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Discovery of a cryptic peptide-binding site on PCSK9 and design of antagonists

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma LDL cholesterol (LDL-c) levels by promoting the degradation of liver LDL receptors (LDLRs). Antibodies that inhibit PCSK9 binding to the EGF(A) domain of the LDLR are effective in lowering LDL-c. However, the discovery of small-molecule therapeutics is hampered by difficulty in targeting the relatively flat EGF(A)-binding site on PCSK9. Here we demonstrate that it is possible to target this site, based on the finding that the PCSK9 P′ helix displays conformational flexibility. As a consequence, the vacated N-terminal groove of PCSK9, which is adjacent to the EGF(A)-binding site, is in fact accessible to small peptides. In phage-display experiments, the EGF(A)-mimicking peptide Pep2-8 was used as an anchor peptide for the attachment of an extension peptide library directed toward the groove site. Guided by structural information, we further engineered the identified groove-binding peptides into antagonists, which encroach on the EGF(A)-binding site and inhibit LDLR binding.

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Figure 1: The Fab33 epitope on PCSK9 overlaps with the LDLR-binding site and shows that the P′ helix is absent.
Figure 2: The conformational flexibility of the P′ helix gives rise to a new strategy to antagonize PCSK9 binding to LDLR.
Figure 3: Experimental design to generate peptides that bind to the N-terminal groove and PCSK9 antagonism of the Fusion1 peptide.
Figure 4: Extension peptides bind to the N-terminal groove but do not inhibit PCSK9 binding to LDLR.
Figure 5: Extension peptides bind to the N-terminal groove and closely mimic the native P′ helix.
Figure 6: Hydrogen bonds of arginine and homo-arginine.
Figure 7: The phage-display-derived peptide MESFPGWNLV(hR)IGLLR is a PCSK9 antagonist, as explained by the structure of the peptide bound to PCSK9ΔP′.

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References

  1. Abifadel, M. et al. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat. Genet. 34, 154–156 (2003).

    Article  CAS  Google Scholar 

  2. Cohen, J.C., Boerwinkle, E., Mosley, T.H. Jr. & Hobbs, H.H. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N. Engl. J. Med. 354, 1264–1272 (2006).

    Article  CAS  Google Scholar 

  3. Horton, J.D., Cohen, J.C. & Hobbs, H.H. PCSK9: a convertase that coordinates LDL catabolism. J. Lipid Res. 50 (Suppl.), S172–S177 (2009).

    Article  Google Scholar 

  4. Seidah, N.G. New developments in proprotein convertase subtilisin-kexin 9's biology and clinical implications. Curr. Opin. Lipidol. 27, 274–281 (2016).

    Article  CAS  Google Scholar 

  5. Chapman, M.J., Stock, J.K., Ginsberg, H.N. & PCSK9 Forum. PCSK9 inhibitors and cardiovascular disease: heralding a new therapeutic era. Curr. Opin. Lipidol. 26, 511–520 (2015).

    Article  CAS  Google Scholar 

  6. Paton, D.M. PCSK9 inhibitors: monoclonal antibodies for the treatment of hypercholesterolemia. Drugs Today (Barc.) 52, 183–192 (2016).

    Article  CAS  Google Scholar 

  7. Stein, E.A. & Swergold, G.D. Potential of proprotein convertase subtilisin/kexin type 9 based therapeutics. Curr. Atheroscler. Rep. 15, 310 (2013).

    Article  Google Scholar 

  8. Ridker, P.M. et al. Cardiovascular efficacy and safety of bococizumab in high-risk patients. N. Engl. J. Med. 376, 1527–1539 (2017).

    Article  CAS  Google Scholar 

  9. Sabatine, M.S. et al. Evolocumab and clinical outcomes in patients with cardiovascular disease. N. Engl. J. Med. 376, 1713–1722 (2017).

    Article  CAS  Google Scholar 

  10. Kwon, H.J., Lagace, T.A., McNutt, M.C., Horton, J.D. & Deisenhofer, J. Molecular basis for LDL receptor recognition by PCSK9. Proc. Natl. Acad. Sci. USA 105, 1820–1825 (2008).

    Article  CAS  Google Scholar 

  11. Zhang, D.W. et al. Binding of proprotein convertase subtilisin/kexin type 9 to epidermal growth factor-like repeat A of low density lipoprotein receptor decreases receptor recycling and increases degradation. J. Biol. Chem. 282, 18602–18612 (2007).

    Article  CAS  Google Scholar 

  12. Schroeder, C.I. et al. Design and synthesis of truncated EGF-A peptides that restore LDL-R recycling in the presence of PCSK9 in vitro. Chem. Biol. 21, 284–294 (2014).

    Article  CAS  Google Scholar 

  13. Stucchi, M. et al. Disrupting the PCSK9/LDLR protein-protein interaction by an imidazole-based minimalist peptidomimetic. Org. Biomol. Chem. 14, 9736–9740 (2016).

    Article  CAS  Google Scholar 

  14. Zhang, Y. et al. Identification of a small peptide that inhibits PCSK9 protein binding to the low density lipoprotein receptor. J. Biol. Chem. 289, 942–955 (2014).

    Article  CAS  Google Scholar 

  15. Zhang, Y. et al. Calcium-independent inhibition of PCSK9 by affinity-improved variants of the LDL receptor EGF(A) domain. J. Mol. Biol. 422, 685–696 (2012).

    Article  CAS  Google Scholar 

  16. Fitzgerald, K. et al. A highly durable RNAi therapeutic inhibitor of PCSK9. N. Engl. J. Med. 376, 41–51 (2017).

    Article  CAS  Google Scholar 

  17. Ray, K.K. et al. Inclisiran in patients at high cardiovascular risk with elevated LDL cholesterol. N. Engl. J. Med. 376, 1430–1440 (2017).

    Article  CAS  Google Scholar 

  18. van Poelgeest, E.P. et al. Antisense-mediated reduction of proprotein convertase subtilisin/kexin type 9 (PCSK9): a first-in-human randomized, placebo-controlled trial. Br. J. Clin. Pharmacol. 80, 1350–1361 (2015).

    Article  CAS  Google Scholar 

  19. Galabova, G. et al. Peptide-based anti-PCSK9 vaccines - an approach for long-term LDLc management. PLoS One 9, e114469 (2014).

    Article  Google Scholar 

  20. Lintner, N.G. et al. Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain. PLoS Biol. 15, e2001882 (2017).

    Article  Google Scholar 

  21. Petersen, D.N. et al. A small-molecule anti-secretagogue of PCSK9 targets the 80S ribosome to inhibit PCSK9 protein translation. Cell Chem. Biol. 23, 1362–1371 (2016).

    Article  CAS  Google Scholar 

  22. Ding, Q. et al. Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing. Circ. Res. 115, 488–492 (2014).

    Article  CAS  Google Scholar 

  23. Seidah, N.G. & Prat, A. The biology and therapeutic targeting of the proprotein convertases. Nat. Rev. Drug Discov. 11, 367–383 (2012).

    Article  CAS  Google Scholar 

  24. Li, J. et al. Secreted PCSK9 promotes LDL receptor degradation independently of proteolytic activity. Biochem. J. 406, 203–207 (2007).

    Article  CAS  Google Scholar 

  25. McNutt, M.C., Lagace, T.A. & Horton, J.D. Catalytic activity is not required for secreted PCSK9 to reduce low density lipoprotein receptors in HepG2 cells. J. Biol. Chem. 282, 20799–20803 (2007).

    Article  CAS  Google Scholar 

  26. Bottomley, M.J. et al. Structural and biochemical characterization of the wild type PCSK9-EGF(AB) complex and natural familial hypercholesterolemia mutants. J. Biol. Chem. 284, 1313–1323 (2009).

    Article  CAS  Google Scholar 

  27. Garvie, C.W. et al. Point mutations at the catalytic site of PCSK9 inhibit folding, autoprocessing, and interaction with the LDL receptor. Protein Sci. 25, 2018–2027 (2016).

    Article  CAS  Google Scholar 

  28. Schechter, I. & Berger, A. On the size of the active site in proteases. I. Papain. Biochem. Biophys. Res. Commun. 27, 157–162 (1967).

    Article  CAS  Google Scholar 

  29. Baruch, A. et al. A phase 1 study to evaluate the safety and LDL cholesterol-lowering effects of RG7652, a fully human monoclonal antibody against proprotein convertase subtilisin/kexin type 9. Clin. Cardiol. 40, 503–511 (2017).

    Article  Google Scholar 

  30. Betzel, C. et al. Structure of a serine protease proteinase K from Tritirachium album limber at 0.98 A resolution. Biochemistry 40, 3080–3088 (2001).

    Article  CAS  Google Scholar 

  31. Liang, L. et al. The crystal structures of two cuticle-degrading proteases from nematophagous fungi and their contribution to infection against nematodes. FASEB J. 24, 1391–1400 (2010).

    Article  CAS  Google Scholar 

  32. Cunningham, D. et al. Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia. Nat. Struct. Mol. Biol. 14, 413–419 (2007).

    Article  CAS  Google Scholar 

  33. Henrich, S. et al. The crystal structure of the proprotein processing proteinase furin explains its stringent specificity. Nat. Struct. Biol. 10, 520–526 (2003).

    Article  CAS  Google Scholar 

  34. Franklin, M.C. et al. Structure and function analysis of peptide antagonists of melanoma inhibitor of apoptosis (ML-IAP). Biochemistry 42, 8223–8231 (2003).

    Article  CAS  Google Scholar 

  35. Zobel, K. et al. Design, synthesis, and biological activity of a potent Smac mimetic that sensitizes cancer cells to apoptosis by antagonizing IAPs. ACS Chem. Biol. 1, 525–533 (2006).

    Article  CAS  Google Scholar 

  36. Andronati, S.A., Karaseva, T.L. & Krysko, A.A. Peptidomimetics - antagonists of the fibrinogen receptors: molecular design, structures, properties and therapeutic applications. Curr. Med. Chem. 11, 1183–1211 (2004).

    Article  CAS  Google Scholar 

  37. Ashkenazi, A., Fairbrother, W.J., Leverson, J.D. & Souers, A.J. From basic apoptosis discoveries to advanced selective BCL-2 family inhibitors. Nat. Rev. Drug Discov. 16, 273–284 (2017).

    Article  CAS  Google Scholar 

  38. Souers, A.J. et al. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat. Med. 19, 202–208 (2013).

    Article  CAS  Google Scholar 

  39. Lee, C.V., Sidhu, S.S. & Fuh, G. Bivalent antibody phage display mimics natural immunoglobulin. J. Immunol. Methods 284, 119–132 (2004).

    Article  CAS  Google Scholar 

  40. Liang, W.C. et al. Function blocking antibodies to neuropilin-1 generated from a designed human synthetic antibody phage library. J. Mol. Biol. 366, 815–829 (2007).

    Article  CAS  Google Scholar 

  41. Lee, C.V. et al. High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold. J. Mol. Biol. 340, 1073–1093 (2004).

    Article  CAS  Google Scholar 

  42. Gallop, M.A., Barrett, R.W., Dower, W.J., Fodor, S.P. & Gordon, E.M. Applications of combinatorial technologies to drug discovery. 1. Background and peptide combinatorial libraries. J. Med. Chem. 37, 1233–1251 (1994).

    Article  CAS  Google Scholar 

  43. Kunkel, T.A., Roberts, J.D. & Zakour, R.A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154, 367–382 (1987).

    Article  CAS  Google Scholar 

  44. Tonikian, R., Zhang, Y., Boone, C. & Sidhu, S.S. Identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries. Nat. Protoc. 2, 1368–1386 (2007).

    Article  CAS  Google Scholar 

  45. Lipari, M.T. et al. Furin-cleaved proprotein convertase subtilisin/kexin type 9 (PCSK9) is active and modulates low density lipoprotein receptor and serum cholesterol levels. J. Biol. Chem. 287, 43482–43491 (2012).

    Article  CAS  Google Scholar 

  46. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  47. Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    Article  CAS  Google Scholar 

  48. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  49. Hampton, E.N. et al. The self-inhibited structure of full-length PCSK9 at 1.9 A reveals structural homology with resistin within the C-terminal domain. Proc. Natl. Acad. Sci. USA 104, 14604–14609 (2007).

    Article  CAS  Google Scholar 

  50. Murshudov, G.N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

    Article  CAS  Google Scholar 

  51. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  52. Bricogne, G. et al. BUSTER version 2.11.2 (Global Phasing Ltd., Cambridge, 2011).

  53. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  54. Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D Biol. Crystallogr. 67, 293–302 (2011).

    Article  CAS  Google Scholar 

  55. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  56. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    Article  CAS  Google Scholar 

  57. Johnson, B.A. & Blevins, R.A. NMR view: a computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614 (1994).

    Article  CAS  Google Scholar 

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Acknowledgements

We would like to thank M. Dillon and S.H. Kim for sequencing Ab7G7, P. Luan for preparing the Fab7G7, P. Liu for N-terminal sequencing, T. Lipari for identifying F.XIa and B. Lazarus for his ingenious suggestions regarding phage-display strategies. Synchrotrons at the ALS and the APS are supported by the Director, Office of Science, Office of Basic Energy Sciences (BES) of the US Department of Energy (DOE) under contracts DE-AC02-05CH11231 and DE-AC02-06CH11357, respectively. The Berkeley Center for Structural Biology is supported in part by the NIH, NIGMS, and HHMI. Supporting institutions of the Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at APS may be found at http://www.ser-cat.org/members.html. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the NIH, NIGMS (including grant no. P41GM103393).

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Authors and Affiliations

Authors

Contributions

Y.Z. identified peptide ligands by phage display, performed phage ELISA and wrote the manuscript. M.U. and S.S. crystallized proteins and solved structures. C.E. coordinated crystallography and solved structures and wrote the manuscript. N.J.S. designed peptides for SAR studies and the antagonist phage library and wrote the manuscript. D.J.B. designed and performed peptide SAR studies and synthesized peptides. M.H.B. developed and conducted TR-FRET assays. A.P. developed experimental strategies for antibody generation and groove validation studies and wrote the manuscript. W.L. developed and conducted PCSK9 P′-helix cleavage assays, protein ELISA and affinity measurements of PCSK9 variants. M.K.-B. performed HepG2 assays, J.Q. conducted SPR studies measuring peptide binding to PCSK9 variants, C.C. and Y.W. generated Ab33 and Ab20 and determined binding affinities by SPR, and P.M. identified inhibitory antibodies and designed PCSK9 open-groove variants and purified protein reagents. P.D.L. conducted NMR studies, and D.K. designed and coordinated the overall study and wrote the manuscript. All authors analyzed and interpreted experimental data.

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Correspondence to Daniel Kirchhofer.

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All authors are current or past employees of Genentech, Inc.

Integrated supplementary information

Supplementary Figure 1 Electron density 2mFo-DFc maps contoured at 1 times rmsd shown for a subset of atoms.

(a) Part of CDR-H2 of Fab33. (b) Fusion2 peptide. (c) Pep1. (d) Pep3. (e) WNLV(hR)IGLLR. (f) MESFPGWNLV(hR)IGLLR.

Supplementary Figure 2 Surface plasmon resonance measurements of PCSK9 variants binding to immobilized LDLR-Fc.

Shown are sensorgrams (different color for each concentration) and fitted curves (black) of (a) PCSK9 (KD 103 ± 3.4 nM), (b) PCSK9-AAA (KD 138 ± 1.3 nM) and (c) PCSK9ΔP' (KD 541 ± 8.2 nM). The KD values, calculated from the determined kon and koff values, were the average ± s.d. of three independent experiments.

Supplementary Figure 3 Binding of phage displaying Pep2-8 (TVFTSWEEYLDWVGSG) or Pep2-8V2A (TAFTSWEEYLDWVGSG) to PCSK9.

The single mutation of valine2 to an alanine reduced binding affinity to PCSK9. The reduced but still detectable binding of Pep2-8V2A to PCSK9 was suitable for its use as an anchor peptide to C-terminally attach an extension peptide library via the GSG linker.

Source data

Supplementary Figure 4 Groove-binding peptides approach Pep2-8 and EGF(A) in a similar fashion as the native P'-helix.

(a) The Pro4 residue of Pep1 is in the same location as Pro155 of the native P'-helix and contacts the side chains of residues Trp6 and Tyr9 of Pep2-8. Analogous contacts are observed for Pep3 (not shown). (b) The structurally analogous PCSK9 P'-helix Pro155 contacts EGF(A) Leu298 in the PCSK9/EGF(A) complex (PDB 2W2M). (c) Cyclic peptides Pep1 (green-yellow) and Pep3 (brown) are closely similar in sequence and structure when bound to PCSK9 and display a residue (Arg2, Phe2) close to the volume occupied by Ser153 in the native P’-helix (Ser153-Ile154-Pro155 in white).

Supplementary Figure 5 Sequential and medium-range NOEs observed for two groove-binding peptides.

(a) MESFPGWNLV(hR)IGLLR peptide in H2O/CD3CN (70:30), at 284K. (b) WNLV(hR)IGLLR peptide in H2O/CD3CN (70:30), at 284K. The thickness of the bars is proportional to the intensities of the NOE signals. The detection of weak, medium-range NOEs indicate a helical propensity for residues W7-L14 of the MESFPGWNLV(hR)IGLLR peptide and for residues W1-L8 of the WNLV(hR)IGLLR peptide.

Supplementary Figure 6 Structure of MESFPGWNLV(hR)IGLLR bound to PCSK9ΔP’ explains antagonism.

Hydrophobic contacts involving side chains from new residues M1 and F4 and residues W7, V10 and hR11 help determine the conformation of the N-terminal extension. Carbon (and sulfur) atoms from these side chains are shown with dotted surfaces.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1–11. (PDF 1635 kb)

Life Sciences Reporting Summary (PDF 172 kb)

Supplementary Data Set 1

Uncropped blot for Figure 2a–d and Figure 4b. (PDF 3594 kb)

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Zhang, Y., Ultsch, M., Skelton, N. et al. Discovery of a cryptic peptide-binding site on PCSK9 and design of antagonists. Nat Struct Mol Biol 24, 848–856 (2017). https://doi.org/10.1038/nsmb.3453

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