Interestingly from our consensus SBVC protocol
Interestingly, from our consensus SBVC protocol only few compounds (less than 150) have been scored with a full ‘ consensus’. That means that these compounds appear in the top 5% of the database when ranked by every scoring function independently. After visual inspection, we have realized that two anthraquinone derivatives were among them. This finding was quite unexpected and intriguing at the same time considering that other anthraquinones were already reported to be modestly active against CK1, such as emodin and 1,4-diamino-5,8-dihydroxy-anthraquinone (DAA). For this reason, we decided to primarily focus our attention to these potential hit compounds. In particular, the 1,4-diamino-anthraquinone (compound , in ) was one of the best ranked compounds of our top 5% list. Considering the encouraging virtual screening results, we have prioritized the acquisition and the biochemical characterization of derivative as new potential CK1δ inhibitor. In particular, derivative is 10-fold more potent compared with the reference CK1δ inhibitor, 3-[(2,4,6-trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261)., As shown in , inhibition of CK1δ by compound is competitive with respect to the phosphodonor substrate ATP, and a 125nM value has been calculated from linear regression analysis of Lineweaver–Burk double reciprocal plots, which is the lowest reported so far a CK1δ inhibitor. On the other hand, according to a preliminary selectivity study (, ), derivative seems to be a quite specific inhibitor of CK1δ respect the other CK1 isoforms, and also with respect to a small panel of different kinases. In particular according to our homology model, derivative makes a stabilizing interaction between the amino group at the 1-position and the backbone carbonyl of Glu83 in the hinge region (). Moreover, one of the carbonyl groups can interact through an H-bonding with the backbone amido moiety of Leu85. These two interactions are the same generally observed in the binding of SB 203580 hydrochloride moiety of ATP into the kinase active site. On the other hand, another hydrogen-bonding interaction has been detected between the amino group 4-position and the carboxylic group of Asp149. Finally, several hydrophobic interactions (Ile15, Ile23, Ala36, Leu135, Ile147) should be taken into account because they may contribute to stabilize the complex between compound and CK1δ. We can also argue that the right balance of both polar and hydrophobic interactions and the appropriate shape complementarity with the ATP-binding cleft might be ultimately responsible for the appreciable selectivity presented by derivative versus other kinases and in particular against the protein kinase CK2. In fact, the shape topology of the ATP-binding cleft of CK2 is clearly different respect CK1 (data not shown). Vice versa, more intriguing is the observation regarding the appreciable selectivity displayed by derivative against the other CK1 isoforms. In fact, analyzing the sequence alignment of the corresponding kinase domains no crucial mutations are detectable and that would be able to justify the observed isoform selectivity spectrum of derivative . Our assumption is that in this specific case the C-terminal domain of CK1 isoforms could play an important role in the control of the inhibitor’s recognition as well as in the corresponding CK1 basal kinase activity. In particular, the C-terminal domain of CK1α and γ may have a deterrent effect on the interaction with derivative , reducing its inhibitory effects as collected in . This experimental evidence has been already reported also for the CK1 inhibitor, IC261. In fact, as suggested by a recent crystallographic information regarding the isoform CK1γ3 the C-terminal domain can closely approach the ATP-binding cleft directly interacting with the inhibitor (PDB code: 2CHL). Beside derivative , the 1-hydroxy-4-amino-anthraquinone (compound in ) also shows an appreciable inhibitory activity against CK1δ (IC=0.6μM). To directly verify the role of the 1,4-diaminobenzene fragment, the corresponding 1,4-dihydroxy-anthraquinone was also tested as potential CK1δ inhibitor (compound in ). Interestingly, compound is practically inactive against the three isoforms CK1δ, γ1 and α supporting the critical role mediated by the aminobenzene fragment into CK1δ recognition. Indeed, a robust and quantitative structure–activity relationship is underway in our laboratories to explore the possibility of decorating or modifying the 1,4-diamino-anthraquinone moiety increasing the pharmacodynamics of the second generation of these CK1δ inhibitors and, at the same time, improving their pharmacokinetics profiles.