DISCOVERY, SYNTHESIS, AND CHARACTERIZATION OF PURINE BASED ISOFORM SELECTIVE INHIBITORS OF INOSITOL HEXAKISPHOSPHATE KINASE 1

Diphosphoinositol phosphates and the enzymes that synthesize them have been increasingly studied as therapeutically interesting molecules. The intercellular pool of the major mammalian diphosphoinositol phosphate, diphosphoinositol pentakisphosphate (5PP-IP5), is formed from inositol hexakisphosphate (IP6) by a family of three inositol hexakisphosphate kinases named IP6K1, IP6K2, and IP6K3. Isoform specific knock out studies in animals have revealed distinct physiological roles for each kinase despite the fact that they catalyze the same reaction. Each kinase has unique subcellular localization, tissue distribution, and network of protein-protein interactions, which allow for the different knockout phenotypes observed. Due to the functional promiscuity of the IP6Ks, it is difficult to know how much of the phenotypes observed in the knock-out studies are driven by a loss of catalytic activity or by the disruption of an important protein-protein interaction. A pan-IP6K inhibitor (N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl) purine, TNP) has been reported and used to inhibit the catalytic activity of IP6K while leaving the scaffolding function intact; however, TNP suffers from poor potency, undesirable physiochemical properties, and its pan-IP6K inhibitory profile. Indeed, it is not possible to use TNP to study the effects of one IP6K isoform in a context where the others are also present. The development of an isoform selective IP6K inhibitor became a priority for our group. Yet, at the conception of this work, there was no assay suitable for measuring IP6K activity in a high enough throughput needed to screen thousands of compounds or support a medicinal chemistry effort. To aid in this effort we developed the first high-throughput homogenous coupled bioluminescence assay for measuring IP6K activity in a 384-well format (Z’ = 0.62±0.05). Using this assay, we commenced a medicinal chemistry effort focused on systematically varying the N2- and N6 positions off of a purine core. The lead compound (24) was identified after multiple rounds of SAR and was found to selectively inhibit IP6K1 over IP6K2 or IP6K3 using biochemical and biophysical approaches. It also boasts increased solubility and IP6K1 potency over TNP. This new compound is a useful tool for additional assay development and exploration of IP6K1 specific cellular biology