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Coumarin as Attractive Casein Kinase 2 (CK2) Inhibitor Scaffold: An Integrate Approach To Elucidate the Putative Binding Motif and Explain Structure–Activity Relationships (Review)

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Cassein Kinase 2 (CK2)n is a tetrameric protein consist of 4 subunits: 2 alpha subunits (catalytic) and 2 beta subunits (regulatory). The alpha subunit is constitutively active either with or without beta subunit. Beta subunit appears to play role in targeting and substrate recruiting.The catalytic subunit of CK2 may behave as oncogenes. Previously, Chilin et al have performed intensive screening program using conventional and in silico methods to discover the novel potent and selective CK2 inhibitors. The research is then documented in an in-house molecular databse (molecular modeling section/MMS database).

Coumarin is a class of benzopyrones compounds commonly found in dietary and showed antitumor activity. This study observed the X-ray diffraction crystal structure of CK2 in complex with the coumarin derivative (DBC). The crystal then was explored for a linear interaction energy (LIE) study to rationalize the different free energies of binding and the key interaction of all coumarin derivatives.

Synthesize and Kinase Activity Assay of Coumarine Derivates

The study started from synthesis of coumarin analogues (67 analogues). The activity of the compounds inhibiting CK2 was then determined by using phosphorylation assay. The results were then summarized in the table I.

fig 1

Figure I. The structure of coumarin

Table I. The summary of coumarin derivates synthesized and its kinase inhibitory activity

tabl 1

Crystallography

In this research, CK2 alfa subunit from Zea mays was used. CK2 alfa from Zea mays possess identical active site with the human protein, as well as close sequence and structural similarities between both of them. The formation of the quartenary complex dose not influence the conformation of the catalytic subunit, thus the CK2 alfa subunit can be used in design of inhibitors of the holoenzyme. In addition, maize enzyme performed better stability compared with the human CK2. The recombinant CK2 alfa subunit from Zea mays was expressed in E.coli, then was isolated and purified. Crystal of the CK2 alfa complex with DBC were obtained by cocrystallization with the sitting drop vapor diffusion technique and then processed to get the X-ray diffraction data.

DBC binds to CK2 inside the ATP binding site, in a position similar to anthraquinone and (MNA) and xanthenone (MNX). The overall structure of the proteins is not affected by the binding of the inhibitor. The position of the DBC hydroxyl group is almost identical to the analogues function in MNX. This OH group establishes two hydrogen bonds, one with the amine function of the Lys68 side chain and another with a water molecule that is conserved in all deposited CK2 structure. The H-bonding network seems to have crucial role in the recognition process of all phenol-like CK2 inhibitors. This finding showed that DBC performed as ATP competitive inhibitor in CK2. The crystal complex of CK2 and DBC has been documented with PDB ID 2QC6.

Figure 2. The top panels shows DBC bound to the CK2 ATP binding site. The hydroxyl function is involved in two hydrogen bonds (at a distance of 2.8 Å) with Lys68 and the conserved water molecule W1. The top panel shows a superimposition of the two inhibitors DBC (yellow carbon atoms) and MNX (green carbon atoms). Bromine atoms are shown in magenta. Note the identical position of the OH function of DBC and MNX. As reference, water molecule W1 and the zone of the hinge region are indicated.

Figure 2. The top panels shows DBC bound to the CK2 ATP binding site. The hydroxyl function is involved in two hydrogen bonds (at a distance of 2.8 Å) with Lys68 and the conserved water molecule W1. The top panel shows a superimposition of the two inhibitors DBC (yellow carbon atoms) and MNX (green carbon atoms). Bromine atoms are shown in magenta. Note the identical position of the OH function of DBC and MNX. As reference, water molecule W1 and the zone of the hinge region are indicated.

 

Linear Interaction Energy Model and Structure-Activity Relationship

The computational method using Molecular Operating Environment (MOE) was used to analyze the linear interaction energy (LIE) and make the qualitative structure-activity relationship (QSAR) between coumarin derivatives and CK2. From the computational methods, there were some groups important in the activity of coumarin derivates inhibiting CK2 kinase activity.

1. Hydrophobic groups in position 3,4 and 8 interacted through can der walls interactions with the CK2 active site.

2. 7-hydroxyl group performed key interaction with Lys68 and water W1, a water molecule very well conserved in all CK2.

3. The presence of an electron-withdrawing group at the 6 position to phenol OH increase the activity of the compound.

4. The pKa of the 7-hydroxyl group has to be lower than 7.

Reference:

Chilin A., Battistutta R., Bortolato A., Cozza G., Zanatta S., Poletto G., Mazzorana M., Zagotto G., Uriarte E., Guiotto A., Pinna L. A., Meggio F., Moro S. (2008) Coumarin as attractive casein kinase 2 (CK2) inhibitor scaffold: an integrate approach to elucidate the putative binding motif and explain structure-activity relationships. J. Med. Chem. 51, 752–759

 

Kontributor:
Yonika Arum Larasati, S.Farm., Apt.