Structural features underlying the selectivity of the kinase inhibitors NBC and dNBC: role of a nitro group that discriminates between CK2 and DYRK1A

Abstract

8-hydroxy-4-methyl-9-nitrobenzo(g)chromen-2-one (NBC) has been found to be a fairly potent ATP site-directed inhibitor of protein kinase CK2 (Ki = 0.22 μM). Here, we show that NBC also inhibits PIM kinases, especially PIM1 and PIM3, the latter as potently as CK2. Upon removal of the nitro group, to give 8-hydroxy-4-methyl-benzo(g)chromen-2-one (here referred to as "denitro NBC", dNBC), the inhibitory power toward CK2 is almost entirely lost (IC(50) > 30 μM) whereas that toward PIM1 and PIM3 is maintained; in addition, dNBC is a potent inhibitor of a number of other kinases that are weakly inhibited or unaffected by NBC, with special reference to DYRK1A whose IC(50) values with NBC and dNBC are 15 and 0.60 μM, respectively. Therefore, the observation that NBC, unlike dNBC, is a potent inducer of apoptosis is consistent with the notion that this effect is mediated by inhibition of endogenous CK2. The structural features underlying NBC selectivity have been revealed by inspecting its 3D structure in complex with the catalytic subunit of Z. mays CK2. The crucial role of the nitro group is exerted both through a direct electrostatic interaction with the side chain of Lys68 and, indirectly, by enhancing the acidic dissociation constant of the adjacent hydroxyl group which interacts with a conserved water molecule in the deepest part of the cavity. By contrast, the very same nitro group is deleterious for the binding to the active site of DYRK1A, as disclosed by molecular docking. This provides the rationale for preferential inhibition of DYRK1A by dNBC.

Synthesis and Properties of a Selective Inhibitor of Homeodomain–Interacting Protein Kinase 2 (HIPK2)

“…It should be noted in this connection that the concentration required for half maximal inhibition is two orders of magnitude higher in cells than it is in vitro (50 µM vs 0.33 µM). This is not unusual among protein kinase inhibitors [42] as exemplified elsewhere [30], [35], [43] and may be accounted for by massive sequestration of lipophilic compounds to cellular structures and to the fact that ATP competitive inhibitors (such as TBID) have to cope with a very high ATP concentration (in the mM range) within the cell.…”

The Selectivity of CK2 Inhibitor Quinalizarin: A Reevaluation

“…All the recombinant α , α ′, and β subunits of CK2 were purified as described in [ 34 , 35 ]. The source of all of the other protein kinases used for selectivity profiling is described in [ 36 ].…”

Recent Advances in the Design, Synthesis, and Biological Evaluation of Selective DYRK1A Inhibitors: A New Avenue for a Disease Modifying Treatment of Alzheimer’s?

“…71 Benzocoumarin 5a (dNBC, Figure 3) has been recently reported as a DYRK1A inhibitor (IC 50 0.60 μM, [ATP] = 20 μM). 73 Authors observe that a 9-substituted nitro functionality (NBC, 5b) is detrimental to DYRK1A activity (IC 50 15.0 μM, [ATP] = 20 μM), yet promotes activity for CK2 (IC 50 5b 0.37 μM vs 5a 32.34 μM), PIM kinase family members, of note PIM3 (IC 50 5b 0.34 μM vs 5a 2.05 μM), and PKB β (IC 50 5b 0.78 μM vs 5a > 30 μM). Indeed, structure based modeling efforts with CK2 suggest 5a is likely buried more deeply than 5b into the binding cleft of DYRK1A where 5a interacts with both the hinge region and the phosphate binding region of the ATP binding site.…”

“…This observation is consistent with the in vitro activity observed for NBC 5b and dNBC 5a. 73 The reader is referred to the original reference containing a detailed depiction of the binding modes of 5a and 5b for further clarification.…”