Biosynthesis and bioengineering of peptidyl epoxyketone proteasome inhibitors — ASN Events
  1. Schedule
  2. SESSION 9: CIPPS
  3. Biosynthesis and bioengineering of peptidyl epoxyketone proteasome inhibitors

Biosynthesis and bioengineering of peptidyl epoxyketone proteasome inhibitors (#48)

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 class of natural products assembled by diverse bacterial taxa that selectively target the catalytically essential N-terminal threonine residue in the β-subunit of the proteasome. This class of natural products has inspired the development of the synthetic peptidyl epoxyketones Carfilzomib (approved injectable drug for multiple myeloma), Oprozomib (orally-active multiple myeloma drug candidate in clinical trials) and Zetomipzomib (undergoing phase 2b clinical trials for treatment of lupus nephritis).

Using a combination of biochemical and genetic approaches, we have extensively investigated the biosynthesis of TMC-86A and eponemycin, two closely related peptidyl epoxyketones produced by Streptomyces species.1,2 TMC-86A and eponemycin are assembled by a nonribosomal peptide synthetase (NRPS) / type I modular polyketide synthase (PKS) multienzyme complex that constructs a peptidyl-α-dimethyl-β-keto acid intermediate, which is converted to the corresponding α-methyl-α, β-epoxyketone by a remarkable flavin-dependent epoxyketone synthase.

In this lecture, I will discuss our efforts to develop a better understanding of the catalytic mechanism and substrate scope of the epoxyketone synthase using a combination of protein structure prediction, substrate docking, molecular dynamics simulations, synthetic substrate analogues, in situ enzymatic deprotection, enzymatic activity assays and site-directed mutagenesis. I will also describe cloning and heterologous expression of the biosynthetic gene cluster for tryptopeptin, a Streptomyces epoxyketone derived from an α-methyl-β-keto acid intermediate. This has enabled efforts to bioengineer hybrid eponemycin / tryptopeptin biosynthetic gene clusters, illuminating the mechanism for productive interaction between the NRPS and PKS multienzymes.

Overall, these studies provide significant new insights into mechanisms for assembly of peptidyl epoxyketones, which can be exploited in biosynthetic engineering approaches to the production of novel natural product analogues with enhanced therapeutic potential.

64da73d91b6c1-TMC-86A+biosynthesis.jpg

  1.  J. Am. Chem. Soc. 2016, 138, 4342.
  2. Nucleic Acids Res. 2023, 51, 1488.
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