Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

An allosteric mechanism for potent inhibition of human ATP-citrate lyase

Nature volume 568, pages 566–570 (2019)Cite this article

Subjects

Abstract

ATP-citrate lyase (ACLY) is a central metabolic enzyme and catalyses the ATP-dependent conversion of citrate and coenzyme A (CoA) to oxaloacetate and acetyl-CoA1,2,3,4,5. The acetyl-CoA product is crucial for the metabolism of fatty acids6,7, the biosynthesis of cholesterol8, and the acetylation and prenylation of proteins9,10. There has been considerable interest in ACLY as a target for anti-cancer drugs, because many cancer cells depend on its activity for proliferation2,5,11. ACLY is also a target against dyslipidaemia and hepatic steatosis, with a compound currently in phase 3 clinical trials4,5. Many inhibitors of ACLY have been reported, but most of them have weak activity5. Here we report the development of a series of low nanomolar, small-molecule inhibitors of human ACLY. We have also determined the structure of the full-length human ACLY homo-tetramer in complex with one of these inhibitors (NDI-091143) by cryo-electron microscopy, which reveals an unexpected mechanism of inhibition. The compound is located in an allosteric, mostly hydrophobic cavity next to the citrate-binding site, and requires extensive conformational changes in the enzyme that indirectly disrupt citrate binding. The observed binding mode is supported by and explains the structure–activity relationships of these compounds. This allosteric site greatly enhances the ‘druggability’ of ACLY and represents an attractive target for the development of new ACLY inhibitors.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: NDI-091143 is a low nanomolar inhibitor of human ACLY.
Fig. 2: Structure of the human ACLY tetramer in complex with NDI-091143 and ADP.
Fig. 3: The structure suggests that the C-terminal domain of ACLY has CCLY activity.
Fig. 4: Binding mode of NDI-091143.

Similar content being viewed by others

Data availability

The atomic coordinates and the electron microscopy maps have been deposited at the Protein Data Bank (PDB) with accession code 6O0H.

References

  1. Chypre, M., Zaidi, N. & Smans, K. ATP-citrate lyase: a mini-review. Biochem. Biophys. Res. Commun. 422, 1–4 (2012).

    CAS  PubMed  Google Scholar 

  2. Zaidi, N., Swinnen, J. V. & Smans, K. ATP-citrate lyase: a key player in cancer metabolism. Cancer Res. 72, 3709–3714 (2012).

    CAS  PubMed  Google Scholar 

  3. Fan, F. et al. On the catalytic mechanism of human ATP citrate lyase. Biochemistry 51, 5198–5211 (2012).

    CAS  PubMed  Google Scholar 

  4. Burke, A. C. & Huff, M. W. ATP-citrate lyase: genetics, molecular biology and therapeutic target for dyslipidemia. Curr. Opin. Lipidol. 28, 193–200 (2017).

    CAS  PubMed  Google Scholar 

  5. Granchi, C. ATP citrate lyase (ACLY) inhibitors: an anti-cancer strategy at the crossroads of glucose and lipid metabolism. Eur. J. Med. Chem. 157, 1276–1291 (2018).

    CAS  PubMed  Google Scholar 

  6. Wakil, S. J., Stoops, J. K. & Joshi, V. C. Fatty acid synthesis and its regulation. Annu. Rev. Biochem. 52, 537–579 (1983).

    CAS  PubMed  Google Scholar 

  7. Tong, L. Structure and function of biotin-dependent carboxylases. Cell. Mol. Life Sci. 70, 863–891 (2013).

    CAS  PubMed  Google Scholar 

  8. Goldstein, J. L. & Brown, M. S. Regulation of the mevalonate pathway. Nature 343, 425–430 (1990).

    ADS  CAS  PubMed  Google Scholar 

  9. Wellen, K. E. et al. ATP-citrate lyase links cellular metabolism to histone acetylation. Science 324, 1076–1080 (2009).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. McTaggart, S. J. Isoprenylated proteins. Cell. Mol. Life Sci. 63, 255–267 (2006).

    CAS  PubMed  Google Scholar 

  11. Hatzivassiliou, G. et al. ATP citrate lyase inhibition can suppress tumor cell growth. Cancer Cell 8, 311–321 (2005).

    CAS  PubMed  Google Scholar 

  12. Warburg, O. On the origin of cancer cells. Science 123, 309–314 (1956).

    ADS  CAS  PubMed  Google Scholar 

  13. Sun, T., Hayakawa, K., Bateman, K. S. & Fraser, M. E. Identification of the citrate-binding site of human ATP-citrate lyase using X-ray crystallography. J. Biol. Chem. 285, 27418–27428 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Sun, T., Hayakawa, K. & Fraser, M. E. ADP-Mg2+ bound to the ATP-grasp domain of ATP-citrate lyase. Acta Crystallogr. F 67, 1168–1172 (2011).

    CAS  Google Scholar 

  15. Hu, J., Komakula, A. & Fraser, M. E. Binding of hydroxycitrate to human ATP-citrate lyase. Acta Crystallogr. D 73, 660–671 (2017).

    CAS  Google Scholar 

  16. Li, J. J. et al. 2-hydroxy-N-arylbenzenesulfonamides as ATP-citrate lyase inhibitors. Bioorg. Med. Chem. Lett. 17, 3208–3211 (2007).

    CAS  PubMed  Google Scholar 

  17. Dufort, F. J. et al. Glucose-dependent de novo lipogenesis in B lymphocytes: a requirement for ATP-citrate lyase in lipopolysaccharide-induced differentiation. J. Biol. Chem. 289, 7011–7024 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Karpusas, M., Branchaud, B. & Remington, S. J. Proposed mechanism for the condensation reaction of citrate synthase: 1.9-Å structure of the ternary complex with oxaloacetate and carboxymethyl coenzyme A. Biochemistry 29, 2213–2219 (1990).

    CAS  PubMed  Google Scholar 

  19. Russell, R. J., Ferguson, J. M., Hough, D. W., Danson, M. J. & Taylor, G. L. The crystal structure of citrate synthase from the hyperthermophilic archaeon Pyrococcus furiosus at 1.9 Å resolution. Biochemistry 36, 9983–9994 (1997).

    CAS  PubMed  Google Scholar 

  20. Francois, J. A. et al. Structure of a NADH-insensitive hexameric citrate synthase that resists acid inactivation. Biochemistry 45, 13487–13499 (2006).

    CAS  PubMed  Google Scholar 

  21. Chittori, S., Savithri, H. S. & Murthy, M. R. N. Crystal structure of Salmonella typhimurium 2-methylcitrate synthase: insights on domain movement and substrate specificity. J. Struct. Biol. 174, 58–68 (2011).

    CAS  PubMed  Google Scholar 

  22. Holm, L., Kääriäinen, S., Rosenström, P. & Schenkel, A. Searching protein structure databases with DaliLite v.3. Bioinformatics 24, 2780–2781 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Alter, G. M. et al. Mutation of essential catalytic residues in pig citrate synthase. Biochemistry 29, 7557–7563 (1990).

    CAS  PubMed  Google Scholar 

  24. Evans, C. T., Kurz, L. C., Remington, S. J. & Srere, P. A. Active site mutants of pig citrate synthase: effects of mutations on the enzyme catalytic and structural properties. Biochemistry 35, 10661–10672 (1996).

    CAS  PubMed  Google Scholar 

  25. Kurz, L. C. et al. Effects of changes in three catalytic residues on the relative stabilities of some of the intermediates and transition states in the citrate synthase reaction. Biochemistry 37, 9724–9737 (1998).

    CAS  PubMed  Google Scholar 

  26. Huang, J., Malhi, M., Deneke, J. & Fraser, M. E. Structure of GTP-specific succinyl-CoA synthetase in complex with CoA. Acta Crystallogr. F 71, 1067–1071 (2015).

    CAS  Google Scholar 

  27. Fraser, M. E., James, M. N., Bridger, W. A. & Wolodko, W. T. A detailed structural description of Escherichia coli succinyl-CoA synthetase. J. Mol. Biol. 285, 1633–1653 (1999).

    CAS  PubMed  Google Scholar 

  28. Wang, L. et al. Accurate and reliable prediction of relative ligand binding potency in prospective drug discovery by way of a modern free-energy calculation protocol and force field. J. Am. Chem. Soc. 137, 2695–2703 (2015).

    CAS  PubMed  Google Scholar 

  29. Wang, L. et al. Modeling local structural rearrangements using FEP/REST: application to relative binding affinity predictions of CDK2 inhibitors. J. Chem. Theory Comput. 9, 1282–1293 (2013).

    CAS  PubMed  Google Scholar 

  30. Ohi, M., Li, Y., Cheng, Y. & Walz, T. Negative staining and image classification — powerful tools in modern electron microscopy. Biol. Proced. Online 6, 23–34 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Suloway, C. et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005).

    CAS  PubMed  Google Scholar 

  32. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).

    CAS  PubMed  Google Scholar 

  33. Yang, Z., Fang, J., Chittuluru, J., Asturias, F. J. & Penczek, P. A. Iterative stable alignment and clustering of 2D transmission electron microscope images. Structure 20, 237–247 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Hohn, M. et al. SPARX, a new environment for Cryo-EM image processing. J. Struct. Biol. 157, 47–55 (2007).

    CAS  PubMed  Google Scholar 

  35. Kimanius, D., Forsberg, B. O., Scheres, S. H. & Lindahl, E. Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5, e18722 (2016).

    PubMed  PubMed Central  Google Scholar 

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

    PubMed  PubMed Central  Google Scholar 

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

    PubMed  PubMed Central  Google Scholar 

  38. Bai, X. C., Rajendra, E., Yang, G., Shi, Y. & Scheres, S. H. Sampling the conformational space of the catalytic subunit of human γ-secretase. eLife 4, e11182 (2015).

    PubMed  PubMed Central  Google Scholar 

  39. Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  41. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002).

    PubMed  Google Scholar 

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

    PubMed  Google Scholar 

  43. Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    CAS  PubMed  Google Scholar 

  44. Harder, E. et al. OPLS3: a force field providing broad coverage of drug-like small molecules and proteins. J. Chem. Theory Comput. 12, 281–296 (2016).

    CAS  PubMed  Google Scholar 

  45. Wang, L., Berne, B. J. & Friesner, R. A. On achieving high accuracy and reliability in the calculation of relative protein-ligand binding affinities. Proc. Natl Acad. Sci. USA 109, 1937–1942 (2012).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  46. Wang, L., Friesner, R. A. & Berne, B. J. Replica exchange with solute scaling: a more efficient version of replica exchange with solute tempering (REST2). J. Phys. Chem. B 115, 9431–9438 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank R. Grassucci, D. Bobe, K. Jordan, L. Yen, A. Raczkowski, W. Rice, E. Eng, M. Kopylov, and B. Carragher for access to the electron microscopy facility and help with data collection at the New York Structural Biology Center; and R. Grassucci and Z. Fu for help with data collection at the EM facility at Columbia University. We thank S. Qian and X. Qiang (Pharmaron) for enzyme-activity assay and kinetics studies, M. Geitman (Beactica) for SPR studies, D. Lakshminarasimhan (Xtal BioStructures) for structural studies on the N-terminal segment, proteolysis and early DSF work, Pharmaron for small-molecule synthesis, and H. Blanchette and K. Kreutter (Nimbus) for discussions. The portion of this research that was conducted in the L.T. laboratory was supported by a grant from Nimbus. Some of this work was performed at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center, supported by grants from the Simons Foundation (349247), NYSTAR, and the NIH National Institute of General Medical Sciences (GM103310).

Reviewer information

Nature thanks Frank M. Raushel and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Authors and Affiliations

  1. Department of Biological Sciences, Columbia University, New York, NY, USA

    Jia Wei & Liang Tong

  2. Nimbus Therapeutics, Cambridge, MA, USA

    Silvana Leit, Jun Kuai, H. James Harwood Jr & Byron DeLaBarre

  3. Schrödinger, LLC, New York, NY, USA

    Eric Therrien & Salma Rafi

Authors
  1. Jia Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Silvana Leit
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jun Kuai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Eric Therrien
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Salma Rafi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. H. James Harwood Jr
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Byron DeLaBarre
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Liang Tong
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.W. carried out protein expression and purification, grid preparation and optimization, electron microscopy data collection, image processing and reconstruction, atomic model building and refinement, and thermal shift assays. S.L. designed the small-molecule inhibitors. J.K. and H.J.H. designed the enzyme assay and kinetics studies. J.K. designed the SPR studies. E.T. and S.R. performed the free-energy perturbation studies. B.D. identified the conditions for stabilizing the ACLY protein. S.L., J.K., H.J.H., B.D., and L.T. supervised the research and analysed the results. L.T., J.W., J.K., B.D., and E.T. wrote the paper. All authors commented on the paper.

Corresponding author

Correspondence to Liang Tong.

Ethics declarations

Competing interests

L.T. is a consultant for Nimbus.

Additional information

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

Extended data figures and tables

Extended Data Fig. 1 Characterization of inhibitor binding to ACLY.

a, The four steps in the reaction catalysed by ACLY. b, Production of ADP is linear over 60 min. Data from duplicate experiments are shown, but the linearity of the progress curve was observed in many additional experiments. c–e, Double reciprocal plots showing that NDI-091143 is competitive versus citrate (Ki = 7.0 ± 0.8 nM) (c); noncompetitive versus ATP (Ki = 218 ± 14 nM) (d); and mixed-type noncompetitive versus CoA (Ki = 5.0 ± 2.1 nM, Ki′ = 156 ± 20 nM) (e). Data from duplicate experiments are shown (n = 2). Although the error on the Ki value versus CoA is relatively high, it does not affect the conclusion regarding the pattern of inhibition versus CoA. The assigned pattern of inhibition has 99.99% probability based on global fitting. The fixed substrate concentrations were 300 μM ATP, 300 μM CoA, and 5 mM citrate. f, Limited proteolysis experiments on full-length human ACLY wild-type (WT), H760A, and H760E mutants in the presence of various substrates and/or NDI-091143. CHY, chymotrypsin. The experiments were repeated at least three times, with similar results.

Extended Data Fig. 2 Negative-stain electron microscopy analysis of ACLY in the absence or presence of inhibitor NDI-091143.

a, A representative negative-stain image of ACLY in the presence of Mg-ATP only. A total of 155 images were examined, with similar results. b, Selected 2D class averages of negatively stained ACLY in the presence of Mg-ATP, generated with the ISAC algorithm. c, A representative negative-stain image of ACLY in the presence of Mg-ATP and NDI-091143. A total of 216 images were examined, with similar results. d, Selected 2D class averages of negatively stained ACLY in the presence of Mg-ATP and inhibitor, generated using the ISAC algorithm. e, The model generated with VIPER and refined with Meridien in SPARX using particles from d.

Extended Data Fig. 3 Processing of single-particle cryo-EM data for ACLY.

Flowchart of data processing showing a representative cryo-EM micrograph, selected reference-free 2D class averages, 3D classification and refinement procedures for the full-length ACLY tetramer as well as two N-terminal segments.

Extended Data Fig. 4 Cryo-EM analysis of the ACLY homo-tetramer and N-terminal segment.

a, Gold-standard FSC curves for masked, unmasked, and high-resolution phase-randomized reconstructions of the ACLY tetramer, as well as its final corrected FSC curve. The correlation is calculated between two independently determined half maps. b, Local-resolution map of the ACLY tetramer. c, Gold-standard FSC curves for the ACLY N-terminal segment, as well as its final corrected FSC curve. The correlation is calculated between two independently determined half maps. d, Local-resolution map of the ACLY N-terminal segment.

Extended Data Fig. 5 Electron microscopy density for selected regions of the ACLY structure.

a, Cryo-EM 3D map of the ACLY tetramer, coloured according to Fig. 1a. b, Electron microscopy density map for a helix in the C-terminal CCLY domain. c, Electron microscopy density of the linker between N-terminal and C-terminal segments (residues 818–830). d, Cryo-EM 3D map of the ACLY N-terminal segment, coloured according to Fig. 1a. e, Electron microscopy density for the β-sheet in the citrate domain. f, Electron microscopy density map for ADP and surrounding residues in the ATP-grasp domain.

Extended Data Fig. 6 Structural comparison between the CCLY domain of ACLY and CS.

a, Overlay of the CCLY tetramer (labelled 1, 2, 3, and 4) with a dimer of chicken CS (light blue and grey). b, Overlay of the CCLY tetramer with a dimer of CS, viewed after a 90° rotation around the horizontal axis from a. Regions where the N-terminal segment of CS overlaps with protomer 3 of CCLY are indicated by red arrows. The second protomer (B) of the CS dimer does not overlay well with CCLY. c, Overlay of the CCLY core (green) with a C-terminal segment (residues 167–366) of 2-methyl CS (grey). d, Overlay of the CCLY tetramer with a dimer of 2-methyl CS. Regions where the N-terminal segment of 2-methyl CS overlaps with protomer 3 of CCLY are indicated by red arrows. The second protomer (B) of the 2-methyl CS dimer has good overlay with protomer 2 of CCLY, suggesting a CCLY dimer (protomers 1 and 2) has similar organization to the 2-methyl CS dimer, although 2-methyl CS dimers can also assemble into decamers.

Extended Data Fig. 7 Catalytic mechanisms of CS and CCLY.

a, Catalytic mechanism of CS. The two steps of the reaction to produce citryl-CoA are labelled. An enol CoA intermediate is produced after the first step. The three arginine residues that recognize OAA are shown. The prime on the label of Arg421 indicates that it is from a different protomer of the enzyme. b, Possible catalytic mechanism of the CCLY domain of ACLY.

Extended Data Fig. 8 Substrate-binding sites in ACLY.

a, Possible binding mode of CoA in the CoA domain. Lys1018 from the CCLY domain of protomer 3 of the tetramer (orange) may contribute to CoA binding. b, Binding sites of NDI-091143 (black) and ADP (brown) as observed in this structure, as well as the possible binding sites of citrate (grey), CoA in the N-terminal CoA domain (CoA1, grey), CoA in the CCLY domain of protomer 3 of the tetramer (CoA2, light blue), and OAA as a mimic of the citryl group (light blue). The translocation of CoA between the two binding sites is indicated by the red arrow. Therefore, step 1 of the ACLY reaction occurs in the ATP-grasp active site, steps 2 and 3 occur at the interface between the citrate and CoA domains, and step 4 occurs in the CCLY domain. The pHis domain provides the His760 residue for both steps 1 and 2.

Extended Data Fig. 9 The inhibitor NDI-091143 is almost completely shielded from the solvent in the binding site.

a, An alternative model for the inhibitor does not fit the density as well and clashes with the protein. The bound conformation of the inhibitor has pseudo two-fold symmetry, around the sulfonamide group. In the alternative model (thin sticks in grey), the distal phenyl group (blue arrow) has weaker density, and an oxygen atom (red arrow) on the sulfonamide has steric clash with the protein (main-chain amide of Gly342). This alternative model is not consistent with the structure–activity relationship of the compounds. The view is related to Fig. 4a by a 40° rotation around the horizontal axis. b, The inhibitor is located in a cavity (grey transparent surface) in the hydrophobic core of the citrate domain. c, The cavity does not exist in the structure of the citrate complex (note the lack of any surfaces near the compound-binding site), and the inhibitor clashes with the enzyme, especially the side chain of Arg379 (red arrow). d, e, The inhibitor is only accessible to the solvent through two small portals. The protein is shown as a molecular surface (cyan).

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Full size table

Supplementary information

Supplementary Information

This file contains Supplementary Text regarding the synthesis of NDI-091143, Scheme 1, and Supplementary Information Figs. 1–3.

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wei, J., Leit, S., Kuai, J. et al. An allosteric mechanism for potent inhibition of human ATP-citrate lyase. Nature 568, 566–570 (2019). https://doi.org/10.1038/s41586-019-1094-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-019-1094-6

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing