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Enzymatic assembly of the salinosporamide γ-lactam-β-lactone anticancer warhead

Nature Chemical Biology volume 18, pages 538–546 (2022)Cite this article

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Abstract

The marine microbial natural product salinosporamide A (marizomib) is a potent proteasome inhibitor currently in clinical trials for the treatment of brain cancer. Salinosporamide A is characterized by a complex and densely functionalized γ-lactam-β-lactone bicyclic warhead, the assembly of which has long remained a biosynthetic mystery. Here, we report an enzymatic route to the salinosporamide core catalyzed by a standalone ketosynthase (KS), SalC. Chemoenzymatic synthesis of carrier protein-tethered substrates, as well as intact proteomics, allowed us to probe the reactivity of SalC and understand its role as an intramolecular aldolase/β-lactone synthase with roles in both transacylation and bond-forming reactions. Additionally, we present the 2.85-Å SalC crystal structure that, combined with site-directed mutagenesis, allowed us to propose a bicyclization reaction mechanism. This work challenges our current understanding of the role of KS enzymes and establishes a basis for future efforts toward streamlined production of a clinically relevant chemotherapeutic.

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Fig. 1: Microbial natural products assembled by terminal cyclization reactions.
Fig. 2: SalC is involved in late-stage salinosporamide A biosynthesis.
Fig. 3: SalC is a γ-lactam-β-lactone bicyclase.
Fig. 4: Transacylation of SalC by acylated SalB.
Fig. 5: SalC structure and active site mutagenesis.
Fig. 6: Abbreviated key bicyclization steps of the proposed SalC mechanism with 15.

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Data availability

Strains and plasmids used in this study are described in Supplementary Table 1. All oligonucleotides (Integrated DNA Technology) are shown in Supplementary Table 2. The salinosporamide (sal) BGC from S. tropica CNB440 is available in the MIBiG database (accession BGC0001041). Other salinosporamide BGCs and the cinnabaramide BGC used for alignments are available in the IMG JGI database. Functional KS and non-elongating KS protein sequences used for alignments can be found in the Supplementary material and through MIBiG. Atomic coordinates and structure factors for the reported crystal structures in this work have been deposited to the PDB under accession number 7S2X(native SalC). Additionally, the following PBD datasets were used for SalC structural comparison: 2HG4, 4WKY, 2QO3 and 4NA2. Source data are provided with this paper. Other relevant data supporting the findings of this study are available in this published article or its Supplementary files.

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Acknowledgements

We are grateful to P.R. Jensen (Scripps Institution of Oceanography) for the S. tropica CNB440 strain, B.M. Dungan (UC San Diego) for assistance with NMR, K.E. Creamer (UC San Diego) for the NaPDoS2.0 analysis and helpful bioinformatics discussions, Y. Su (UC San Diego Molecular Mass Spectrometry Facility) for intact proteomics experiments and J.P. Noel and G. Louie (Salk Institute for Biological Studies) for beamline coordination. Beamline 8.2.2 of the Advanced Light Source, a U.S. DOE Office of Science User Facility under contract number DE-AC02-05CH11231, is supported in part by the ALS-ENABLE program funded by the National Institutes of Health, National Institute of General Medical Sciences, grant P30-GM124169-01. This work was supported by NIH grants R01CA127622 (B.S.M.), R01AI047818 (B.S.M.) and F31HD101307 (K.D.B.), R35GM134910 (D.R.), the Robert A. Welch Foundation (grant number AA-1280 (D.R.)) and the São Paulo Research Foundation (FAPESP; grant number 2011/21358-5 (D.B.B.T.)).

Author information

Author notes

  1. Percival Yang-Ting Chen

    Present address: Morphic Therapeutics, Waltham, MA, USA

  2. Daniela B. B. Trivella

    Present address: Brazilian Biosciences National Laboratory, National Center for Research in Energy and Materials, Campinas, Brazil

  3. Daniela B. B. Trivella

    Present address: Institute of Chemistry, University of Campinas, Campinas, Brazil

  4. Tobias A. M. Gulder

    Present address: Chair of Technical Biochemistry, Technical University of Dresden, Dresden, Germany

Authors and Affiliations

  1. Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA, USA

    Katherine D. Bauman, Vikram V. Shende, Percival Yang-Ting Chen, Daniela B. B. Trivella, Tobias A. M. Gulder & Bradley S. Moore

  2. Department of Chemistry and Biochemistry, Baylor University, Waco, TX, USA

    Sreekumar Vellalath & Daniel Romo

  3. Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA

    Bradley S. Moore

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Contributions

K.D.B., V.V.S., T.A.M.G. and B.S.M. designed the study. K.D.B. performed protein expression and purification, enzymatic assays, mutagenesis and data analysis. V.V.S. synthesized and characterized all substrates in this study. P.Y.-T.C., D.B.B.T. and K.D.B. performed all crystallography experiments and subsequent data analysis. T.A.M.G. performed all gene inactivation experiments, preliminary protein expressions and initial substrate synthesis. S.V. and D.R. provided helpful initial synthetic discourse. K.D.B. and B.S.M. wrote the manuscript with input from all coauthors.

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Correspondence to Bradley S. Moore.

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Nature Chemical Biology thanks Andrew Gulick, Yi Tang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Salinosporamide A hydrolysis and subsequent THF ring formation is accelerated by the presence of SalC.

a, LCMS chromatograms of salinosporamide A (1) hydrolysis assay with and without SalC. * indicates compound retains chloride and no THF ring formation has occurred as evidenced by characteristic isotope pattern. b, Structures of compounds putatively identified by LCMS in a.

Extended Data Fig. 2

Naturally produced analogs of salinosporamide A that served as inspiration for simplisporamide.

Extended Data Fig. 3 SalC activity assay with diffusible substrates.

a, Reaction scheme depicting chemoenzymatic synthesis and subsequent SalB-PCP acylation assay to generate (15). b, LCMS chromatograms (BPCs and EICs m/z 266.18) of SalC activity assay with diffusible substrates shown in part (a) including pantetheine-activated (13), CoaA product-activated (19), CoaD product-activated (20), CoA activated (14), and carrier protein-activated substrate (15). Substrates were activated via in vitro CoA enzyme biosynthesis. All SalC activity assays contained apo-SalB-PCP as well.

Extended Data Fig. 4 Proteasome inhibitory activity of SalC assay product using purified human 20S proteasome.

a, Proteasome activity determined by reading fluorescence (AFUs) of the cleaved substrate (Suc-LLVY-AMC) at 355 nm (excitation) and 460 nm (emission) every five minutes after substrate was added for 30 min in the presence of various inhibitors. Blank (no proteasome added) = black, control (no inhibitor) = gray, epoxomicin (0.5 μM) = green, SalC reaction product = red, no SalC control = dark blue, no substrate control = orange, salinosporamide A (0.5 μM) = light blue. Samples run in duplicate, all data points shown. b, Magnification of y-axis of plot from a to examine successful inhibition of the 20S proteasome by epoxomicin, SalC reaction product, and salinosporamide A. c, Percent proteasome inhibition at 30 min, relative to control (no inhibitor, 0% inhibition). See Supplementary Fig. 14 for corresponding LCMS traces of extracts used in these assays.

Source data

Extended Data Fig. 5 Acylation of SalC with diffusible substrates.

a, Reaction scheme depicting chemoenzymatic synthesis to generate (22) b, UV chromatograms (215 nm) of intact protein LCMS for transacylation assay with diffusible substrates. Transacylation assay utilized linear mechanistic probe (21) activated in different ways (CoA-precursor-activated (23, 24), CoA-activated (25), and SalB-PCP-tethered, all generated in situ) and SalC. Transacylation assay with column purified 22 shown for comparison.

Extended Data Fig. 6 SalC overlay with a trans-AT KS.

SalC structure aligned with closest Dali server homolog, the trans-AT KS OzmN KS2 (PDB ID: 4WKY) from the hybrid NRPS/PKS oxazolomycin pathway, RMSD 0.842 Å. Overall structure of SalC dimer (colors shown as previous, KS monomers in brown and green, flanking subdomains in yellow and teal) with OzmN KS2 (gray).

Extended Data Fig. 7 SalC overlay with functional type I KS.

SalC KS aligned with DEBS KS3 (PDB: 2QO3), RMSD 1.225 Å. a, Overall structure of SalC dimer (colors shown as previous, KS monomers in brown and green, flanking subdomains in yellow and teal) with DEBS KS3 (gray). b, Active site overlay of SalC (green) and DEBS KS3 (gray).

Extended Data Fig. 8 Tyr284 is conserved in SalC homologs.

Condensed alignment showing conservation of Tyr284 in all SalC homologs from Salinispora and Streptomyces cinnabarigriseus JS360 (CinC) but not in canonical elongating KSs. All SalC homologs from Salinispora strains found in JBI IMG database. For functional KS sequences refer to Supplementary Figure 5.

Extended Data Fig. 9 SalC structure overlaid with bacillaene PKS (bae) KS2 bound to its natural intermediate.

SalC is shown in green and BaeKS2 is shown in grey, bae intermediate in gold. PDB ID: 4NA2, RMSD 1.071 Å.

Extended Data Fig. 10 Proposed active site mechanism of SalC.

Catalysis is initiated by deprotonation of Cys180 followed by transacylation of the SalB-PCP tethered substrate through a tetrahedral intermediate (not shown). Lys348 deprotonates His353, and hydrogen bonding of the thioamide carbonyl to Tyr284 facilitates deprotonation of the thioester α-proton by His353. An intramolecular aldol reaction forms the γ-lactam; the oxyanion is presumably stabilized by dipole interactions with backbone amides, as is hypothesized for KSs25. Subsequent β-lactonization through a tetrahedral intermediate leads to release of simplisporamide from SalC. Finally, Cys180 is reprotonated by His353.

Supplementary information

Supplementary Information

Supplementary Figs. 1–30, Tables 1–4, Notes Figs. 1–29 and Notes Table 1.

Reporting Summary

Source data

Source Data Extended Data Fig. 4

Unprocessed Arbitrary Fluorescence Units (AFUs) for proteasome inhibition assay.

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Bauman, K.D., Shende, V.V., Chen, P.YT. et al. Enzymatic assembly of the salinosporamide γ-lactam-β-lactone anticancer warhead. Nat Chem Biol 18, 538–546 (2022). https://doi.org/10.1038/s41589-022-00993-w

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