An unconventional gatekeeper mutation sensitizes inositol hexakisphosphate kinases to an allosteric inhibitor

Reviewer #1 (Public Review):

The manuscript by Aguirre et al. describes an elegant approach for developing selective inhibitors of inositol hexakisphosphate kinases (IP6Ks). There are 3 IP6K isozymes (IP6K1-3) in humans, which catalyze the synthesis of inositol pyrophosphates. The lack of isozyme-selective inhibitors has hampered efforts to understand their individual physiological roles. While several inhibitors of IP6Ks have been described, their either lack isozyme selectivity or inhibit other kinases. To address this gap, Aguirre et al. used an analog-sensitive approach, which involves the identification of a mutant that, in an ideal world, doesn't impact the activity of the enzyme but renders it sensitive to an inhibitor that is absolutely selective for the engineered (analog-sensitive) enzyme. Initially, they generated the canonical gatekeeper (Leu210 in IP6K1) mutations (glycine and alanine); unfortunately, these mutations had a deleterious effect on the enzymatic activity of IP6K1. Interestingly, mutation of Leu210 to a valine, a subtly smaller amino acid, didn't affect enzymatic activity. The authors then designed a clever high-throughput assay to identify compounds that show selectivity for L210V IP6K1 versus WT IP6K1. The assay monitors the reverse reaction catalyzed by IP6Ks, monitoring the formation of ATP using a luminescence-based readout. After validating the screen, the authors screened 54,912 compounds. After culling the list of compounds using several criteria, the authors focused on one particular compound, referred to as FMP-201300. FMP-201300 was ~10-fold more potent against L210V IP6K1 compared to WT IP6K1. This selectivity was maintained for IP6K2. Mechanistic studies showed that FMP-201300 is an allosteric inhibitor of IP6K1. The authors also did a small SAR campaign to identify key functional groups required for inhibition.

Overall, this manuscript describes a unique and useful strategy for developing isozyme-selective inhibitors of IP6Ks. The serendipitous finding that subtle changes to the gatekeeper position can sensitize the IP6K1 mutant to allosteric inhibitors will undoubtedly inspire other analog-sensitive inhibitor studies. The manuscript is well-written and the experiments are generally well-controlled.

Reviewer #2 (Public Review):

Fiedler and colleagues set out to establish an analog-sensitive approach for selective inhibition of the mammalian IP6K isozymes. IP6Ks are inositol hexakisphosphate kinases, and the authors found that the classic glycine and alanine gatekeeper mutation (established by Kevan Shokat as the "bump and hole approach" for various protein kinases) resulted in limited catalytic efficiency. Therefore, the authors decided to use a leucine-to-valine mutation, which did not affect kinase activity, but, unfortunately, was less amenable to any of the well-established analog-sensitive kinase inhibitors such as PP1 and naphthyl-PP1. To overcome this limitation, the authors performed an elegant HT screen and identified a benzimidazole-based mutant-selective small molecule inhibitor. A focused SAR analysis combined with detailed kinetic studies revealed the hit molecule FMP-201300 as an allosteric inhibitor of IP6K mutants. While co-crystallization experiments failed, the authors used high-end HDX-MS measurements to gain insight into the structural and conformational determinants of mutant selectivity.

Overall, this is an excellent study of high quality. The identified FMP-201300 has the potential for further compound and probe development. My only minor comment is that the authors could spend more time discussing the proposed allosteric binding mode of FMP-201300 and provide more detailed figures to highlight the proposed interactions with the protein and the conformational changes that must ultimately take place to accommodate the allosteric modulator. I appreciate that the co-crystallization experiments did not yield bound inhibitor structures, but perhaps the authors could consider MD simulations to complete their study.