Mechanism of BIP-4 mediated inhibition of InsP(3)Kinase-A

Overexpression of the neuronal InsP(3)kinase-A increases malignancy of different tumor types. Since InsP(3)kinase-A highly selectively binds Ins(1,4,5)P(3), small molecules competing with Ins(1,4,5)P(3) provide a promising approach for the therapeutic targeting of InsP(3)kinase-A. Based on this consideration, we analyzed the binding mechanism of BIP-4 (2-[3,5-dimethyl-1-(4-nitrophenyl)-1H-pyrazol-4-yl]-5, 8-dinitro-1H-benzo[de]isoquinoline-1,3(2H)-dione), a known competitive small-molecule inhibitor of Ins(1,4,5)P(3). We tested a total of 80 BIP-4 related compounds in biochemical assays. The results of these experiments revealed that neither the nitrophenyl nor the benzisochinoline group inhibited InsP(3)kinase-A activity. Moreover, none of the BIP-4 related compounds competed for Ins(1,4,5)P(3), demonstrating the high selectivity of BIP-4. To analyze the inhibition mechanism of BIP-4, mutagenesis experiments were performed. The results of these experiments suggest that the nitro groups attached to the benzisochinoline ring compete for binding of Ins(1,4,5)P(3) while the nitrophenyl group is associated with amino acids of the ATP-binding pocket. Our results now offer the possibility to optimize BIP-4 to design specific InsP(3)Kinase-A inhibitors suitable for therapeutic targeting of the enzyme.