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

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.

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.

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 KSs. Subsequent β-lactonization through a tetrahedral intermediate leads to release of simplisporamide from SalC. Finally, Cys180 is reprotonated by His353.

Fig. 1:. Microbial natural products assembled by terminal cyclization reactions.

a, salinosporamide biosynthetic gene cluster (sal BGC) and proposed bicyclization of the SalB-PCP-tethered linear intermediate to assemble the salinosporamide A (1) pharmacophore. Bonds formed by unknown cyclization enzyme(s) are highlighted in gray. b, Representative additional examples of microbial natural products assembled via terminal cyclization reactions: obafluorin (2), fumiquinazoline F (3), vibralactone (4), nocardicin A (5), tenuazonic acid (6). Bond(s) formed in cyclization reaction and catalyzing enzyme class shown in red.

Fig. 2:. SalC is involved in late-stage salinosporamide A biosynthesis.

LCMS chromatograms of salinosporamide A standard and extracts from Salinispora cultures: wild type Salinispora tropica CNB440; S. tropica CNB440△salO; S. tropica CNB440△salA; S. tropica CNB440△salD; S. tropica CNB440△salC. 7 = salinosporamide B, 8 = salinosporamide J.

Fig. 3:. SalC is a γ-lactam-β-lactone bicyclase.

a, Reaction scheme for the chemoenzymatic synthesis of PCP-tethered linear substrate (15) from pantetheine-activated simplisporamide precursor (13) via in vitro CoA biosynthesis and Sfp-mediated loading. b, Intact protein LCMS chromatogram of SalB-PCP acylation assay (UV, 215 nm). See Supplementary Fig. 11 for intact proteomics HRMS data. c, LCMS chromatogram of SalB-PCP tryptic digest post acylation assay and MS2 Ppant ejection assay. d, Reaction scheme for SalC activity assay. e, LCMS chromatograms of linear hydrolysis product (16) standard, SalC activity assay with no SalC (3 h), SalC activity assay (3 h), and SalC activity assay (overnight). See Supplementary Fig. 12 for HRMS data for R-17 and Supplementary Note for full characterization.

Fig. 4:. Transacylation of SalC by acylated-SalB.

a, Reaction scheme for the chemoenzymatic synthesis of 22 and subsequent transacylation assay with SalC. b, UV chromatograms (215 nm) of intact protein LCMS of apo-SalB-PCP and SalC (bottom trace; small amount of holo-SalB-PCP present likely due to native E. coli PPTase modification), SalB-PCP acylated with the mechanistic probe (21) and purified by size exclusion chromatography to yield 22 (middle trace), and the complete transacylation assay using 22 and SalC (top trace). See Supplementary Figs. 16 and 17 for intact proteomics HRMS data.

Fig. 5:. SalC structure and active site mutagenesis.

a, SalC overall tetrameric structure with each monomeric domain colored differently (KS domains in green and brown, flanking subdomain in teal and yellow). b, SalC active site with proposed catalytic residues labeled. c, UV (215 nm) chromatograms from intact protein LCMS transacylation assays using column purified 22 and SalC mutants. d, LCMS chromatograms of SalC activity assays (3 h) with WT SalC and SalC active site variants using 15. See Supplementary Figs. 27–30 for intact proteomics HRMS data.

Fig. 6:. Abbreviated key bicyclization steps of the proposed SalC mechanism with 15.

Once substrate undergoes transacylation from acylated SalB (15) to Cys180 of SalC (not shown), hydrogen bonding by Tyr284 facilitates deprotonation of the thioester α-proton by His353. An intramolecular aldol reaction forms the γ-lactam and the resulting oxyanion is presumably stabilized by dipole interactions with backbone amides (not shown). Subsequent β-lactonization through a tetrahedral intermediate leads to release of simplisporamide (R-17) from SalC. See Extended Data Fig. 10 for further details.