SUMMARY The human TEAD family of transcription factors (TEAD1-4) is required for YAP-mediated transcription in the Hippo pathway. Hyperactivation of TEAD’s co-activator YAP contributes to tissue overgrowth and human cancers, suggesting that pharmacological interference of TEAD-YAP activity may be an effective strategy for anticancer therapy. Here we report the discovery of a central pocket in the YAP-binding domain (YBD) of TEAD that is targetable by small molecule inhibitors. Our X-ray crystallography studies reveal that flufenamic acid, a non-steroidal anti-inflammatory drug (NSAID), binds to the central pocket of TEAD2 YBD. Our biochemical and functional analyses further demonstrate that binding of NSAIDs to TEAD inhibits TEAD-YAP-dependent transcription, cell migration and proliferation, indicating that the central pocket is important for TEAD function. Therefore, our studies discover a novel way of targeting TEAD transcription factors and set the stage for therapeutic development of specific TEAD-YAP inhibitors against human cancers.

CK2 is a ubiquitous Ser/Thr protein kinase involved in the control of various signaling pathways and is known to be constitutively active. In the present study, we identified aryl 2-aminothiazoles as a novel class of CK2 inhibitors, which displayed a non-ATP-competitive mode of action and stabilized an inactive conformation of CK2 in solution. Enzyme kinetics studies, STD NMR, circular dichroism spectroscopy, and native mass spectrometry experiments demonstrated that the compounds bind in an allosteric pocket outside the ATP-binding site. Our data, combined with molecular docking studies, strongly suggested that this new binding site was located at the interface between the αC helix and the flexible glycine-rich loop. A first hit optimization led to compound 7, exhibiting an IC 50 of 3.4 μM against purified CK2α in combination with a favorable selectivity profile. Thus, we identified a novel class of CK2 inhibitors targeting an allosteric pocket, offering great potential for further optimization into anticancer drugs.

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

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