Synthesis facilitates an understanding of the structural basis for translation inhibition by the lissoclimides

Abstract

The lissoclimides are unusual succinimide-containing labdane diterpenoids that were reported to be potent cytotoxins. Our short semisynthesis and analogue-oriented synthesis approaches provide a series of lissoclimide natural products and analogues that expand the structure-activity relationships (SARs) in this family. The semisynthesis approach yielded significant quantities of chlorolissoclimide (CL) to permit an evaluation against the National Cancer Institute's 60-cell line panel and allowed us to obtain an X-ray co-crystal structure of the synthetic secondary metabolite with the eukaryotic 80S ribosome. Although it shares a binding site with other imide-based natural product translation inhibitors, CL engages in a particularly interesting and novel face-on halogen-π interaction between the ligand's alkyl chloride and a guanine residue. Our analogue-oriented synthesis provides many more lissoclimide compounds, which were tested against aggressive human cancer cell lines and for protein synthesis inhibitory activity. Finally, computational modelling was used to explain the SARs of certain key compounds and set the stage for the structure-guided design of better translation inhibitors.

Figure 1

Natural product inhibitors of eukaryotic translation are important as potential drugs and biochemical tools. Pictured are Synribo^®, a translation inhibitor and approved drug for CML; cycloheximide, a common laboratory reagent for translation inhibition; representative lissoclimides and their cytotoxicity toward P388 murine leukemia cells (IC₅₀); and lactimidomycin, a recently discovered cytotoxin and translation inhibitor. For a complete list of lissoclimide natural products, a few derivatives, and their cytotoxicity toward P388 cells, see ref . All data above compiled from refs –.

Figure 2

Our two synthesis approaches to the lissoclimides are designed to access a broad range of structurally perturbed analogues for a better understanding of structure-activity relationships. a. Previous short but limited semi-synthesis of lissoclimides from sclareolide; b. An analogue-oriented synthesis plan (* denotes sites of possible variant substitution and configurations)

Figure 3

The X-ray co-crystal structure of CL with the eukaryotic 80S ribosome reveals the molecular basis of translation inhibition. a. Location of CL in a putative in silico model of an actively translating 80S ribosome. b. CL clashes with the CCA-end of the E-site tRNA. The three tRNAs and mRNA (coloured in red) for this model were taken from the PDB entry 4V6F. The 60S (light blue) and 40S (light orange) subunits are represented as surfaces. c. CL (yellow) binds to the 60S E-site. CL shares the same binding pocket as cycloheximide (pink) and lactimidomycin (cyan). Eukaryotic specific ribosomal protein eL42 is shown in green and 25S rRNA in grey. d. Detail of the interactions occurring between the CL molecule (yellow, represented as sticks) and the neighboring residues. Direct contacts take place with nucleotides G92, C93, U2763, A2802 and G2794 of the 25S rRNA. A hydrogen bond is also observed between CL and the ribosomal protein eL42.

Figure 4

Our analogue-oriented synthesis approaches to the lissoclimides and congeners are designed to access point changes in the natural product structures at key positions. a. A readily diversifiable synthesis of C18-unoxygenated lissoclimides is exemplified by a second, independent synthesis of hatQ. b. Synthesis of highly functionalized lissoclimide analogues with C18 oxygenation as found in highly cytotoxic natural product hatJ.

Figure 5

Crystallographically derived binding arrangement of CL in the 80S ribosome and predicted binding poses of three analogue structures to rationalize aspects of the experimentally determined SAR. a. Co-crystal structure of CL with the 80S ribosome. b. Docking-based structure of analogue 39 bound to the 80S ribosome. c. Docking-based structure of analogue 45 bound to the 80S ribosome. d. Docking-based structure of analogue 37 bound to the 80S ribosome.