Leibniz-Prize and GRK1721 to K.P.H.) and a PhD fellowship from Boehringer Ingelheim Fonds (BIF) to E.v.d.L. This research was supported by the healthcare business of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID 10.13039/100009945), which provided solid material of inhibitor M4076, free of charge.

Author information

Author notes

  1. A. Alt

    Present address: Proteros Biostructures GmbH, Martinsried, Germany

  2. These authors contributed equally: K. Stakyte, M. Rotheneder, K. Lammens, J. D. Bartho.

Authors and Affiliations

  1. Gene Center, Department of Biochemistry, Ludwig-Maximilians-Universität, Munich, Germany

    K. Stakyte, M. Rotheneder, K. Lammens, J. D. Bartho, A. Alt, E. van de Logt & K. P. Hopfner

  2. Merck KGaA, Darmstadt, Germany

    U. Grädler, T. Fuchß & U. Pehl

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  1. K. Stakyte
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Contributions

J.D.B., K.S., M.R., A.A. and E.v.d.L. were involved in protein production and kinase assays. K.S., M.R., K.L. and J.D.B. carried out structural studies. T.F. was the project leader and principal inventor of M4076 at Merck KGaA, Darmstadt, Germany. U.G. was involved in the structure-based design of M4076 and cryo-EM structural refinement efforts. T.F. and U.P. contributed biochemical assay and kinase selectivity results and graphs. Together with K.L., K.S., M.R., J.D.B., T.F. and U.G. contributed to manuscript preparation. K.P.H. and all authors were involved in the interpretation of data, final manuscript preparation and approval.

Corresponding authors

Correspondence to K. Lammens or K. P. Hopfner.

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Competing interests

K.S., M.R., K.L., J.D.B., E.v.d.L. and K.-P.H. declare no competing interests. A.A. contributed as an employee at the Gene Center, Department of Biochemistry, LMU at the time of the study and is currently an employee of Proteros Biostructures. U.G., T.F. and U.P. are employees of Merck KGaA, Darmstadt, Germany. Proteros Biostructures was contracted by Merck KGaA, Darmstadt, Germany, to determine the cryo-EM structure of ATM in complex with M4076.

Additional information

Peer reveiw information Nature Structural & Molecular Biology thanks Xiaodong Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Anke Sparmann and Beth Moorefield were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Features of the N-terminal Spiral domain of human ATM.

(a) Cryo-EM density maps and models representing three conformational states of N-terminal Spiral-Pincer domains. Left: N-terminal Spiral domains are touching. Middle: N-terminal Spiral domains are not touching. Right: One N-terminal Spiral domain is resolved, the other one is more flexible. (b) Sequence alignment of zinc-binding motifs of ATM from different species. ATM residues involved in zinc coordination are highlighted and coloured by conservation. (c) Kinase activity time-course of zinc-binding motif mutant (C1899A, C1900A, left) and WT (right) ATM. Kinase activity on CtIP C-terminus (650–897, phosphomimic mutation T847E) visualized by phosphoprotein SDS-PAGE staining. Uncropeed gel image is available in Source Data: Extended Data Fig. 1. (d)Effects of chelating agents on the thermal stability of ATM. 1,10-phenanthroline (black) and TPEN (grey) are more specific to Zn2+. ATM only (red) and EGTA (pink) were used as controls. (e)Comparison of map to model FSC curves of previously published Spiral (PDB ID: 5NP0) and here presented Spiral domain model.

Source data

Extended Data Fig. 2 N-terminal alpha-solenoid structure of human ATM.

(a) Overview of organization of N-terminal spiral, pincer and TRD2 domains, helices depicted as cylinders. TAN motif labelled. (b) Interaction between unstructured loop 825–879 and R35 (helix 2). Density map contoured at 0.0034 and R35 colored in magenta. (c)Top view of spiral solenoid ring. Helices 25–28 and ring-crossing loop (630–643) colored in orange. Helices 29–30 and partially resolved loop (657–683) colored in purple. (d) Interaction between Spiral loop (549–570) joining helices 24 and 25 (blue), and TRD2 loops 2084–2092 and 2107–2123 (green). Density map contoured at 0.0032.

Extended Data Fig. 3 Mapping of cancer-associated human ATM missense mutations.

(a) Positions of commonly reported (count > 5) cancer mutations from COSMIC database depicted on to the ATM protomer. Approximate locations of F858L and T1880R mutations are shown based on unmodelled density. One ATM protomer coloured using domain colour-code, another–shown as a grey surface. Mutated residues are shown as red spheres with mutations indicated. (b) Model of KU-55933-bound ATM kinase with commonly (count > 5) mutated amino acid side chains coloured in red. Opposite protomer shown in grey. (c) Table summarising cancer-associated human ATM missense mutations. Mutations grouped based on their locations and coloured using domain colour-code.

Extended Data Fig. 4 Conformation of KU-55933-bound human ATM.

(a) Superimposition of KU-55933-bound human ATM dimer (color) and ‘closed dimer’ of human ATM (PDB ID: 5NP0; grey). Side and bottom views of C-terminal halves shown, helices depicted as cylinders. TRD3 α21-α22 indicated by black dotted line. (b) Sequence alignments of PRD loops from PIKKs from different organisms (top) and PRD loops of human ATM and its orthologs (bottom). PRD loops are colored by conservation and substrate-mimicking Q2971 of human PRD is highlighted. (c) Superimposition of kinase and PRD domains of inhibitor-bound (color) and apo human ATM (PDB ID: 6K9L; grey) models. (d) Cryo-EM density (grey, contoured at 0.0346) and model of the KU-55933 inhibited ATM active site. Clearly visible inhibitor proximal side chains labelled. (e) KU-55933 consists of three different ring systems: Thianthrene (orange), Pyran-4-one (red), Morpholine (green). (f) Detailed view of ATM active site with KU-55933. Van der Waals interactions indicated by dotted lines and interacting residues labelled.

Extended Data Fig. 5 Inhibition of ATM by M4076.

(a) Inhibition of basal ATM kinase activity by M4076. Phosphorylation of CtIP C terminus (650–897, phosphomimetic mutation T847E) visualized by phosphoprotein SDS-PAGE staining, same gel as in Fig. 1a. The experiment was repeated twice with similar results. Uncropped gel image is presented in Source Data Fig. 1. (b) M4076 chemical structure and assignment of functional groups. (c) Detailed view of M4076 (magenta) bound ATM active site. Van der Waals interactions illustrated as black dotted lines and interacting residues labelled. (d) Dose-response curves of enzymatic DNA-PK kinase inhibition by M4076 and KU-55933. Data represent the mean ± s.e.m. from n = 16 independent experiments performed in duplicates for M4076 (IC50 = 600 ± 40 nM) and from one experiment performed in duplicates for KU-55933 (IC50 = 1,400 ± 30 nM). Data behind graph in panel d are available in Source Data Extended Data Fig. 5d.

Source data

Extended Data Fig. 6 Kinase specificity of KU-55933 and M4076 inhibitors.

(a)In vitro cell-based IC50 for inhibition of 2 Gy-induced phosphorylation of CHK2 (pThr68) in the human colon carcinoma cell line HCT116 for M4076 (IC50 = 10 ± 2 nM) from a Luminex assay. Data represent the mean ± s.e.m. from n = 3 independent experiments performed in singlicates. (b) In vitro cell-based IC50 for inhibition of bleomycin-induced phosphorylation of CHK2 (pThr68) in the human colon carcinoma cell line HCT116 for KU-55933 (IC50 = 1,100 ± 260 nM) from an ELISA. Data represent the mean ± s.e.m. from n = 2 independent experiments performed in singlicates. (c) Dose-response curves of enzymatic ATR kinase inhibition by M4076. Data represent the mean ± s.e.m. from n = 7 independent experiments performed in duplicates for M4076 (IC50 = 10,000 ± 320 nM). (d) Dose-response curves of enzymatic mTOR kinase inhibition by the racemate of M4076. Data represent the mean ± s.e.m. from n = 1 experiment performed in duplicate for the racemate of M4076 (IC50 > 30,000 nM). (e)Dose-response curves of enzymatic inhibition of recombinant PIK3 kinases p110α/p85α, p110β/p85α, p120γ, and p110δ/p85α by the racemate of M4076. Data represent the mean ± s.e.m. from n = 1 experiment performed in duplicates for the racemate of M4076 (IC50 values above 8.5 µM). (f)Dose-response curves of enzymatic inhibition of recombinant PIK3 kinases p110α/p85α, p110β/p85α, p120γ, and p110δ/p85α by KU-55933. Data represent the mean ± s.e.m. from n = 1 experiment performed in duplicates (PIK3 isoforms α, β and δ: IC50 values ~2 µM; PI3Kγ: IC50 = 14 µM). (g) Reaction Biology Corporation (RBC) kinase selectivity profiles of 583 kinases for M4076 tested at 1 µM. Segment sizes of pie chart represent numbers of 583 kinases from RBC panel and ATM in addition with IC50 ranges (cyan: 96% of the tested 583 kinases showed kinase activity values above 50% corresponding to IC50 values above 1,000 nM; dark blue: 10 kinases and their mutants, 22 kinases in total (3.8% out of 583 kinases tested) showed kinase activity values below 50% corresponding to IC50 values between 100–1000 nM). ATM is the only kinase with an IC50 value below 100 nM (0.2%). (h) IC50 values of M4076 determined for 10 kinases and their mutants, which showed kinase activity values below 50% tested at 1 µM M4076 in the RBC kinase selectivity profile against 583 kinases (22 kinases in total, corresponding to the dark blue segment in the Reaction Biology kinase selectivity pie chart in Extended Data Fig. 6g). 1: M4076 was tested in duplicate in a 10-dose IC50 mode with 3-fold serial dilution starting at 10 µM. Reactions were carried out at Km ATP concentrations of individual kinases according to the RBC Km binning structure. 2: The biochemical ATM IC50 value of 0.2 nM for M4076 was used for IC50-split calculation of individual kinases. (i) Sequence alignment of human ATM and PIK3 kinases p110α/p85α, p110β/p85α, p120γ, and p110δ/p85α active sites, coloured by conservation. Glycine-rich, catalytic and activation loops labelled. ATM residues forming hydrogen interactions with the inhibitor are marked with red dots, van der Waals interactions - yellow dots, and π-stacking interactions - black dot. Data for panels a-h are available as Source Data (Source Data Extended Data Fig. 6a-h).

Source data

Extended Data Fig. 7 Comparison of ATPγS-bound to KU-55933 and M4076 inhibited ATM kinase active sites.

(a) Active site of KU-55933 (cyan) bound ATM (yellow) with highlighted surrounding residues. (b) Gold-standard Fourier shell correlation (FSC) curve from RELION-3.0 of the KU-55933 full ATM map. (c)Same as (A) with ATPγS (green, orange). (d) FSC curve from RELION-3.0 of the ATPγS bound ATM FATKIN map. (e)Same as (A) with M4076 (magenta). (f) FSC curve from CryoSPARC-2.14 of the M4076-bound ATM FATKIN map. (g) Superimposition of ATPγS (ATM in grey) and M4076 inhibitor (ATM in yellow) bound ATM active sites. (h) ATM active site density (grey, contoured at 0.0169) at 2.8 Å resolution with a fitted ATM model and ATPγS (orange, green).

Extended Data Fig. 8 N-terminal conformations of M4076 and ATPγS bound ATM.

(a) Masked N-terminal 3D classification of M4076-bound ATM followed by further processing of dominant class and comparison to KU-55933 bound ATM conformation. (b) Masked N-terminal 3D classification of ATPγS bound ATM followed by further processing of dominant class and comparison to KU-55933 bound ATM conformation.

Supplementary information

Supplementary Information

Supplementary Figs. 1–7 and notes.

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Supplementary Data 1

Raw data for Table 2.

Supplementary Video 1

General architecture of human ATM bound to KU-55933. The video shows a general overview of domain organization of the KU-55933-inhibited ATM dimer. Locations and conformations of newly identified zinc-binding motifs are highlighted. Domains are colored as in Fig. 1.

Supplementary Video 2

Flexibility of the human ATM N terminus. The video shows the transition between three distinct conformations of ATM N-terminal solenoid structures. The most prominent class has touching N termini (N-term touch), the second class has more distant symmetric N termini (N-term no-touch) and the least abundant class shows one flexibly detached spiral domain (open N-term). Domains are colored as in Fig. 1.

Supplementary Video 3

Comparison of the inhibitor-bound active site conformations. Conformational changes in the ATM kinase active site are visualized by morphing between KU-55933, ATPγS and M4076 bound structures. The active site cleft geometry of the presented ATM structures is nearly identical except the hinge region residue C2770 and minor changes in the P-loop.

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Stakyte, K., Rotheneder, M., Lammens, K. et al. Molecular basis of human ATM kinase inhibition. Nat Struct Mol Biol 28, 789–798 (2021). https://doi.org/10.1038/s41594-021-00654-x

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