2-Aminothiazole Derivatives as Selective Allosteric Modulators of the Protein Kinase CK2. 2. Structure-Based Optimization and Investigation of Effects Specific to the Allosteric Mode of Action

Abstract

Protein CK2 has gained much interest as an anti-cancer drug target in the last decade. We had previously described the identification of a new allosteric site on the catalytic α-subunit, along with first small molecule ligands based on the 4-(4-phenylthiazol-2-ylamino) benzoic acid scaffold. In the present work, structure optimizations guided by a binding model led to the identification of the lead compound 2-hydroxy-4-((4-(naphthalen-2-yl)thiazol-2yl)amino)benzoic acid (27), showing a submicromolar potency against purified CK2α (IC 50 = 0.6 µM). Furthermore, 27 induced apoptosis and cell death in 786-O renal cell carcinoma cells (EC 50 = 5 µM) and inhibited STAT3 activation even more potently than the ATPcompetitive drug candidate CX-4945 (EC 50 s: 1.6 µM vs. 5.3 µM). Notably, the potencies of our allosteric ligands to inhibit CK2 varied depending on the individual substrate. Altogether, the novel allosteric pocket was proved a druggable site, offering an excellent perspective to develop efficient and selective allosteric CK2 inhibitors. Recently, we identified 2-aminothiazole derivatives as novel allosteric inhibitors of CK2α; 20 exemplarily shown are compounds 1-4 (Figure 1). Using complementary methods, it was demonstrated that compound 3 binds in an allosteric pocket adjacent to the ATP binding site, between the glycine-rich loop and the αC-helix. A preliminary hit optimization led to compound 4, exhibiting an IC 50 of 3.4 µM. However, it had yet to be shown that the new CK2 modulator class can be developed further into more potent drugs, which is often a limitation with allosteric target sites that were not evolutionary designed for high-affinity interactions with small molecules. 21, 22 Therefore, we carried out a compound optimization guided by a binding model, as will be described below. The cellular effects of the allosteric CK2

Proposed allosteric inhibitors bind to the ATP site of CK2α

“…We have used two representative compounds from Series A for further investigation, which we denote 1 and 2 (these were referred to as 6 and 27 respectively in the original papers 35,36 ). 1 was chosen as much of the validation of Series A and Site A relied upon this compound 35 . 2 was chosen as it was the highest affinity inhibitor reported in Series A.…”

“…The original publication contained no structural data on the inhibitor binding mode. In fact, the authors state that 'cocrystallisation attempts aimed at elucidating the non-ATP competitive binding mode of 2-aminothiazole derivatives with CK2α were not successful' 35,36 . We have rectified this and have determined structures of both 1 and 2 in complex with CK2α (Fig.…”

“…1), proposed to bind in a novel site (Site A, SI Fig. 2) just outside of the highly conserved ATP site, has been published 35,36 . The authors used enzymatic assays, native mass spectrometry and competitive ligand-based NMR studies to characterize the mode of inhibition.…”

“…While their data were presented as supporting non-ATP-competitive inhibition, there were several reasons why we speculated that the mechanism of inhibition by these molecules was not necessarily allosteric. In particular: the enzyme assays showed mixed mode inhibition; ATP analogues were replaced in NMR studies; high affinity ligands showed unexpected signal in ligand-based NMR experiments; and, mutations close to the ATP site affected inhibitory potential 35,36 . Our motivation to investigate these inhibitors in more detail arose from the somewhat conflicting data presented, 35,36 as well as the chemical properties of these new inhibitors, which are not unlike known ATP competitive kinase inhibitors.…”

Identification and Biological Evaluation of CK2 Allosteric Fragments through Structure-Based Virtual Screening

“…The αD pocket named site 3 was demonstrated as the allosteric pocket to accommodate the biaryl rings of CAM4712 analogues [17][18][19]. Recently, Bestgen et al discovered aminothiazole derivatives as the allosteric modulators of CK2 by targeting the interface between the αC helix and glycine-rich loop [20,21]. However, most allosteric modulators were discovered by high-throughput screening experiment, which is time-consuming and serendipitous to some extent due to the lack of the comprehensive understanding about the allosteric sites and mechanisms [22].…”