Oral Presentation The 16th Australian Peptide Conference 2025

Peptidyl epoxyketone proteasome inhibitor biosynthesis: Substrate scope and catalytic mechanism of epoxyketone synthase EpnF (129445)

Gregory L Challis 1 2 3
  1. Department of Chemistry, University of Warwick, Coventry, UK
  2. Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia
  3. ARC Centre of Excellence for Innovations in Peptide and Protein Science, Monash University, Clayton, VIC, Australia

Peptidyl epoxyketones are potent proteasome inhibitors produced by Actinomycetes and other classes of bacteria. They consist of a variable peptidyl cap, assembled by a nonribosomal peptide synthetase, fused to a conserved epoxyketone pharmacophore introduced by the successive action of a polyketide synthase and a multifunctional flavoenzyme. This family of natural products has inspired the development of several synthetic peptidyl epoxyketones, including the constitutive proteasome inhibitor carfilzomib, which is approved for the treatment of multiple myeloma,1 and immunoproteasome inhibitors with promise for the treatment of inflammatory diseases such as lupus nephritis and Alzheimer's.2,3

We have previously identified the biosynthetic gene clusters for TMC-86A and tryptopeptin,4,5 two peptidyl epoxyketones produced by Streptomyces species, and investigated several key aspects of TMC-86A/eponemycin and tryptopeptin biosynthesis using a combination of molecular genetics, enzymology, and organic synthesis.4,5,6 A key challenge in investigating the multifunctional flavoenzymes responsible for installation of the epoxyketone pharmacophore is that their beta-ketoacid substrates are intrinsically unstable.4 To overcome this problem, we have developed an approach for in situ substrate generation using the corresponding beta-keto methyl esters and commercially available pig liver esterase. Using this system, we have examined the substrate scope of the epoxyketone synthase EpnF with a wide range of synthetic analogues. These studies reveal that EpnF is remarkably substrate tolerant, underscoring its potential to be harnessed for chemoenzymatic synthesis of diverse epoxyketones. A combination of AlphaFold models, molecular dynamics simulations, and site directed mutagenesis has also been used to probe the mechanism of EpnF, which employs a catalytic flavin cofactor and molecular oxygen as the sole stoichiometric co-substrate to effect successive decarboxylative-desaturation and enantiospecific enone epoxidation reactions.   

  1. K.B. Kim and C.M. Crews. From epoxomicin to carfilzomib: chemistry, biology, and medical outcomes. Nat. Prod. Rep., 2013, 30, 600-604.
  2. T. Muchamuel, R.A. Fan, J.L. Anderl, D.J. Bomba, H.W.B. Johnson, E. Lowe, B.B. Tuch, D.L McMinn, B.Millare, and C.J Kirk. Zetomipzomib (KZR-616) attenuates lupus in mice via modulation of innate and adaptive immune responses. Front. Immunol. 2023, 14, 1043680.
  3. J.E. Park, C.L. Chaudhary, D. Bhattarai, K.B. Kim. Brain-Permeable Immunoproteasome-Targeting Macrocyclic Peptide Epoxyketones for Alzheimer’s Disease. J. Med. Chem. 2024, 67, 7146–7157.
  4. D. Zabala, J.W. Cartwright, D.M. Roberts, B.J.C. Law, L. Song, M. Samborskyy, P.F. Leadlay, J. Micklefield, and G.L. Challis. A flavin-dependent decarboxylase-dehydrogenase-monooxygenase assembles the warhead of α, β-epoxyketone proteasome inhibitors. J. Am. Chem. Soc. 2016, 138, 4342-4345
  5. C. Huang, D. Zabala, E.L.C. de los Santos, L. Song, C. Corre, L.M. Alkhalaf and G.L. Challis. Parallelized gene cluster editing illuminates mechanisms of epoxyketone proteasome inhibitor biosynthesis. Nucleic Acids Res. 2023, 51, 1488–1499.
  6. B.R. Terlouw, C. Huang, D. Meijer, J.D.D. Cediel-Becerra, M.L. Rothe, M. Jenner, S. Zhou, Y. Zhang, C.D. Fage, Y. Tsunematsu, G.P. van Wezel, S.L. Robinson, F. Alberti, L.M. Alkhalaf, M.G. Chevrette, G.L. Challis and M.H. Medema. PARAS: high-accuracy machine-learning of substrate specificities in nonribosomal peptide synthetases. BioRxiv, 2025, DOI: 10.1101/2025.01.08.631717.