Abstract
ATX is a plasma lysophospholipase D that hydrolyzes lysophosphatidylcholine (LPC) and produces lysophosphatidic acid. To date, no ATX-inhibition-mediated treatment strategies for human diseases have been established. Here, we report anti-ATX DNA aptamers that inhibit ATX with high specificity and efficacy. We solved the crystal structure of ATX in complex with the anti-ATX aptamer RB011, at 2.0-Å resolution. RB011 binds in the vicinity of the active site through base-specific interactions, thus preventing the access of the choline moiety of LPC substrates. Using the structural information, we developed the modified anti-ATX DNA aptamer RB014, which exhibited in vivo efficacy in a bleomycin-induced pulmonary fibrosis mouse model. Our findings reveal the structural basis for the specific inhibition of ATX by the anti-ATX aptamer and highlight the therapeutic potential of anti-ATX aptamers for the treatment of human diseases, such as pulmonary fibrosis.
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Acknowledgements
We thank the beamline staff at BL32XU and BL41XU of SPring-8, Japan, for assistance with data collection. We thank T. Kishimoto for the LPA assays, S. Yamazaki for in vitro assays, E. Inomata for aptamer stability experiments and all other Ribomic members for discussions. We thank T. Nagano (University of Tokyo) for 3BoA. This work was supported by a grant from the Core Research for Evolutional Science and Technology Program, the Creation of Basic Chronic Inflammation, from the Japan Science and Technology Agency, to O.N. This work was also supported in part by a grant from NEDO.
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K. Kato prepared and crystallized the protein and determined the crystal structure; H.I., S.F., Y. Nonaka, M.F., S.M., S.O., K. Kano and J.A. performed in vitro and in vivo experiments; H.I. and S.M. designed aptamers; J.M. prepared the protein; R.I. and H.N. assisted with the structural analysis; and K. Kato, R.I., H.N., Y. Nakamura and O.N. wrote the manuscript with help from all authors. Y. Nakamura and O.N. directed and supervised all of the research.
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Except for K. Kato, J.A., J.M., R.I., H.N. and O.N., all authors are either employees of Ribomic Inc. and/or hold equity in Ribomic Inc. The remaining authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Characterization of RB011.
a, b, RB011 binding to ATX measured by SPR. Sensorgrams of different concentrations of RB011 (50, 25, 12.5, 6.25 and 3.12 nM from upper to lower sensorgrams) binding to human ATX (a) and mouse ATX (b). c, Sensorgrams of RB011 binding to mouse ATX (ENPP2) and ENPP1. d, Plasma pharmacokinetic profiles of the PEGylated RB011. The PEGylated RB011 was administered intravenously, subcutaneously, or intraperitoneally to C57BL/6J mice at 10 mg/kg. Data are mean ± S.E.M. (n = 3, number of mice). e, Pharmacokinetic parameters.
Supplementary Figure 2 Electron density map.
The 2mFO – DFC electron density map around the ATX–RB011 interface is shown as a gray mesh, contoured at 1σ. Water molecules are shown as red spheres.
Supplementary Figure 3 Multiple sequence alignment of ATX proteins from different animal species.
The zinc-coordinating and catalytic residues, gray triangles; the residues interacting with the stem loop, red triangles; the residues interacting with the corner junction, yellow triangles; and the residues interacting with the terminal stem, blue triangles.
Supplementary Figure 4 Multiple sequence alignment of the mouse ENPP family proteins.
The zinc-coordinating and catalytic residues and the residues interacting with RB011 are shown as in Supplementary Fig. 3.
Supplementary Figure 5 Recognition of ATX by RB011.
a, The catalytic domain of ATX in complex with RB011. b, The catalytic domain of ENPP1 with RB011 (model). The ATX–RB011 complex was superimposed on ENPP1 (PDB ID 4GTW). The yellow circle indicates predicted steric clashes between RB011 and a loop region (red) in the insertion subdomain. c, Sensorgrams of RB011 binding to the wild type and mutants of mouse ATX. d, Inhibition of the LysoPLD activities of the wild type and mutants of mouse ATX by RB011 (0.1 μM). Data are mean ± S.D (n = 4). Results are from two independent experiments with two technical replicates each.
Supplementary Figure 6 Structure-based optimization of anti-ATX aptamers.
a, Inhibition of LPA production in human serum after 3 h incubation at 37°C in the presence of 1 or 0.1 μM of the indicated inhibitors. Data are mean ± S.D. (n = 3, number of samples). b, Lung pharmacokinetic profiles of RB014. RB014 was administered intranasally to C57BL/6J mice at 20 μg/mouse. Data are mean ± S.D. (n = 3, number of mice). c, Inhibition of ATX activity by RB014 in BALF of BLM-induced pulmonary fibrosis model mice. The BALF of BLM-induced pulmonary fibrosis model mice was prepared, as in the experiments in Fig. 5, and then the LysoPLD activities were monitored. Data are mean ± S.E.M. (n = 5, number of mice). d, Inhibition of LPA production by RB014 in BALF of BLM-induced pulmonary fibrosis model mice. RB014 was administered intranasally to C57BL/6J mice at 20 μg/mouse. Data are mean ± S.E.M. (n = 5, number of mice). The variances between the groups were tested by the f-test. The p value was obtained by the Student’s t-test for the 18:2-LPA levels of Day 7.
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Kato, K., Ikeda, H., Miyakawa, S. et al. Structural basis for specific inhibition of Autotaxin by a DNA aptamer. Nat Struct Mol Biol 23, 395–401 (2016). https://doi.org/10.1038/nsmb.3200
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DOI: https://doi.org/10.1038/nsmb.3200
