Abstract
The prenyl-binding protein PDEδ is crucial for the plasma membrane localization of prenylated Ras. Recently, we have reported that the small-molecule Deltarasin binds to the prenyl-binding pocket of PDEδ, and impairs Ras enrichment at the plasma membrane, thereby affecting the proliferation of KRas-dependent human pancreatic ductal adenocarcinoma cell lines. Here, using structure-based compound design, we have now identified pyrazolopyridazinones as a novel, unrelated chemotype that binds to the prenyl-binding pocket of PDEδ with high affinity, thereby displacing prenylated Ras proteins in cells. Our results show that the new PDEδ inhibitor, named Deltazinone 1, is highly selective, exhibits less unspecific cytotoxicity than the previously reported Deltarasin and demonstrates a high correlation with the phenotypic effect of PDEδ knockdown in a set of human pancreatic cancer cell lines.
Highlights
The prenyl-binding protein PDEd is crucial for the plasma membrane localization of prenylated Ras
To examine whether PDEd inhibition by Deltarasin and Deltazinone 1 downregulates the signal transduction via these pathways, we examined the phosphorylation of extracellular signal regulated kinase (Erk) and S6 Ribosomal Protein (S6P) in the oncogenic KRasdependent Panc-Tu-1 and KRas-independent PANC-1 cells
Deltarasin was the first small molecule effectively targeting the spatial organization of Ras by competing for the farnesyl-binding pocket of PDEd and thereby resulting in a reduced proliferation of oncogenic KRas-dependent pancreatic ductal adenocarcinoma cells[13]
Summary
The prenyl-binding protein PDEd is crucial for the plasma membrane localization of prenylated Ras. Subsequent detailed analysis of the dose–response curves characteristic for Deltarasin revealed that this PDEd ligand displays a ‘switch-like’ inhibition of proliferation; that is, the corresponding dose–response curve is very steep in the 3–8 mM range with a Hill coefficient of À 5.3 to À 10.8 (Supplementary Fig. 1) Such behaviour could arise from non-linear effects of Ras plasma membrane localization on signalling or could be indicative of general cytotoxicity by Deltarasin at high concentration and/or of interaction with additional target proteins in cells causing undesired side effects. Analysis of Deltarasin binding to additional proteins revealed that the compound binds to different G-protein-coupled receptors, ion channels and transporters (Supplementary Table 1) Given this undesirable property of Deltarasin at concentrations 45 mM, validation of PDEd as potential target for small-molecule interference with Ras localization and thereby signalling activity, called for the development of a novel chemotype for inhibition of the Ras–PDEd interaction, which would not display a comparable general cytotoxicity
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