Identification of pyrazolopyridazinones as PDEδ inhibitors

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.

RAS Proteins and Their Regulators in Human Disease

“…Depletion of PDEδ results in RAS mis-localization and consequently in attenuated signaling. Earlier deltarasin and recently deltazinone 1, which shows relatively less nonspecific cytotoxicity than deltarasin, were developed as inhibitors of the KRAS4b-PDEδ interaction (Papke et al, 2016; Zimmermann et al, 2013). …”

Structure-based development of PDEδ inhibitors

“…This suggests that Deltarasin eventually modulates several off-targets and so the 'switch-like' behavior in growth inhibition assays may be due to general cytotoxicity, rather than dependent on the approach to block the PDEδ/KRas interaction. Indeed, Deltazinone 1 inhibited the cell growth of KRas dependent cell lines in a clear dose-dependent manner, and did not show unspecific cytotoxicity even at 24 μm concentrations (Papke et al, 2016). Because Deltazinone 1 needed at least 10 μm concentrations to show an effect in cell-based assays, this limitation may not be chemotype-but systemdependent.…”

“…This prompted us to develop a novel PDEδ inhibitor chemotype to address the question whether those limitations were chemotype-dependent or intrinsic to the PDEδ-inhibition approach. To identify alternative chemotypes, further analysis of the previously developed high-throughput Alpha Screen hit set pointed towards pyrrolopyridazinone 14 and pyrazolopyridazinone 15 as inhibitors of the PDEδ/KRas interaction (Figure 4) (Papke et al, 2016;Murarka et al, 2017).…”

Plasma membrane phosphatidylinositol 4-phosphate and 4,5-bisphosphate determine the distribution and function of K-Ras4B but not H-Ras proteins

“…Structural analysis, showed that these transport proteins provide with a hydrophobic grove suitable to adopt the hydrophobic farnesyl group on the K-Ras protein, thereby increasing its water solubility (26)(27)(28). To test how this process affects the translocation of the protein from the PM to the Golgi, we pretreated cells with deltarasin, a compound that competes to Ras binding by mimicking the structure of farnesyl groups (26).…”