Research paperA novel class of selective CK2 inhibitors targeting its open hinge conformation
Graphical abstract
Introduction
CK2 is a ubiquitous Ser/Thr protein kinase acting on several substrates whose phosphorylation is involved in a wide range of cellular processes, spanning from cell differentiation to apoptosis [1]. It is a tetramer, formed by two catalytic α and two regulatory β subunits. Historically considered a constitutively active kinase [2], it has been proposed that CK2 is instead kept in a non-active state by a peculiar inhibition mechanism, where the tetramers supramolecularly organize into trimers and other more complex structures, and then revert to the active state upon necessity [[3], [4], [5]].
CK2 is expressed at abnormally high levels in various cancers and hematological malignancies [6,7]. Its increased activity has been related with a wide range of responses, such as augmented neovascularisation [8], multidrug resistance [9] and reduced apoptosis [10], all traits that contribute to increased tumour aggressiveness and poorer overall prognosis.
Consistently with the protective role of CK2, tumour cells undergo apoptosis if its catalytic activity is reduced [11]. Furthermore, treatment with CK2 inhibitors induces apoptosis more efficiently in tumour cells than in healthy control cells [12]. Drug discovery efforts devoted at interfering with CK2 activity culminated in the development of CX-4945 [13]; CX-4945 (silmitasertib) has recently been designated as orphan drug for the treatment of cholangiocarcinoma [14] and is in clinical trials for the treatment of various cancers [15].
Potency and selectivity of CK2 inhibitors rely on their interactions with both the hinge region, on one side of the kinase ATP-binding pocket, and the peculiar positively charged region, on the other side. Here we focus on a new class of CK2 inhibitors, based on a central 2-cyano-2-propenamide scaffold variously substituted on both sides to optimize interactions with the two CK2 regions named above. We first determined the crystal structure of CK2α in complex with SRPIN803, which contains a 6-methylene-5-imino-1,3,4-thiadiazolopyrimidin-7-one scaffold where the 2-cyano-2-propenamide is cyclised and fused to a thiadiazole ring (Fig. 1a). SRPIN803 was previously identified in a drug development campaign against SRPK1 and kindly provided to us by the authors of this study [16]. SRPK1 is a protein kinase involved in the regulation of various RNA-processing pathways including RNA translation, stability and alternative splicing [17]. SRPIN803, tested against a panel of 306 kinases, showed stronger inhibition of CK2 than of its original target SRPK1, with IC50 values of 203 nM and 2.4 μM, respectively, and much lower activity on all other kinases tested [16]. Due to the involvement of SRPK1 in angiogenesis [18] and of CK2 in neovascularisation [8], the combined inhibitory effect of SRPIN803 has been successfully used in the treatment of a mouse model of age-related macular degeneration, a progressive degenerative condition resulting in vision loss [16].
Unexpectedly, the structure of SRPIN803, as determined by crystallography in the complex with CK2α, was in the open form as shown in Fig. 1b (i.e., different from the closed form, where the thiadiazole nitrogen reacted with the nitrile carbon, as reported in Ref. [16] and in Fig. 1a). SRPIN803 was de novo synthesized in our laboratory according to the published protocol [16] and the product obtained fully characterized, as described below, and named SRPIN803-rev (SRPIN803-revised), confirming the open form (Fig. 1b). With the correct structure elucidated for the parent inhibitor, we initiated its optimization by producing and characterizing various derivatives of SRPIN803-rev with increased inhibitory potency against CK2. High-resolution crystallographic structures of CK2α in complex with the various inhibitors presented here showed interactions with side chains of the CK2α hinge region. This unique feature among the CK2 inhibitors developed so far, all interacting with main chain atoms of the hinge region, constitutes the basis of the noticeable selectivity of this series of compounds. Moreover, their binding is only compatible with the open hinge conformation, extremely rare among protein kinases [19]. The best inhibitor (compound 4) has IC50 = 280 nM against CK2α and only inhibits this kinase and its paralogue CK2α’ by more than 50% when tested at 1 μM on a panel of 320 kinases. The most promising compounds, when tested on a cell-based assay, demonstrated to be cell permeable and to inhibit endocellular CK2, thus promoting tumour cells death.
Section snippets
Synthesis and characterization of SRPIN803-rev
SRPIN803-inp (SRPIN803, initially proposed), first developed and kindly provided to us by the Prof. Hagiwara laboratory, was soaked into CK2 apo crystal and the crystallographic structure of the complex determined at 1.5 Å resolution. Clear positive electron density was present in the kinase ATP pocket, not compatible with the structure of SRPIN803-inp (Fig. 1a) but instead corresponding to compound SRPIN803-rev (Fig. 1b and S1). The observed electron density did not change when only the first
Conclusions
The structural bases for the binding mode of SRPIN803-inp to CK2 and its reported specificity [16] were puzzling. Its co-crystallization with CK2α identified a chemical structure different from the one reported (SRPIN803-rev). De novo synthesis of SRPIN803-inp also resulted in SRPIN803-rev. Based on our characterization of SRPIN803-rev and SRPIN803-inp, and on previous reports describing very similar discrepancies, in a very similar molecular context, between the expected and the obtained
Chemistry
The synthetic procedures for the preparation, isolation and characterization of SRPIN803-rev and compounds 2–11 are reported in the SI. Compounds 12–14 were purchased from VITAS-M Laboratory. All compounds were negative as PAINS or aggregators when tested on dedicated web servers [42,43].
Protein production
The expression of human CK2 α and β subunits was induced in E. coli BL21-DE3 with 0,5 mM IPTG for 4 h at 30 °C. CK2α pellet mixed with an equal amount of pellet expressing β subunit was resuspended in buffer A
Associated content
The Supporting Information contains further experimental details, tables and figures, as consecutively outlined. Protein Crystallography: collection and refinement statistics, electron density maps, comparison with SRPIN803 docking pose. Small molecule crystallography: methods, results and collection and refinement statistics. Kinase assay: IC50 curves. Kinase panel profiling. Synthetic methods, 1H NMR, 13C NMR, IR and HPLC traces for SRPIN803-inp, SRPIN803-rev and compounds 2–11.
Declaration of interest
The authors declare no competing financial interest.
Accession codes
Structures were deposited to the PDB with accession numbers 6RB1 (SRPIN803-rev), 6RCM (cmp 3), 6RFE (cmp 4), 6RFF (cmp 7) and 6RCB (cmp 14). Authors will release the atomic coordinates and experimental data upon article publication. CCDC 1910754 contains the supplementary crystallographic data obtained from SRPIN803-rev ligand crystals. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via https://www.ccdc.cam.ac.uk/structures/.
Acknowledgments
We thank Dr. Massimo Bellanda and Prof. Danilo Pedron (University of Padua, Italy) for their help with NMR and IR spectra acquisition and analysis. We are grateful to the staff of the XRD1 beamline at Elettra (Trieste, Italy) and of the ID30-A1 and ID30-A3 beamlines at ESRF (Grenoble, France), for on-site assistance. G.L. is supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC “MFAG 2017”, code 19882). We are grateful to Prof. Masatoshi Hagiwara (Kyoto University, Japan) for
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The authors contributed equally to the work.