Targeting the PI5P4K lipid kinase family in cancer using novel covalent inhibitors

Abstract

The PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of potent, specific small molecules available. Here we present the discovery and characterization of a novel pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4K//. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4K's reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders.

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

“…As previously reported, the selectivity profiling of THZ-P1-2 with KINOMEscan has shown that this compound exhibits cross activity with the type I lipid kinase PIKFYVE, as well as several protein kinases including BRK, TYK2, and Abl. We next questioned whether the bicyclic pyrrolo­[2,3- d ]­pyrimidine scaffold on compound 30 could dial out these off-targets, making it a more selective PI5P4K inhibitor.…”

“…Inspired by the SAR information we obtained above, as well as the cocrystal structure of our inhibitor THZ-P1-2 with PI5P4Kα, we cyclized the pyrimidine ring from carbon-4 and -5 to yield a bicyclic pyrrolo­[2,3- d ]­pyrimidine and introduced a 4-methylphenyl, a favored residue (e.g., compound 9 ), attached to the N atom of the pyrrolo ring system. We hypothesized that this could potentially improve the overall selectivity of the scaffold while maintaining on-target activity.…”

“…Our recently reported irreversible inhibitor of PI5P4Ks, THZ-P1-2, displays submicromolar biochemical potency against PI5P4Kα kinase activity. 17 Although the THZ-P1-2/ PI5P4Kα cocrystal structure (PDB-ID: 6OSP) did not resolve the loop that contains the covalently targeted cysteine (Cys293), the structure clearly revealed key noncovalent interactions, including two hydrogen bonds between the aminopyrimidine, as well as the benzamide carbonyl and Asn198, a hydrogen bond forming between the pyrimidine and Val199, and T-shaped π-stacking between the phenylenediamine group and Phe134 in the p-loop (Figure 2). In addition, Phe134 and Phe200 form a narrow passage encaging the phenylenediamine of THZ-P1-2 (Supporting Figure 7).…”

Discovery and Structure–Activity Relationship Study of (Z)-5-Methylenethiazolidin-4-one Derivatives as Potent and Selective Pan-phosphatidylinositol 5-Phosphate 4-Kinase Inhibitors

“…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-P1-2 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.…”

“…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 inhibitor AZD9291 and renders tumors resistant. 18 To find novel, reversible PI5P4K inhibitor scaffolds, we performed a high-throughput screen of our internal kinase inhibitor library, containing approximately 6,000 structurally diverse compounds developed and synthesized in our lab against PI5P4Kα, using an ADP-Glo assay to measure the PI5P4Kα kinase activity.…”