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.
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The atomic coordinates and the electron microscopy maps have been deposited at the Protein Data Bank (PDB) with accession code 6O0H.
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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).
Nature thanks Frank M. Raushel and the other anonymous reviewer(s) for their contribution to the peer review of this work.
L.T. is a consultant for Nimbus.
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Extended data figures and tables
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.
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.
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.
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.
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.
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.
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).
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Wei, J., Leit, S., Kuai, J. et al. An allosteric mechanism for potent inhibition of human ATP-citrate lyase. Nature 568, 566–570 (2019) doi:10.1038/s41586-019-1094-6
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