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Small molecule inhibition of the KRAS–PDEδ interaction impairs oncogenic KRAS signalling

Abstract

The KRAS oncogene product is considered a major target in anticancer drug discovery1,2,3. However, direct interference with KRAS signalling has not yet led to clinically useful drugs3,4,5,6,7,8. Correct localization and signalling by farnesylated KRAS is regulated by the prenyl-binding protein PDEδ, which sustains the spatial organization of KRAS by facilitating its diffusion in the cytoplasm9,10,11. Here we report that interfering with binding of mammalian PDEδ to KRAS by means of small molecules provides a novel opportunity to suppress oncogenic RAS signalling by altering its localization to endomembranes. Biochemical screening and subsequent structure-based hit optimization yielded inhibitors of the KRAS–PDEδ interaction that selectively bind to the prenyl-binding pocket of PDEδ with nanomolar affinity, inhibit oncogenic RAS signalling and suppress in vitro and in vivo proliferation of human pancreatic ductal adenocarcinoma cells that are dependent on oncogenic KRAS. Our findings may inspire novel drug discovery efforts aimed at the development of drugs targeting oncogenic RAS.

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Figure 1: Structure-based development of inhibitors.
Figure 2: In-cell measurements of the effect of deltarasin on the interaction of RAS with PDEδ and resulting delocalization of KRAS.
Figure 3: Inhibition of PDEδ–KRAS interaction suppresses proliferation and MAPK-signalling in oncogenic KRAS-dependent PDAC cells.
Figure 4: Deltarasin impairs dose-dependent in vivo growth of xenografted pancreatic carcinoma in nude mice.

Accession codes

Accessions

Protein Data Bank

Data deposits

The atomic coordinates of PDEd in complex with inhibitor 1, rac-S1, rac-2 and (S)-5 are deposited in the Protein Data Bank with accession numbers 4JV6, 4JV8, 4JVB and 4JVF, respectively.

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Acknowledgements

The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Program (FP7/2007-2013)/ERC Grant agreement no. 268309 to H.W., and no. 268782 to A.W. The Compound Management und Screening Center (COMAS), Dortmund, Germany, is acknowledged for carrying out high-throughput screening and data analysis. G.Z. acknowledges the Fonds der Chemischen Industrie for a Kekulé Scholarship. We thank K. Michel for help with western blot analysis. We are grateful to C. Degenhart, A. Wolf, S. Baumann and A. Choidas for help with screening assay development and for the determination of solubility, membrane permeability and stability of deltarasin.

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A.W., P.I.H.B. and H.W. conceived the project. H.W., G.T. and G.Z. developed the screen and chemistry to generate the PDEδ inhibitor, P.I.H.B. designed the cell biological experiments and A.W. with S.I. solved the structures by X-ray crystallography. A.W., H.W., S.I. and G.Z. designed the PDEδ structure-based inhibitor development. S.A.H. developed the inducible shRNA systems and the xenograft model, G.Z. synthesized the inhibitors and performed the biochemical and biophysical characterization experiments, B.P. performed the real-time cell analysis, FACS and western blot analysis. N.V., B.P. and A.C. performed the microscopy experiments. M.H. performed the shRNA real-time cell analysis and xenograft experiments. H.W. and P.I.H.B. wrote the manuscript with help from A.W., S.I., B.P., G.T. and G.Z.

Corresponding authors

Correspondence to Alfred Wittinghofer or Philippe I. H. Bastiaens or Herbert Waldmann.

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Competing interests

G.Z., B.P., S.I., N.V., G.T., A.W., P.I.H.B. and H.W. are inventors on an MPG patent application.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-19 (pages 1-20), Supplementary Table 1 (page 21), Supplementary Methods for Biochemistry and Crystallography (pages 22-25), Supplementary Tables 2-3 (page 26-27), Supplementary Methods for Cell Biology (pages 28-33), Chemical Synthesis (pages 34-72) and additional references (page 73). (PDF 4793 kb)

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Zimmermann, G., Papke, B., Ismail, S. et al. Small molecule inhibition of the KRAS–PDEδ interaction impairs oncogenic KRAS signalling. Nature 497, 638–642 (2013). https://doi.org/10.1038/nature12205

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