Figure 1.
Structures of all possible halogenated derivatives of benzotriazole.
Table 1.
Experimental values for triazole proton dissociation (pKa), aqueous solubility (Cw), and inhibitory activity (IC50) against CK2α compared with molecular volumes of brominated Bt derivatives, ab initio derived free energies of proton dissociation (ΔGdiss), free energies of solvation of the anionic forms, free energy of binding to CK2α estimated with the aid of Autodock for ligands both in neutral and monoanionic state (ΔGbind), average ligand movement upon 3 ns Molecular Dynamics of the complex with CK2α in aqueous solution (RMFS), and the displacement between average ligand location estimated from 3 ns MD simulations in aqueous medium from the location of TBBt in the crystal structure with CK2α (RMSD).
Figure 2.
Inhibitory activities (IC50) of brominated Bt derivatives predicted on the basis of: (A) Vmol and experimental pKa; (B) ab initio derived ΔGsolv(anion) and ΔGdiss free energies; and (C) autodock-derived free energy of binding (ΔGbind).
All relations point to predominance of hydrophobic interactions (Vmol or ΔGsolv(anion)), accompanied by protonation of the anionic form of the ligand upon binding to CK2α (pKa or ΔGdiss) (see text for details).
Figure 3.
Location of all nine halogenated Bt derivatives in complex with CK2α.
For each ligand the average location determined from the 3 ns trace of Molecular Dynamics performed in the presence of explicit aqueous solvent is presented in relation to X-ray structure of CK2α (pdbj91). All ligands were found to bind in the same orientation (see panel A), in the position almost identical to that found for TBBt in the crystal structure of the complex with CK2α (see panel B, TBBt, from PDB, in magenta and putative location of Bt in green). The lowest-energy structures identified in 3 ns MD traces are presented in Figure S3.
Figure 4.
Distribution of short halogen-acceptor (O, N, S, π system) contacts identified in 21 accessible structures of complexes of CK2α with halogenated ligands.
The Gaussian cumulative distribution was fitted to the crystallographic data for halogen to donor distance (solid line in panel A) and, according to the Anderson-Darling test, experimental data up to 3.7 Å agrees with a normal distribution (panel B). However, pairs separated by more than 3.7 Å are overrepresented, clearly limiting the maximal distance for eventual halogen-bonding interactions to the sum of donor and acceptor VdW radii. Note that an isolated water molecule (red triangles in panels A, C) is an equally favorable acceptor to the protein (O, N, S, π-electrons). The cumulative distribution of the experimental data is visibly better represented by a bi-normal distribution (panel C), in which the contribution of an additional narrow peak represents putative halogen-bonding (panel D). This is additionally supported by the distribution of angles X…Acc-C and C-X…Acc, which, for short halogen-acceptor distances, are substantially restricted to the regions favoring halogen bond formation (see also Figure S2).
Figure 5.
Schematic representation of the effects of stepwise bromination of the benzene ring of benzotriazole (Bt), including interdependence of the molecular volume (A) and pKa (B) of the products with their IC50 for inhibition of protein kinase CK2α: (a) Green arrows follow sequential bromination of the central vicinal C(5)/C(6) atoms, leading to a moderate decrease of pKa and a large decrease in IC50; (b) Red arrows illustrate the lesser effects of bromination of the peripheral C(4)/C(7) atoms, resulting in a significant decrease of pKa, with virtually no gain in inhibitory activity; (c) Black lines link products with the same number of bromine atoms, but substantially differing in pKa and IC50, culminating in the di- and tri-brominated derivatives with inhibitory activities comparable to that of TBBt.
Note that the parent Bt (pKa 8.56), which is not an inhibitor (IC50>2 mM), is located outside the diagrams.