Journal of Medicinal Chemistry
Article
manner further validated the interaction of both biotinylated and free
deltarasin with PDEδ in MDCK cell lysates.
ABBREVIATIONS USED
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ITC, isothermal titration calorimetry; DtBAD, di-tert-butyl
azadicarboxylate; MDCK, Madin−Darby canine kidney; NMP,
N-methyl pyrrolidone; PD, pull down; PDEδ, protein originally
identified as δ-subunit of phosphodiesterase 6, prenyl binding
protein; Ras, rat sarcoma (protein); SPR, surface plasmon
resonance; TAMRA, tetramethylrhodamine; Tm, protein melting
point; TMAD, N,N,N′,N′-tetramethylazodicarboxamide
Inihibition of the Ras−PDEδ Interaction by Deltarasin.
To characterize the interaction of deltarasin with PDEδ in human
pancreatic adenocarcinoma cells, we analyzed the effect of
deltarasin on the Ras−PDEδ interaction in oncogenic K-Ras
dependent Panc-Tu-I cells.34 GST−PDEδ was immobilized on
magnetic beads and treated with Panc-Tu-I cell lysates. The
fraction of endogenous Ras that bound to the immobilized
GST−PDEδ was analyzed by means of SDS-PAGE and Western
blot using an H/K-Ras specific antibody. GST−PDEδ clearly
enriched Ras at the solid phase (Figure 13), and deltarasin
decreased the amount of Ras−PDEδ complex on the solid phase
in a dose-dependent manner. At compound concentrations as
low as 500 nM, the amount of endogenous Ras at the solid phase
was clearly decreased. These experiments with endogenous Ras
clearly validate previous FRET−FLIM experiments in living cells
using labeled Ras−PDEδ.16
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CONCLUSION AND OUTLOOK
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We have described the structure guided development of PDEδ
inhibitors based on a bis-benzimidazole scaffold, which target the
farnesyl binding pocket of PDEδ with nanomolar affinity. Our
results demonstrate that the prenyl binding pocket of PDEδ is
druggable and that logical, structure-guided design allows us to
increase inhibitor potency and properties in a stepwise
continuous manner. Our findings suggest that also other
inhibitor classes with different scaffold structures and chemo-
types may be developed following this structure guided
approach. We have provided proof for this notion by the
identification of potent atorvastatin derivatives. A detailed kinetic
analysis of the interaction between potent bis-benzimidazoles
and PDEδ revealed that off rates are at least 3 orders of
magnitude lower than the rate constants koff for farnesylated
Ras-family proteins. Investigation of target engagement in cell
lysates showed that bis-benzimidazoles like deltarasin bind at
nanomolar concentrations to endogenous PDEδ.
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ASSOCIATED CONTENT
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* Supporting Information
Detailed experimental procedures for biochemistry, cell biology,
and chemical synthesis. This material is available free of charge
AUTHOR INFORMATION
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Corresponding Author
+49 (231) 133-2400.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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The Compound Management und Screening Center (COMAS),
Dortmund, is acknowledged for carrying out high-throughput
screening and data analysis. G.Z. acknowledges the Fonds der
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Chemischen Industrie for a Kekule Scholarship. We are grateful
to Carsten Degenhart and Dr. Alexander Wolf, LDC, for help
with initial Alpha screen assay development. Biaffin GmbH,
Kassel, is acknowledged for carrying out SPR measurements.
G.Z., C.S.F., G.T., S.I., A.W., and H.W. are inventors on a
Max-Planck patent application concerning PDED inhibitors.
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dx.doi.org/10.1021/jm500632s | J. Med. Chem. XXXX, XXX, XXX−XXX