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Systematic Analyses of Substrate Preferences of 20S Proteasomes Using Peptidic Epoxyketone Inhibitors.

Huber EM, de Bruin G, Heinemeyer W, Paniagua Soriano G, Overkleeft HS, Groll M
J Am Chem Soc 137 7835-42 (2015)

Cited: 22 times
EuropePMC logo PMID: 26020686

Abstract

Cleavage analyses of 20S proteasomes with natural or synthetic substrates allowed to infer the substrate specificities of the active sites and paved the way for the rational design of high-affinity proteasome inhibitors. However, details of cleavage preferences remained enigmatic due to the lack of appropriate structural data. In a unique approach, we here systematically examined substrate specificities of yeast and human proteasomes using irreversibly acting α',β'epoxyketone (ep) inhibitors. Biochemical and structural analyses provide unique insights into the substrate preferences of the distinct active sites and highlight differences between proteasome types that may be considered in future inhibitor design efforts. (1) For steric reasons, epoxyketones with Val or Ile at the P1 position are weak inhibitors of all active sites. (2) Identification of the β2c selective compound Ac-LAE-ep represents a promising starting point for the development of compounds that discriminate between β2c and β2i. (3) The compound Ac-LAA-ep was found to favor subunit β5c over β5i by three orders of magnitude. (4) Yeast β1 and human β1c subunits preferentially bind Asp and Leu in their S1 pockets, while Glu and large hydrophobic residues are not accepted. (5) Exceptional structural features in the β1/2 substrate binding channel give rise to the β1 selectivity of compounds featuring Pro at the P3 site. Altogether, 23 different epoxyketone inhibitors, five proteasome mutants, and 43 crystal structures served to delineate a detailed picture of the substrate and ligand specificities of proteasomes and will further guide drug development efforts toward subunit-specific proteasome inhibitors for applications as diverse as cancer and autoimmune disorders.

Reviews citing this publication (3)

  1. Site-Specific Proteasome Inhibitors. Kisselev AF. Biomolecules 12 54 (2021)
  2. A Nut for Every Bolt: Subunit-Selective Inhibitors of the Immunoproteasome and Their Therapeutic Potential. Huber EM, Groll M. Cells 10 1929 (2021)
  3. Covalent-reversible peptide-based protease inhibitors. Design, synthesis, and clinical success stories. Feral A, Martin AR, Desfoux A, Amblard M, Vezenkov LL. Amino Acids 55 1775-1800 (2023)

Articles citing this publication (19)

  1. A Set of Activity-Based Probes to Visualize Human (Immuno)proteasome Activities. de Bruin G, Xin BT, Kraus M, van der Stelt M, van der Marel GA, Kisselev AF, Driessen C, Florea BI, Overkleeft HS. Angew Chem Int Ed Engl 55 4199-4203 (2016)
  2. A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome. Huber EM, Heinemeyer W, Li X, Arendt CS, Hochstrasser M, Groll M. Nat Commun 7 10900 (2016)
  3. Brief treatment with a highly selective immunoproteasome inhibitor promotes long-term cardiac allograft acceptance in mice. Sula Karreci E, Fan H, Uehara M, Mihali AB, Singh PK, Kurdi AT, Solhjou Z, Riella LV, Ghobrial I, Laragione T, Routray S, Assaker JP, Wang R, Sukenick G, Shi L, Barrat FJ, Nathan CF, Lin G, Azzi J. Proc Natl Acad Sci U S A 113 E8425-E8432 (2016)
  4. A humanized yeast proteasome identifies unique binding modes of inhibitors for the immunosubunit β5i. Huber EM, Heinemeyer W, de Bruin G, Overkleeft HS, Groll M. EMBO J 35 2602-2613 (2016)
  5. 20S Proteasome as a Drug Target in Trichomonas vaginalis. O'Donoghue AJ, Bibo-Verdugo B, Miyamoto Y, Wang SC, Yang JZ, Zuill DE, Matsuka S, Jiang Z, Almaliti J, Caffrey CR, Gerwick WH, Eckmann L. Antimicrob Agents Chemother 63 e00448-19 (2019)
  6. Discovery of dehydroabietic acid sulfonamide based derivatives as selective matrix metalloproteinases inactivators that inhibit cell migration and proliferation. Huang RZ, Liang GB, Huang XC, Zhang B, Zhou MM, Liao ZX, Wang HS. Eur J Med Chem 138 979-992 (2017)
  7. Roseltide rT7 is a disulfide-rich, anionic, and cell-penetrating peptide that inhibits proteasomal degradation. Kam A, Loo S, Fan JS, Sze SK, Yang D, Tam JP. J Biol Chem 294 19604-19615 (2019)
  8. Structural Basis for the Species-Selective Binding of N,C-Capped Dipeptides to the Mycobacterium tuberculosis Proteasome. Hsu HC, Singh PK, Fan H, Wang R, Sukenick G, Nathan C, Lin G, Li H. Biochemistry 56 324-333 (2017)
  9. Competitive Metabolite Profiling of Natural Products Reveals Subunit Specific Inhibitors of the 20S Proteasome. Pawar A, Basler M, Goebel H, Alvarez Salinas GO, Groettrup M, Böttcher T. ACS Cent Sci 6 241-246 (2020)
  10. Structure-Based Design of Inhibitors Selective for Human Proteasome β2c or β2i Subunits. Xin BT, Huber EM, de Bruin G, Heinemeyer W, Maurits E, Espinal C, Du Y, Janssens M, Weyburne ES, Kisselev AF, Florea BI, Driessen C, van der Marel GA, Groll M, Overkleeft HS. J Med Chem 62 1626-1642 (2019)
  11. The Architecture of the Anbu Complex Reflects an Evolutionary Intermediate at the Origin of the Proteasome System. Fuchs ACD, Alva V, Maldoner L, Albrecht R, Hartmann MD, Martin J. Structure 25 834-845.e5 (2017)
  12. Total synthesis and absolute stereochemistry of the proteasome inhibitors cystargolides A and B. Tello-Aburto R, Hallada LP, Niroula D, Rogelj S. Org Biomol Chem 13 10127-10130 (2015)
  13. Noncytotoxic Inhibition of the Immunoproteasome Regulates Human Immune Cells In Vitro and Suppresses Cutaneous Inflammation in the Mouse. Ah Kioon MD, Pierides M, Pannellini T, Lin G, Nathan CF, Barrat FJ. J Immunol 206 1631-1641 (2021)
  14. Yeast PI31 inhibits the proteasome by a direct multisite mechanism. Rawson S, Walsh RM, Velez B, Schnell HM, Jiao F, Blickling M, Ang J, Bhanu MK, Huang L, Hanna J. Nat Struct Mol Biol 29 791-800 (2022)
  15. Cell-Based Optimization of Covalent Reversible Ketoamide Inhibitors Bridging the Unprimed to the Primed Site of the Proteasome β5 Subunit. Stubba D, Bensinger D, Steinbacher J, Proskurjakov L, Salcedo Gómez Á, Schmidt U, Roth S, Schmitz K, Schmidt B. ChemMedChem 14 2005-2022 (2019)
  16. Design, synthesis, and evaluation of cystargolide-based β-lactones as potent proteasome inhibitors. Niroula D, Hallada LP, Le Chapelain C, Ganegamage SK, Dotson D, Rogelj S, Groll M, Tello-Aburto R. Eur J Med Chem 157 962-977 (2018)
  17. Synthesis and Biochemical Evaluation of Warhead-Decorated Psoralens as (Immuno)Proteasome Inhibitors. Schiffrer ES, Proj M, Gobec M, Rejc L, Šterman A, Mravljak J, Gobec S, Sosič I. Molecules 26 E356 (2021)
  18. Brain-Derived 11S Regulator (PA28αβ) Promotes Proteasomal Hydrolysis of Elongated Oligoglutamine-Containing Peptides. Kriachkov VA, Gotmanova NN, Tashlitsky VN, Bacheva AV. Int J Mol Sci 24 13275 (2023)
  19. Rational design of proteasome inhibitors based on the structure of the endogenous inhibitor PI31/Fub1. Velez B, Razi A, Hubbard RD, Walsh R, Rawson S, Tian G, Finley D, Hanna J. Proc Natl Acad Sci U S A 120 e2308417120 (2023)


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