Ubiquitin proteasome system UPS responsible for much of the

Ubiquitin-proteasome system (UPS), responsible for much of the regulated proteolysis in the cell, mainly contains ubiquitin (Ub), Ub-activating enzyme E1, Ub-conjugating enzymes E2s (UBCs), Ub-protein ligands E3s, and 26S proteasome [9]. E3 ligand is known as substrate-specific, which can catalyze transfer of ubiquitin from E2s to an amide linkage with the substrate or with a polyubiquitin chain already anchored to it, then the tagged substrate is degraded by the 26S proteasome [10]. Because the UPS represents a major system controlling many cellular processes, drugs suppressing the UPS can be efficacious in the treatment of cancers. So far, several small molecules like bortezomib and carfilzomib have been approved as proteasome inhibitors for clinical use [11], [12]. Proteolysis targeting chimeras (PROTACs), owning full advantage of the function of protein degradation by UPS, is a heterobifunctional 5720 sale with two recruiting ligands connected via a linker, in which one ligand is specific to the protein of interest while the other one specifically recruits E3 ligase [13], [14], [15]. Generally, an E3 ligase needs a specific recognition signal to recruit and ubiquitinate its natural substrate, however, PROTAC acts in a way that can effectively tether the E3 ligase to the substrate and result in the ubiquitination and subsequent proteasomal degradation of the target protein (Fig. 1) [16]. Typically, a small molecule protein inhibitor is usually selected as the ligand specific to the protein of interest, and so far thalidomide, lenalidomide, pomalidomide and their derivatives have been chosen as the recruiters of E3 ligase [17], [18], [19], [20]. At present, PROTAC has been widely applied to induce the degradation of various proteins, such as BRD4, Sirt2, and BCR-ABL [21], [22], [23]. Based on the above, we hypothesized that a PROTAC strategy would be effective to induce CK2 protein degradation. Herein reported is an approach for the preparation of novel PROTACs via “click reaction” for degradation of CK2 protein (Fig. 2). Importantly, “click reaction” is a very facile, selective and “green” reaction with the aim of binding two molecules together under mild water-tolerant conditions. CX-4945, a selective CK2 inhibitor with a strong binding force stemming from hydrophobic interactions of flat tricyclic core with binding pocket residues, hydrogen bonding of pyridine nitrogen with the NH of Val116 in the hinge region, and an ionic bridge with Lys68, was first elected as CK2 targeting ligand [24]. And pomalidomide was applied as a recruiter of E3 ligase to conjugate with CX-4945 via different linkers by click reaction. It was worth noting that the number of alkyl chains was important for PROTACs, because the length of the chain has a great influence on the interaction between E3 ligase and CK2 protein.
Results and discussion
Conclusions We have reported the design and synthesis of four CK2 targeting compounds composed of a CK2 inhibitor CX-4945 and pomalidomide, which are connected by alkyl linkers via click reactions. Among them, compound 2 exhibited a strong PROTAC feature. As a CK2 protein degrader, compound 2 degrades the protein in a dose and time-dependent manner, and can maintain CK2 at a low basal level. And the degrader degrades the protein through an ubiquitin and proteasome-dependent proteolysis pathway. The degradation of CK2 by compound 2 resulted in a series of changes in its downstream proteins which include the reduced phosphorylation of Akt (a phosphorylated substrate of CK2) and the up-regulation of p53 (a tumor suppressor protein). Significantly, 2 also possessed similar cytotoxicity to CX-4945 toward CK2 overexpressed cancer cells like MDA-MB-231, but its mechanism of action is quite different from the inhibitor. Owing to the overexpression of CK2 in most tumors with highly pleiotropic features, and more obvious influence on the downstream proteins of CK2 during a shorter time period triggered by PROTAC, targeting CK2 degradation will have potential applications for cancer treatments.