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Antiviral drug recognition and elevator-type transport motions of CNT3

Nature Chemical Biology (2024)Cite this article

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Abstract

Nucleoside analogs have broad clinical utility as antiviral drugs. Key to their systemic distribution and cellular entry are human nucleoside transporters. Here, we establish that the human concentrative nucleoside transporter 3 (CNT3) interacts with antiviral drugs used in the treatment of coronavirus infections. We report high-resolution single-particle cryo-electron microscopy structures of bovine CNT3 complexed with antiviral nucleosides N4-hydroxycytidine, PSI-6206, GS-441524 and ribavirin, all in inward-facing states. Notably, we found that the orally bioavailable antiviral molnupiravir arrests CNT3 in four distinct conformations, allowing us to capture cryo-electron microscopy structures of drug-loaded outward-facing and drug-loaded intermediate states. Our studies uncover the conformational trajectory of CNT3 during membrane transport of a nucleoside analog antiviral drug, yield new insights into the role of interactions between the transport and the scaffold domains in elevator-like domain movements during drug translocation, and provide insights into the design of nucleoside analog antiviral prodrugs with improved oral bioavailability.

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Fig. 1: NAAs interact with hCNT3.
Fig. 2: Cryo-EM structures of IFS bCNT3 in lipid nanodiscs.
Fig. 3: Alternating access of MPV-loaded bCNT3.
Fig. 4: All-atom MD simulations of CNT3 transport dynamics.
Fig. 5: Cryo-EM ensembles of bCNT3 trimers.
Fig. 6: Scaffold domain and local lipid bilayer deformations during transport domain movements.

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Data availability

Atomic coordinates have been deposited in the PDB with the following ID numbers: 8TZ2 (apo), 8TZ5 (NHC), 8TZ6 (PSI-6206), 8TZ1 (ribavirin), 8TZ3 (GS4 consensus), 8TZ4 (GS4 subset), 8TZD (INT1–INT1–OFS; MPV condition 1), 8TZ7 (INT1 homotrimer; MPV condition 1 ensemble), 8TZ8 (INT1–INT1–INT3; MPV condition 1 ensemble), 8TZA (INT1–INT1–INT2; MPV condition 2 ensemble) and 8TZ9 (INT2 homotrimer; MPV condition 2 ensemble). The reconstructed cryo-EM maps have been deposited in the Electron Microscopy Data Bank with the following ID numbers: EMD-41731 (apo), EMD-41734 (NHC), EMD-41735 (PSI-6206), EMD-41730 (ribavirin), EMD-41732 (GS4 consensus), EMD-41733 (GS4 subset), EMD-41755 (INT1–INT1–OFS; MPV condition 1), EMD-41736 (INT1 homotrimer; MPV condition 1 ensemble), EMD-41737 (INT1–INT1–INT3; MPV condition 1 ensemble), EMD-41738 (INT2 homotrimer; MPV condition 2 ensemble), EMD-41739 (INT1–INT1–INT2; MPV condition 2 ensemble), EMD-41740 (consensus; MPV condition 1), EMD-41752 (consensus; MPV condition 2), EMD-41747 (INT3 homotrimer; MPV condition 1 ensemble), EMD-41746 (INT3–INT3–INT1; MPV condition 1 ensemble), EMD-41745 (INT3–INT3–OFS; MPV condition 1 ensemble), EMD-41744 (INT1–INT1–OFS; MPV condition 1 ensemble), EMD-41741 (OFS–OFS–OFS; MPV condition 1 ensemble), EMD-41742 (OFS–OFS–INT1; MPV condition 1 ensemble), EMD-41743 (OFS–OFS–INT3; MPV condition 1 ensemble), EMD-41748 (OFS–INT1–INT3 clockwise; MPV condition 1 ensemble), EMD-41749 (OFS–INT1–INT3 counterclockwise; MPV condition 1 ensemble), EMD-41751 (INT1 homotrimer; MPV condition 2 ensemble) and EMD-41750 (INT1–INT2–INT2; MPV condition 2 ensemble). Source data are provided with this paper. Additional data relevant to this paper are available upon reasonable request to S.-Y. L.

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Acknowledgements

Cryo-EM data were screened and collected at the Duke University SMIF, the National Cancer Institute’s NCEF at the Frederick National Laboratory for Cancer Research, the Pacific Northwest Center for Cryo-EM (PNCC) at Oregon Health and Science University and the National Institute of Environmental Health Sciences. We thank J. Myers at PNCC, A. Wier, T. Fox and U. Baxa at NCEF and N. Bhattacharya at SMIF for assistance with microscope operation. We thank M. Hirschi for initial biochemistry of bCNT3 and K. Tsolova for help with part of the radioactive tracer uptake assay and manuscript reading. This research was supported by a National Institutes of Health grant R21AI166134 (S.-Y.L.) and the National Institute of Health Intramural Research Program, the US National Institutes of Environmental Health Science (ZIC ES103326 to M.J.B.) and the National Science Foundation (MCB-2111728 to W.I.). A portion of this research was supported by National Institutes of Health grant U24GM129547, was performed at the PNCC at Oregon Health and Science University and was accessed through EMSL (grid.436923.9), a Department of Energy Office of Science User Facility sponsored by the Office of Biological and Environmental Research. The Duke University SMIF is affiliated with the North Carolina Research Triangle Nanotechnology Network, which is, in part, supported by the National Science Foundation (ECCS-2025064).

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Authors and Affiliations

  1. Department of Biochemistry, Duke University School of Medicine, Durham, NC, USA

    Nicholas J. Wright, Feng Zhang, Yang Suo, Ying Yin, Justin G. Fedor & Seok-Yong Lee

  2. Departments of Biological Sciences, Chemistry, and Bioengineering, Lehigh University, Bethlehem, PA, USA

    Lingyang Kong & Wonpil Im

  3. Department of Health and Human Services, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, Durham, NC, USA

    Kedar Sharma & Mario J. Borgnia

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Contributions

N.J.W. conducted biochemical preparation, sample freezing, single-particle 3D reconstruction, model building and radiotracer uptake assays. F.Z. performed electrophysiological recordings. Y.S. collected data and performed part of structural analysis. Y.Y. performed biochemical characterization of bCNT3, and J.F. performed part of the radiotracer uptake assay, all under the guidance of S.-Y.L. L.K. performed all MD simulations under the guidance of W.I. K.S. performed part of the cryo-EM sample screening under the guidance of M.J.B. N.J.W. and S.-Y.L. wrote the paper with input from the rest of authors.

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Correspondence to Seok-Yong Lee.

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Nature Chemical Biology thanks Raimund Dutzler and the other, anonymous, reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Functional characterization, protein biochemistry, and cryo-EM sample preparation of bCNT3.

a, b, c, Time-dependent uptake of 1.0 µM [3H]-ribavirin at room temperature is linear within 30 min for hCNT1 (a, background corrected uptake rate of 0.0036 ± 0.0002 pmole/min), hCNT2 (b, background corrected uptake rate of 0.41 ± 0.01 pmole/min), hCNT3 (b, background corrected uptake rate of 0.87 ± 0.04 pmole/min), hENT1 (c, background corrected uptake rate of 0.056 ± 0.004 pmole/min) and hENT2 (c, background corrected uptake rate of 0.098 ± 0.007 pmole/min). Data shown in panels a, b, c are from the same series of experiments (n = 3 biological replicates, individual replicates shown) with the water-injected control measurements re-shown in each figure panel to convey signal-to-background for each transporter subtype. d, Cold-competition of WT hCNT3 or WT bCNT3 mediated [3H]-ribavirin uptake by cold NHC or GS4. (ChoCl – sodium-free negative control condition with 96 mM choline chloride; 15-minute uptake with 0.1 μM [3H]-ribavirin; n = 3 biological replicates with individual replicates and mean ± s.e.m. shown). e, Representative size-exclusion chromatography profile and corresponding SDS-PAGE analysis of purified nanodisc reconstituted bCNT3. Fractions pooled for cryo-EM analysis indicated with an asterisk (*). Purification and nanodisc reconstitution of bCNT3 was repeated routinely with similar results for each cryo-EM structure reported in this study. f, Representative cryo-EM micrograph (top) and 2D class averages (bottom) of nanodisc reconstituted bCNT3. Micrographs of similar quality were recorded for each cryo-EM dataset.

Source data

Extended Data Fig. 2 Cryo-EM image processing of drug-bound and drug-free IFS states of bCNT3.

a, Cryo-EM image processing procedures. b, Fourier-shell correlation plots (top) and angular orientation distributions (bottom) from the final reconstructions. c, Local resolution of the final reconstructions (local resolution determined in cryoSPARC).

Extended Data Fig. 3 Local cryo-EM map quality.

Local cryo-EM maps for secondary structure segments. From top to bottom are NHC, PSI-6206, Ribavirin, GS441524 (subset), GS441524 (consensus) and apo, respectively. Cryo-EM densities are shown at map threshold values of 6σ for NHC, PSI-6206, ribavirin, GS4 sub., apo, or 7σ for GS4 cons.

Extended Data Fig. 4 Sodium ion coordination in the inward-facing state.

a, Location of the two sodium ion binding sites relative to the nucleoside binding site within the NHC bound IFS bCNT3 structure. b, Architecture and coordination geometries of the two sodium ion binding sites in the 2.31 Å cryo-EM map (map thresholds used denoted in panel insets).

Extended Data Fig. 5 TEVC studies of molnupiravir interaction with CNT3.

a, TEVC recordings of inward currents elicited by application of 100 μM uridine, or current block by co-application of 100 μM uridine + 1 mM MPV in WT hCNT3 expressing oocytes (representative trace at left, summary of peak currents from n = 6 biological replicates at right with individual measurements and mean ± s.e.m. shown; values are baseline corrected to leak current per oocyte). b, TEVC recordings of inward currents elicited by application of 100 μM uridine, or current block by co-application of 100 μM uridine + 1 mM MPV in WT bCNT3 expressing oocytes (representative trace at left, summary of peak currents from n = 4 biological replicates at right with individual measurements and mean ± s.e.m. shown; values are baseline corrected to leak current per oocyte).

Source data

Extended Data Fig. 6 Cryo-EM image processing of molnupiravir-bound OFS, INT1, INT2, INT3 conformational states of bCNT3.

a, Initial cryo-EM image processing procedure for the ‘MPV condition 1’ dataset. b, Cryo-EM ensemble analysis of ‘MPV condition 1’. Final Fourier shell correlation plots and angular orientation distribution plots shown next to every reconstruction obtained.

Extended Data Fig. 7 Cryo-EM image processing of molnupiravir-bound INT1 and INT2 conformational states of bCNT3.

a, Initial cryo-EM image processing procedure for the ‘MPV condition 2’ dataset. b, Cryo-EM ensemble analysis of ‘MPV condition 2’. Final Fourier shell correlation plots and angular orientation distribution plots shown next to every reconstruction obtained.

Extended Data Fig. 8 MPV ligand densities in representative OFS, INT1, INT2, and INT3 cryo-EM reconstructions.

Cryo-EM densities around the nucleoside binding site from representative reconstructions containing OFS, INT1, INT2, or INT3 protomers (reconstructions used: OFS-INT1-INT1 highest res; INT1-INT1-INT1 ‘condition 1’ ensemble analysis; INT2-INT2-INT2 ‘condition 2’ ensemble analysis; INT1-INT1-INT3 ‘condition 1’ ensemble analysis). Sharpened maps shown (threshold = 6σ).

Extended Data Fig. 9 Comparison of conformational trajectories in MPV-loaded bCNT3 and apo nwCNT.

a, Structural superposition of bCNT3 (top left) and nwCNT (bottom left) conformers. Alignment based on the trimerization interface, TM3 and TM6. Relative positions of HP1 (middle) and HP2 (right), with lines denoting a marker residue for each conformational state. b, Transport domain movement quantified by its tilt angle relative to a representative IFS structure (X-axis) and distance displacement of nucleoside binding site center of mass (Y-axis). For bCNT3, coordinates corresponding to the highest resolution instance of each conformer used for the analysis (OFS-INT1-INT1 highest res; INT1-INT1-INT1 ‘condition 1’ ensemble analysis; INT2-INT2-INT2 ‘condition 2’ ensemble analysis; INT1-INT1-INT3 ‘condition 1’ ensemble analysis; all relative to the GS4 consensus IFS structure). c, Structural superposition of the transport domain for OFS, INT1, INT3, INT3 and IFS (NHC) structures. d, [3H]-ribavirin uptake (1.0 μM) in 10 minutes into oocytes expressing WT or mutant bCNT3 (n = 3; mean ± s.e.m. and individual replicates shown).

Source data

Extended Data Fig. 10 Map-to-model analysis for reconstructions obtained from the cryo-EM ensemble analysis.

a, Map-to-model of every trimer reconstruction obtained from MPV ‘condition 1’ with the ensemble analysis. Map-to-model correlation analysis shown below each reconstruction (for details see Methods). b, Map to model of every trimer reconstruction obtained from MPV ‘condition 2’ with the ensemble analysis. Map-model correlation analysis shown below each reconstruction (for details see Methods).

Source data

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Wright, N.J., Zhang, F., Suo, Y. et al. Antiviral drug recognition and elevator-type transport motions of CNT3. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01559-8

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  • DOI: https://doi.org/10.1038/s41589-024-01559-8

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