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Chemoselective small molecules that covalently modify one lysine in a non-enzyme protein in plasma

Abstract

A small molecule that could bind selectively to and then react chemoselectively with a non-enzyme protein in a complex biological fluid, such as blood, could have numerous practical applications. Herein, we report a family of designed stilbenes that selectively and covalently modify the prominent plasma protein transthyretin in preference to more than 4,000 other human plasma proteins. They react chemoselectively with only one of eight lysine ε-amino groups within transthyretin. The crystal structure confirms the expected binding orientation of the stilbene substructure and the anticipated conjugating amide bond. These covalent transthyretin kinetic stabilizers exhibit superior amyloid inhibition potency compared to their noncovalent counterparts, and they prevent cytotoxicity associated with amyloidogenesis. Though there are a few prodrugs that, upon metabolic activation, react with a cysteine residue inactivating a specific non-enzyme, we are unaware of designed small molecules that react with one lysine ε-amine within a specific non-enzyme protein in a complex biological fluid.

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Figure 1: RP-HPLC analysis of the chemoselectivity of compounds 1–4 in recombinant WT TTR versus K15A TTR homotetramer solutions and human plasma.
Figure 2: Comparison of the potency of covalent kinetic stabilizers and their noncovalent counterparts and an assessment of WT TTR tetramer dissociation kinetics in the presence of a covalent kinetic stabilizer.
Figure 3: Inhibition of WT TTR cytotoxicity in human IMR-32 neuroblastoma cells as a function of the dose of covalent and noncovalent TTR kinetic stabilizers.
Figure 4: Crystal structure of the WT TTR–(benzoyl substructure of 4)2 conjugate, showing the amide bond linkage to the Lys15 ε-amino group.

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References

  1. Savi, P. et al. Identification and biological activity of the active metabolite of clopidogrel. Thromb. Haemost. 84, 891–896 (2000).

    Article  CAS  Google Scholar 

  2. Estebanez-Perpina, E. et al. Structural insight into the mode of action of a direct inhibitor of coregulator binding to the thyroid hormone receptor. Mol. Endocrinol. 21, 2919–2928 (2007).

    Article  CAS  Google Scholar 

  3. Guo, F. et al. Breaking the one antibody-one target axiom. Proc. Natl. Acad. Sci. USA 103, 11009–11014 (2006).

    Article  CAS  Google Scholar 

  4. Cohen, E. et al. Opposing activities protect against age-onset proteotoxicity. Science 313, 1604–1610 (2006).

    Article  CAS  Google Scholar 

  5. Hardy, J. & Selkoe, D.J. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297, 353–356 (2002).

    Article  CAS  Google Scholar 

  6. Balch, W.E., Morimoto, R.I., Dillin, A. & Kelly, J.W. Adapting proteostasis for disease intervention. Science 319, 916–919 (2008).

    Article  CAS  Google Scholar 

  7. Johnson, S.M. et al. Native state kinetic stabilization as a strategy to ameliorate protein misfolding diseases: a focus on the transthyretin amyloidoses. Acc. Chem. Res. 38, 911–921 (2005).

    Article  CAS  Google Scholar 

  8. Westermark, P., Sletten, K., Johansson, B. & Cornwell, G.G. Fibril in senile systemic amyloidosis is derived from normal transthyretin. Proc. Natl. Acad. Sci. USA 87, 2843–2845 (1990).

    Article  CAS  Google Scholar 

  9. Coelho, T. Familial amyloid polyneuropathy: new developments in genetics and treatment. Curr. Opin. Neurol. 9, 355–359 (1996).

    Article  CAS  Google Scholar 

  10. Jacobson, D.R. et al. Variant-sequence transthyretin (isoleucine 122) in late-onset cardiac amyloidosis in black Americans. N. Engl. J. Med. 336, 466–473 (1997).

    Article  CAS  Google Scholar 

  11. Sekijima, Y. et al. The biological and chemical basis for tissue selective amyloid disease. Cell 121, 73–85 (2005).

    Article  CAS  Google Scholar 

  12. Holmgren, G. et al. Clinical improvement and amyloid regression after liver transplantation in hereditary transthyretin amyloidosis. Lancet 341, 1113–1116 (1993).

    Article  CAS  Google Scholar 

  13. Klabunde, T. et al. Rational design of potent human transthyretin amyloid disease inhibitors. Nat. Struct. Biol. 7, 312–321 (2000).

    Article  CAS  Google Scholar 

  14. Monaco, H.L., Rizzi, M. & Coda, A. Structure of a complex of two plasma proteins: transthyretin and retinol-binding protein. Science 268, 1039–1041 (1995).

    Article  CAS  Google Scholar 

  15. Wojtczak, A., Luft, J. & Cody, V. Mechanism of molecular recognition. Structural aspects of 3,3′-diiodo-L-thyronine binding to human serum transthyretin. J. Biol. Chem. 267, 353–357 (1992).

    CAS  PubMed  Google Scholar 

  16. Colon, W. & Kelly, J.W. Partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro. Biochemistry 31, 8654–8660 (1992).

    Article  CAS  Google Scholar 

  17. Liu, K. et al. A glimpse of a possible amyloidogenic intermediate of transthyretin. Nat. Struct. Biol. 7, 754–757 (2000).

    Article  CAS  Google Scholar 

  18. Jiang, X. et al. An engineered transthyretin monomer that is nonamyloidogenic, unless it is partially denatured. Biochemistry 40, 11442–11452 (2001).

    Article  CAS  Google Scholar 

  19. Hammarstrom, P., Wiseman, R.L., Powers, E.T. & Kelly, J.W. Prevention of transthyretin amyloid disease by changing protein misfolding energetics. Science 299, 713–716 (2003).

    Article  Google Scholar 

  20. Hammarstrom, P., Schneider, F. & Kelly, J.W. Trans-suppression of misfolding in an amyloid disease. Science 293, 2459–2462 (2001).

    Article  CAS  Google Scholar 

  21. Hurshman, A.R., White, J.T., Powers, E.T. & Kelly, J.W. Transthyretin aggregation under partially denaturing conditions is a downhill polymerization. Biochemistry 43, 7365–7381 (2004).

    Article  CAS  Google Scholar 

  22. Hurshman Babbes, A.R., Powers, E.T. & Kelly, J.W. Quantification of the thermodynamically linked quaternary and tertiary structural stabilities of transthyretin and its disease-associated variants: The relationship between stability and amyloidosis. Biochemistry 47, 6969–6984 (2008).

    Article  CAS  Google Scholar 

  23. Adamski-Werner, S.L., Palaninathan, S.K., Sacchettini, J.C. & Kelly, J.W. Diflunisal analogues stabilize the native state of transthyretin. Potent inhibition of amyloidogenesis. J. Med. Chem. 47, 355–374 (2004).

    Article  CAS  Google Scholar 

  24. Johnson, S.M., Connelly, S., Wilson, I.A. & Kelly, J.W. Toward optimization of the linker substructure common to transthyretin amyloidogenesis inhibitors using biochemical and structural studies. J. Med. Chem. 51, 6348–6358 (2008).

    Article  CAS  Google Scholar 

  25. Johnson, S.M. et al. Bisaryloxime ethers as potent inhibitors of transthyretin amyloid fibril formation. J. Med. Chem. 48, 1576–1587 (2005).

    Article  CAS  Google Scholar 

  26. Miroy, G.J. et al. Inhibiting transthyretin amyloid fibril formation via protein stabilization. Proc. Natl. Acad. Sci. USA 93, 15051–15056 (1996).

    Article  CAS  Google Scholar 

  27. Oza, V.B. et al. Synthesis, structure, and activity of diclofenac analogues as transthyretin amyloid fibril formation inhibitors. J. Med. Chem. 45, 321–332 (2002).

    Article  CAS  Google Scholar 

  28. Razavi, H. et al. Benzoxazoles as transthyretin amyloid fibril inhibitors: synthesis, evaluation, and mechanism of action. Angew. Chem. Int. Ed. Engl. 42, 2758–2761 (2003).

    Article  CAS  Google Scholar 

  29. Wiseman, R.L. et al. Kinetic stabilization of an oligomeric protein by a single ligand binding event. J. Am. Chem. Soc. 127, 5540–5551 (2005).

    Article  CAS  Google Scholar 

  30. Foss, T.R. et al. Kinetic stabilization of the native state by protein engineering: implications for inhibition of transthyretin amyloidogenesis. J. Mol. Biol. 347, 841–854 (2005).

    Article  CAS  Google Scholar 

  31. Foss, T.R., Wiseman, R.L. & Kelly, J.W. The pathway by which the tetrameric protein transthyretin dissociates. Biochemistry 44, 15525–15533 (2005).

    Article  CAS  Google Scholar 

  32. Johnson, S.M., Connelly, S., Wilson, I.A. & Kelly, J.W. Biochemical and structural evaluation of highly selective 2-arylbenzoxazole-based transthyretin amyloidogenesis inhibitors. J. Med. Chem. 51, 260–270 (2008).

    Article  CAS  Google Scholar 

  33. Petrassi, H.M., Klabunde, T., Sacchettini, J.C. & Kelly, J.W. Structure-based design of N-phenyl phenoxazine transthyretin amyloid fibril inhibitors. J. Am. Chem. Soc. 122, 2178–2192 (2000).

    Article  CAS  Google Scholar 

  34. Purkey, H.E. et al. Hydroxylated polychlorinated biphenyls selectively bind transthyretin in blood and inhibit amyloidogenesis: rationalizing rodent PCB toxicity. Chem. Biol. 11, 1719–1728 (2004).

    Article  CAS  Google Scholar 

  35. Baures, P.W., Oza, V.B., Peterson, S.A. & Kelly, J.W. Synthesis and evaluation of inhibitors of transthyretin amyloid formation based on the non-steroidal anti-inflammatory drug, flufenamic acid. Bioorg. Med. Chem. 7, 1339–1347 (1999).

    Article  CAS  Google Scholar 

  36. Green, N.S., Palaninathan, S.K., Sacchettini, J.C. & Kelly, J.W. Synthesis and characterization of potent bivalent amyloidosis inhibitors that bind prior to transthyretin tetramerization. J. Am. Chem. Soc. 125, 13404–13414 (2003).

    Article  CAS  Google Scholar 

  37. Miller, S.R., Sekijima, Y. & Kelly, J.W. Native state stabilization by NSAIDs inhibits transthyretin amyloidogenesis from the most common familial disease variants. Lab. Invest. 84, 545–552 (2004).

    Article  CAS  Google Scholar 

  38. Burgi, H.B., Dunitz, J.D., Lehn, J.M. & Wipff, G. Stereochemistry of reaction paths at carbonyl centers. Tetrahedron 30, 1563–1572 (1974).

    Article  Google Scholar 

  39. Hammarstrom, P., Jiang, X., Deechongkit, S. & Kelly, J.W. Anion shielding of electrostatic repulsions in transthyretin modulates stability and amyloidosis: insight into the chaotrope unfolding dichotomy. Biochemistry 40, 11453–11459 (2001).

    Article  CAS  Google Scholar 

  40. Purkey, H.E., Dorrell, M.I. & Kelly, J.W. Evaluating the binding selectivity of transthyretin amyloid fibril inhibitors in blood plasma. Proc. Natl. Acad. Sci. USA 98, 5566–5571 (2001).

    Article  CAS  Google Scholar 

  41. Lashuel, H.A., Wurth, C., Woo, L. & Kelly, J.W. The most pathogenic transthyretin variant, L55P, forms amyloid fibrils under acidic conditions and protofilaments under physiological conditions. Biochemistry 38, 13560–13573 (1999).

    Article  CAS  Google Scholar 

  42. Baures, P.W., Peterson, S.A. & Kelly, J.W. Discovering transthyretin amyloid fibril inhibitors by limited screening. Bioorg. Med. Chem. 6, 1389–1401 (1998).

    Article  CAS  Google Scholar 

  43. Zhang, Q. & Kelly, J.W. Cys10 mixed disulfides make transthyretin more amyloidogenic under mildly acidic conditions. Biochemistry 42, 8756–8761 (2003).

    Article  CAS  Google Scholar 

  44. Reixach, N. et al. Tissue damage in the amyloidoses: transthyretin monomers and nonnative oligomers are the major cytotoxic species in tissue culture. Proc. Natl. Acad. Sci. USA 101, 2817–2822 (2004).

    Article  CAS  Google Scholar 

  45. Reixach, N. et al. Cell based screening of inhibitors of transthyretin aggregation. Biochem. Biophys. Res. Commun. 348, 889–897 (2006).

    Article  CAS  Google Scholar 

  46. O'Brien, J., Wilson, I., Orton, T. & Pognan, F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 267, 5421–5426 (2000).

    Article  CAS  Google Scholar 

  47. Wojtczak, A., Cody, V., Luft, J.R. & Pangborn, W. Structure of rat transthyretin (rTTR) complex with thyroxine at 2.5 angstrom resolution: first non-biased insight into thyroxine binding reveals different hormone orientation in two binding sites. Acta Crystallogr. D Biol. Crystallogr. 57, 1061–1070 (2001).

    Article  CAS  Google Scholar 

  48. Marinec, P.S. et al. FK506-binding protein (FKBP) partitions a modified HIV protease inhibitor into blood cells and prolongs its lifetime in vivo. Proc. Natl. Acad. Sci. USA 106, 1336–1341 (2009).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  50. Storoni, L.C., McCoy, A.J. & Read, R.J. Likelihood-enhanced fast rotation functions. Acta Crystallogr. D Biol. Crystallogr. 60, 432–438 (2004).

    Article  Google Scholar 

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Acknowledgements

We acknowledge US National Institutes of Health grants DK46335 to J.W.K. and CA58896 and AI42266 to I.A.W. We also thank the Skaggs Institute for Chemical Biology and the Lita Annenberg Hazen Foundation for financial support. Technical support from M. Saure and G. Dendle is greatly appreciated. We are grateful to C. Fearns for carefully reading and editing the manuscript. We thank the General Clinical Research Center of the Scripps Research Institute for providing human blood. The authors also acknowledge R. Stanfield, X. Dai, S. Yoon, R. Xu and D. Ekiert for assisting with X-ray data collection. X-ray diffraction data were collected at GM/CA-CAT 23-IDB beamline at the Advanced Photon Source, Argonne National Laboratory. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Basic Energy Sciences, under contract number W-31-109-Eng-38.

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Contributions

S. Choi performed the chemical syntheses and all biochemical assays and wrote the majority of the paper. S. Connelly performed the crystallization, crystallographic structure determination and the structural analyses and wrote the crystallography section of the paper. N.R. carried out all the cell-based assays and wrote that section of the manuscript. I.A.W. supervised the crystallographic work and edited the manuscript. J.W.K. supervised the chemical biology and edited the paper.

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Correspondence to Jeffery W Kelly.

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Competing interests

J.W.K. is a founder, shareholder and paid consultant for Foldrx Pharmaceuticals, Inc., a biotechnology company that specializes in the discovery and development of drug therapies for transthyretin amyloidoses.

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Supplementary Methods, Supplementary Scheme 1, Supplementary Figures 1–14 and Supplementary Table 1 (PDF 629 kb)

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Choi, S., Connelly, S., Reixach, N. et al. Chemoselective small molecules that covalently modify one lysine in a non-enzyme protein in plasma. Nat Chem Biol 6, 133–139 (2010). https://doi.org/10.1038/nchembio.281

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