Structure–Activity Relationship Study of Covalent Pan-phosphatidylinositol 5-Phosphate 4-Kinase Inhibitors

Abstract

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are important molecular players in a variety of diseases, such as cancer. Currently available PI5P4K inhibitors are reversible small molecules, which may lack selectivity and sufficient cellular on-target activity. In this study, we present a new class of covalent pan-PI5P4K inhibitors with potent biochemical and cellular activity. Our designs are based on THZ-P1-2, a covalent PI5P4K inhibitor previously developed in our lab. Here, we report further structure-guided optimization and structure–activity relationship (SAR) study of this scaffold, resulting in compound 30, which retained biochemical and cellular potency, while demonstrating a significantly improved selectivity profile. Furthermore, we confirm that the inhibitors show efficient binding affinity in the context of HEK 293T cells using isothermal CETSA methods. Taken together, compound 30 represents a highly selective pan-PI5P4K covalent lead molecule.

Pharmacological inhibition of PI5P4Kα/β disrupts cell energy metabolism and selectively kills p53-null tumor cells

“…While this paper was under review, two new classes of chemical probes targeting PI5P4K were reported (62)(63)(64). Although these inhibitors appear to have weaker affinities than CC260 in vitro, one of them can react covalently with a free cysteine adjacent to the lipid kinase's active site, which should enhance its effectiveness and selectivity.…”

Targeting the PI5P4K Lipid Kinase Family in Cancer Using Covalent Inhibitors

“…JNK-IN-7 was found to inhibit the kinase activity of PI5P4Ka/b/g in a radiometric thin-layer chromatography (TLC) assay (Figures S1B and S1C), and mass spectrometry revealed labeling of cysteine residues located on a disordered loop that is not observed in the available PI5P4K crystal structures (Figures S1D and S1E). We pursued a focused medicinal chemistry campaign guided by biochemical kinase assays and cellular pulldowns to optimize the potency and selectivity of phenylamino-pyrimidine, which resulted in the development of THZ-P1-2 (Figure 1A) (full structure-activity relationship study described in Manz et al, 2019). THZ-P1-2 demonstrated inhibition of PI5P4Ka kinase activity (Figure 1B), with a half maximal inhibitory concentration (IC 50 ) of 190 nM using an ADP-Glo (Promega) bioluminescence assay.…”

“…Originally inspired by the imatinib core structure, this ''privileged'' kinase scaffold served as a promising starting point for developing a selection of analogs with chemical modifications with which to explore structure-activity relationships. Among this panel of molecules (Manz et al, 2019), we found a critical feature resulting in superior potency to be a switch from a 2,4-pyrimidine to a 4,6-pyrimidine hinge-interacting motif. We established THZ-P1-2's inhibitory activity with an apparent IC 50 of 190 nM on PI5P4Ka in a fixed time point assay, the isoform with the highest kinase activity and thus easiest to analyze using an ATPase assay (ADP-Glo).…”

Crucial Players for Inter-Organelle Communication: PI5P4Ks and Their Lipid Product PI-4,5-P2 Come to the Surface

“…All things considered, in the recent past, PI5P4Ks and their product PI-4,5-P 2 have risen from insignificance to being without a doubt one of the key metabolic sensors and regulators within the cell as well as pivotal players for interorganelle communication necessary for cell survival. With drugs being developed against these kinases (Davis et al, 2013;Clarke et al, 2015;Al-Ramahi et al, 2017;Kitagawa et al, 2017;Manz et al, 2020;Sivakumaren et al, 2020;Chen et al, 2021), targeting them in the near future in various diseases is looking brighter.…”