Abstract
The discovery of several hundred different protein kinases involved in highly diverse cellular signaling pathways is in stark contrast to the much smaller number of known modulators of cell signaling. Of these, the H series protein kinase inhibitors (1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H8) N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89)) are frequently used to block signaling pathways in studies of cellular regulation. To elucidate inhibition mechanisms at atomic resolution and to enable structure-based drug design of potential therapeutic modulators of signaling pathways, we determined the crystal structures of corresponding complexes with the cAPK catalytic subunit. Complexes with H7 and H8 (2.2 A) and with H89 (2.3 A) define the binding mode of the isoquinoline-sulfonamide derivatives in the ATP-binding site while demonstrating effects of ligand-induced structural change. Specific interactions between the enzyme and the inhibitors include the isoquinoline ring nitrogen ligating to backbone amide of Val-123 and an inhibitor side chain amide bonding to the backbone carbonyl of Glu-170. The conservation of the ATP-binding site of protein kinases allows evaluation of factors governing general selectivity of these inhibitors among kinases. These results should assist efforts in the design of protein kinase inhibitors with specific properties.
Highlights
The discovery of several hundred different protein kinases involved in highly diverse cellular signaling pathways is in stark contrast to the much smaller number of known modulators of cell signaling
There is evidence that isoquinolinesulfonyl inhibitors may be promising in this field (9 –14)
In all H inhibitor co-crystals the enzyme is in an essentially closed conformation (Fig. 2) with two differences: a very small rotation of the N lobe by 2° toward a slightly more open conformation compared with the AMPPNP-cAPK1⁄7PKI structure, and a large variability of the conformation of the first two -strands in the region of the glycine flap (Gly-50 to Val-57)
Summary
The H series protein kinase inhibitors (1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H8) N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89)) are frequently used to block signaling pathways in studies of cellular regulation. The conservation of the ATP-binding site of protein kinases allows evaluation of factors governing general selectivity of these inhibitors among kinases. These results should assist efforts in the design of protein kinase inhibitors with specific properties. The crystal structures of kinase bound inhibitor molecules described here show the mode of inhibitory action and the factors governing selectivity and will provide a firm basis for the design of new protein kinase inhibitors
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