Amazingly, compound 24 gained back some PI5P4Kactivity with an IC50 of 26 activity, whereas it is unfavorable in the context of PI5P4Kactivity, which is in contrast to the high similarity of the two binding sites (Supporting Figure 7). Similarly, compounds 26 and 27 introduce either a dimethylamine or a methoxy group, respectively, in the para-position of the phenyl moiety on the right-hand side. with an indole, a potent pan-PI5P4K inhibitor with excellent kinome-wide selectivity. Finally, we employed isothermal cellular thermal shift assays (CETSAs) to demonstrate the effective cellular target engagement of PI5P4Kand -by the inhibitors in HEK 293T cells. Graphical Abstract INTRODUCTION Phosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2), are key players in many cell-regulating and -signaling processes. Inositide function is controlled by lipid kinases, which alter their phosphorylation pattern, thus generating a range of signaling molecules with different downstream effects. Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) represent a family of these lipid kinases, which have been shown to phosphorylate phosphatidylinositol 5-phosphate (PI5P) on its 4-position to produce PI-4,5-P2.1 In healthy cells, PI5P4Ks play a role in regulating cellular metabolism, stress response, and immunological processes,2?7 while the abnormal function of these lipid kinases has been linked to diseases, such as diabetes and cancer.8?11 As a result, PI5P4K inhibitors pose potentially important therapeutic agents, for example, in oncology. To date, several small molecule PI5P4K inhibitors with low- or sub-micromolar potency have been reported in the literature,12?17 including our recently reported phenylamino pyrimidine-based, covalent pan-PI5P4K inhibitors, THZ-P1C2 and compound 32 (labeled compound 30 in the original publication; Supporting Figure 1).16,17 Here, we report our efforts to find chemically distinct PI5P4K inhibitors that can serve as chemical probes for further drug development efforts. We focused on developing reversible inhibitors that would not be susceptible to potentially acquired resistance through mutation of the active site cysteine residue, which is modified by irreversible PI5P4K inhibitors.16,17 A prominent example of this type of resistance mechanism is the EGFR C797S mutation that develops in nonsmall cell lung cancer following treatment with irreversible small molecule EGFR Rabbit Polyclonal to APLP2 (phospho-Tyr755) inhibitor AZD9291 and renders tumors resistant.18 To find novel, reversible PI5P4K inhibitor scaffolds, we performed a high-throughput WH 4-023 screen of our internal kinase inhibitor library, containing approximately 6, WH 4-023 000 structurally diverse compounds developed and synthesized in our lab against PI5P4Kkinase activity. This screen identified potent hit compound CVM-05C002 (IC50 270 nM). CVM-05C002 contains a distinct (inhibition have been previously described,12 we adapted this WH 4-023 assay to establish a protocol for a miniaturized high-throughput screen. We optimized the assay to perform in a 384-well format with respect to PI5P4Kenzyme concentration and ADP-Glo reagent volumes to obtain a robust signal-to-background ratio, a stable inhibitory activity (IC50 0.27 activity was not due to compound interactions with assay reagents. This was done by counterscreening at a fixed ADP/ATP concentration ratio representing 20% conversion but excluding the PI5P4Kenzyme, as well as the lipid substrate, confirming no assay interference with CVM-05C002 (Figure 1B). Open in a separate window Figure 1. (A) Scatter plot of high-throughput screen for the PI5P4Kactivity of in-house small molecule library with ~6000 compounds (shown are hits at <120% DMSO control). The results of two replicates are plotted against each other (normalized to DMSO). Negative controls (DMSO) are shown in dark gray, CVM-05C002 is depicted in green, and the 10% DSMO control cutoff (90% inhibition) is labeled. (B) ConcentrationC response curves of CVM-05C002, including counterscreen results. (C) Structure of identified lead compound, CVM-05C002. Interestingly, several structurally similar molecules to CVM-05C002 were present in the library, while only four analogues were found among the top hits identified in the high-throughput screen. Subsequent testing of these hits in our ADP-Glo-based PI5P4Kassay revealed that these analogues are ~30-fold less potent than CVM-05C002, or more (data not shown). Furthermore, the closest analogues of CVM-05C002, only differing in the 3,5-substituted pyridine moiety of the hit compound, showed strong PIM kinase off-targets (Supporting Table 1). This was not surprising since similar scaffolds have been shown to be potent PIM kinase inhibitors.26 However, since CVM-05C002 was the only compound of this scaffold without any PIM kinase off-target activity, carrying a unique 3,5-substituted pyridine moiety, while showing potent on-target activity, we were highly motivated to further optimize this screening hit and explore its SAR. Finally, knowing that the scaffold of CVM-05C002 could potentially have nonspecific effects due to its similarity to compounds with reported reactivity,22 we performed mass spectrometry (MS) analysis of recombinant PI5P4Kand -showed no detectable reaction after 2 h, while the was cocrystallized in complex with CVM-05C002 at a 1.7 ? resolution, confirming a noncovalent binding mode of the inhibitor in the active site of the lipid kinase (Figure 2) (PDB-ID: 6UX9). Open in a separate window Figure 2. (A) Binding site of cocrystal structure of PI5P4Kwith bound inhibitor CVM-05C002 (dark gray). Key hydrogen bond interactions.