An Improved PDE6D Inhibitor Combines with Sildenafil To Inhibit KRAS Mutant Cancer Cell Growth

Abstract

The trafficking chaperone PDE6D (or PDEδ) was proposed as a surrogate target for K-Ras, leading to the development of a series of inhibitors that block its prenyl binding pocket. These inhibitors suffered from low solubility and suspected off-target effects, preventing their clinical development. Here, we developed a highly soluble, low nanomolar PDE6D inhibitor (PDE6Di), Deltaflexin3, which has the lowest off-target activity as compared to three prominent reference compounds. Deltaflexin3 reduces Ras signaling and selectively decreases the growth of KRAS mutant and PDE6D-dependent cancer cells. We further show that PKG2-mediated phosphorylation of Ser181 lowers K-Ras binding to PDE6D. Thus, Deltaflexin3 combines with the approved PKG2 activator Sildenafil to more potently inhibit PDE6D/K-Ras binding, cancer cell proliferation, and microtumor growth. As observed previously, inhibition of Ras trafficking, signaling, and cancer cell proliferation remained overall modest. Our results suggest reevaluating PDE6D as a K-Ras surrogate target in cancer.

A general Bioluminescence Resonance Energy Transfer (BRET) protocol to measure and analyze protein interactions in mammalian cells

“…From this representation one can also identify the optimal A/D-plasmid ratio (here at A/D = ~10:1) for dose-response experiments (Figure 2A). Under these conditions the BRET-ratio response of the BRETbiosensor depends pseudo-linearly on manipulations that affect the interaction, such as drugtreatments 12 . The preferred format employs the expression signal ratio on the x-axis, as it is derived from measured signal values (Figure 2B).…”