Small-molecule tools have enabled mechanistic investigations and therapeutic targeting of the protein kinase-like (PKL) superfamily. However, such tools are still lacking for many PKL members, including the highly conserved and disease-related UbiB family. Here, we sought to develop and characterize an inhibitor for the archetypal UbiB member COQ8, whose function is essential for coenzyme Q (CoQ) biosynthesis. Guided by crystallography, activity assays and cellular CoQ measurements, we repurposed the 4-anilinoquinoline scaffold to selectively inhibit human COQ8A in cells. Our chemical tool promises to lend mechanistic insights into the activities of these widespread and understudied proteins and to offer potential therapeutic strategies for human diseases connected to their dysfunction.
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LC–MS lipid measurement data that support the findings of this study were deposited into the MassIVE data repository under accession number MSV000090082. Structural data generated in this study have been deposited in the PDB with accession codes 7UDP and 7UDQ. Structural data that were used to support the findings in this study are available in the PDB with accession codes 5I35 and 4PED. Source data are provided with this paper.
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We thank members of the Pagliarini lab for helpful discussions throughout this project. This work was supported by NIH awards R35GM131795 (D.J.P.) and T32GM008505 (N.H.M), funds from the BJC Investigator Program (D.J.P.) and NSF DGE-1747503 (N.H.M.). We would like to thank K. Overmyer, B. Paulson, E. Trujillo and J. Coon for assistance with LC–MS method development. We thank G. Hicks for graphic design assistance. This study made use of the Washington University in St. Louis Genome Engineering and iPSC Center for instrument use and the Washington University in St. Louis Center for Drug Discovery for synthesis services. The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative Medicines Initiative (EU/EFPIA; ULTRA-DD grant number 115766), Janssen, Merck KGaA Darmstadt Germany, MSD, Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation-FAPESP, Takeda and Wellcome (106169/ZZ14/Z). The NIH is acknowledged for support (1U24DK11604-01). We thank Biocenter Finland/DDCB for financial support and the CSC-IT Center for Science, Ltd. (Finland), for allocation of computational resources. We also thank B. Ehrmann and D.E. Weatherspoon for LC–MS/HRMS support provided by the Mass Spectrometry Core Laboratory at the University of North Carolina at Chapel Hill. The core is supported by the National Science Foundation under grant number CHE-1726291. We thank T. Pantsar (University of Eastern Finland) for useful discussions. This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract number DE-AC02-06CH11357. GM/CA@APS has been funded by the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006, P30GM138396). The Eiger 16M detector at GM/CA-XSD was funded by NIH grant S10 OD012289. We thank C. Ogata for beamline support. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant 085P1000817). The Collaborative Crystallography Core in the Department of Biochemistry, Univeristy of Wisconsin–Madison, received support from the Department of Biochemistry endowment.
J.D.V., C.R.C., C.A.Z. and M.B.R. are employed by Promega Corporation. The remaining authors declare no competing interests.
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a, Inhibition of COQ8AN∆250 ATPase activity by the top candidate inhibitors (n = 3 independent samples from one experiment, mean ± SD) (IC50 data are available in the supporting information). b, Top candidate inhibitor binding curves, as determined by DSF (n = 3 independent samples from one experiment, mean ± SD) (Kd,app data are available in the supporting information).
a, Schematic outlining the experimental plan to measure de novo CoQ production. b, De novo production of 13C6-PPHB10 in WT, COQ8AKO, COQ8BKO, and COQ8A/BDKO HAP1 cells after treatment with 10 µM 13C6-4-HB and either DMSO or 20 µM UNC-CA157 (grey = DMSO, red=UNC-CA157, n = 3 independent samples from one experiment, mean ± SD) (two sided students t-test, no adjustment for multiple comparisons). c, Unlabeled PPHB10 levels in WT, COQ8AKO, COQ8BKO, and COQ8A/BDKO HAP1 cells after treatment with 10 µM 13C6-4-HB and either DMSO or 20 µM UNC-CA157 (grey = DMSO, red=UNC-CA157, n = 3 independent samples from one experiment, mean ± SD) (two sided students t-test, no adjustment for multiple comparisons). d, Unlabeled CoQ10 levels in WT, COQ8AKO, COQ8BKO, and COQ8A/BDKO HAP1 cells after treatment with 10 µM 13C6-4-HB and either DMSO or 20 µM UNC-CA157 (grey = DMSO, red=UNC-CA157, n = 3 independent samples from one experiment, mean ± SD) (two sided students t-test, no adjustment for multiple comparisons).
a-c, Protein-ligand interaction networks for three independent complexes of COQ8AN∆254 and UNC-CA157 generated in LigPlot + (PDB: 7UDQ and 7UDP) (hydrogen bond = dashed line; hydrophobic contact = arc with spokes). d, Protein-ligand interaction network for COQ8AN∆254 R611K and AMPPNP generated in LigPlot + (PDB: 5I35) (hydrogen bond = dashed line; hydrophobic contact = arc with spokes). e, SDS-PAGE analysis of COQ8AN∆250 purifications. f, COQ8AN∆250 WT and binding pocket mutant thermal stabilization by UNC-CA157, as determined by DSF (n = 3 independent samples from one experiment, mean ± SD) (two sided students t-test, no adjustment for multiple comparisons, ***p = 1.2 × 10−10 F336A, ***p = 4.5 × 10−5 A356G, ***p = 2.4 × 10−9 K358A, ***p = 4.7 × 10−11 K358E, ***p = 1.7 × 10−6 L443A, *p = 0.0264 T445A, ***p = 1.8 × 10−10 L447A, ***p = 1.4 × 10−9 V448P, ***p = 9.5 × 10−12 F495A; all six WT-1 and WT-2 data points were used for statistical analysis). g, Stick representation of UNC-CA157 and adjacent F495 residue. h, Conservation of F495 across the UbiB protein family. i, Thermal stabilization of COQ8AN∆250 WT and F495L as well as Coq8pN∆41 WT and L353F by UNC-CA157 (n = 3 independent samples from one experiment, mean ± SD) (two sided students t-test, no adjustment for multiple comparisons, *p = 0.0126 COQ8A WT vs. F495L, ***p = 5.6 × 10−7 Coq8p WT vs. L353F). j, Inhibition of COQ8AN∆250 WT and F495L ATPase activity by UNC-CA157 (n = 3 independent samples from one experiment, mean ± SD).
Extended Data Fig. 4 Results from de novo CoQ production with TPP-UNC-CA157 and inhibitor toxicity analysis.
a, Unlabeled CoQ10 and PPHB10 levels in WT, COQ8AKO, COQ8BKO, and COQ8A/BDKO HAP1 cells after treatment with 10 µM 13C6-4-HB and either DMSO or 17.6 µM TPP-UNC-CA157 (n = 3 independent samples from one experiment, mean ± SD) (two sided students t-test, no adjustment for multiple comparisons, *p = 0.0228 WT PPHB10, ***p = 0.0003 COQ8AKO PPHB10). b-d, 13C6-CoQ10 levels in WT HAP1 cells after treatment with 10 µM 13C6-4-HB and indicated concentrations of UNC-CA157, TPP-UNC-CA157 or Alkyl-TPP (n = 3 independent samples from one experiment, mean ± SD) (two sided students t-test, no adjustment for multiple comparisons, *p = 0.0482 DMSO vs. 43.9 µM UNC-CA157, *p = 0.0118 DMSO vs. 22.0 µM TPP-UNC-CA157, **p = 0.0030 DMSO vs. 43.9 µM TPP-UNC-CA157, **p = 0.0026 DMSO vs. 87.8 µM TPP-UNC-CA157). e, Growth analysis of WT HAP1 cells in either 10 mM glucose or 10 mM galactose with indicated concentrations of TPP-UNC-CA157 (n = 3 biologically independent wells of cells in one experiment, mean ± SEM).
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Murray, N.H., Asquith, C.R.M., Fang, Z. et al. Small-molecule inhibition of the archetypal UbiB protein COQ8. Nat Chem Biol 19, 230–238 (2023). https://doi.org/10.1038/s41589-022-01168-3
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