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Small-molecule inhibition of the archetypal UbiB protein COQ8

Nature Chemical Biology volume 19, pages 230–238 (2023)Cite this article

Subjects

Abstract

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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Fig. 1: Characterization of in vitro COQ8A inhibitors.
Fig. 2: UNC-CA157 decreases de novo CoQ production in COQ8BKO HAP1 cells.
Fig. 3: Mitochondrial targeting of TPP-UNC-CA157 increases cellular efficacy in HAP1 cells.
Fig. 4: TPP-UNC-CA157 kinome specificity.

Data availability

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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Acknowledgements

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.

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Authors and Affiliations

  1. Department of Biochemistry, University of Wisconsin–Madison, Madison, WI, USA

    Nathan H. Murray, Naomi Ptak, Robert W. Smith, Craig A. Bingman & David J. Pagliarini

  2. Morgridge Institute for Research, Madison, WI, USA

    Nathan H. Murray & David J. Pagliarini

  3. Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, USA

    Nathan H. Murray, Zixiang Fang & David J. Pagliarini

  4. Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Christopher R. M. Asquith, Michael P. East & Gary L. Johnson

  5. Structural Genomics Consortium and Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Christopher R. M. Asquith

  6. School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland

    Christopher R. M. Asquith

  7. Promega Corporation, Madison, WI, USA

    James D. Vasta, Chad A. Zimprich, Cesear R. Corona & Matthew B. Robers

  8. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, USA

    David J. Pagliarini

  9. Department of Genetics, Washington University School of Medicine, St. Louis, MO, USA

    David J. Pagliarini

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Contributions

N.H.M. and D.J.P. wrote the manuscript. N.H.M., C.R.M.A. and D.J.P. conceived the overall project and its design. C.R.M.A. performed compound synthesis and contributed to inhibitor development. Z.F. performed and analyzed MS experiments. N.P. contributed reagents (cloning) and developed DSF methods. R.W.S. and C.A.B. performed crystallization trials and solved the crystal structure. J.D.V., C.A.Z., C.R.C. and M.B.R. performed NanoBRET analyses. M.P.E. and G.L.J. developed and performed the MIBS–MS analyses. All authors reviewed and edited the manuscript.

Corresponding author

Correspondence to David J. Pagliarini.

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Competing interests

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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Extended data

Extended Data Fig. 1 COQ8A inhibition in vitro.

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).

Source data

Extended Data Fig. 2 De novo CoQ10 biosynthesis measurements.

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).

Source data

Extended Data Fig. 3 F495 is required for potent inhibition.

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).

Source data

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).

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–6, Figs. 1–4 and Note TPP-UNC-CA157 synthesis.

Reporting Summary

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Source Data Extended Data Fig. 1

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Statistical source data and uncropped gel.

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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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