CHARACTERIZATION OF NOVEL INHIBITORS OF THE TRAFFICKING CHAPERONE PDE6D

The RAS protein has been considered undruggable for more than 30 years due to the absence of evident target sites. However, recent developments have paved the way for novel approaches in targeting Ras.
In the first part of my thesis, a concise review is provided of different strategies that focus on directly targeting four distinct druggable regions within Ras. Particular focus is given to the insights gained from the development of allele-specific covalent and non-covalent inhibitors against Ras. Furthermore, we address the utility of macromolecular binders to identify and validate targetable regions on Ras and we discuss the promising development of small molecule binders into degraders of Ras.
The second, main part of my thesis is dedicated to the characterization and evaluation of novel PDE6D inhibitors named Deltaflexins. The KRAS-trafficking chaperone protein PDE6D, which is also known as PDE𝛿 (PDEdelta), was initially proposed as a surrogate therapeutic target for K-Ras. However, off-target effects, solubility issues and inefficient cell penetration have hampered the advancement of previously established PDE6D inhibitors to the clinical stage. In our study, we have developed Deltaflexins that display potent inhibitory effects against PDE6D at nanomolar concentrations and enhanced selectivity to PDE6D. Our findings indicate that increasing the affinity to the PDE6D hydrophobic pocket is linked to poorer solubility and also increased off-target binding to PDE6D-related UNC119. Therefore, we decided to take a combinatorial treatment approach to target K-Ras.
To this end, we combined Deltaflexin3 with the clinically approved drug Sildenafil. Deltaflexin3 was chosen among all investigated compounds as it displays the highest selectivity towards PDE6D and a high solubility. Sildenafil is known to promote phosphorylation of the K-Ras residue Ser181 thereby reducing the affinity between K-Ras and PDE6D. As a result, this combinatorial treatment efficiently suppressed K-Ras-dependent signaling and led to synergistic inhibitory effects on the cancer cell proliferation as well as tumor growth in chorioallantoic membrane assay.
Recently, we demonstrated that oncogenic K-Ras hinders cellular differentiation in our research project. Knowing that cell stemness is linked to primary cilia and that PDE6D can facilitate the trafficking of K-Ras to the primary cilium, another focus of our research was to study the novel molecular mechanisms of how PDE6D and K-Ras collaborate to impact cell differentiation, which may have significant implications for cancer evolution. Therefore, in the third part of my thesis, I will introduce K-Ras chimera that can be orthogonally trafficked into the primary cilium to validate ciliary functions of K-Ras and the impact of K-Ras localization on cell differentiation.