A Chemical Biology Approach Towards Targeting Understudied Lipid Kinases in Cancer and Ebola

Abstract

Cellular processes are orchestrated in an intricate and highly coordinated fashion in order to facilitate proper metabolism, cell cycle progression, gene expression, and ultimately survival. Phosphoinositides, lipid moieties which exist in tiny proportions within the cell, are examples of important messengers whose complex signaling is controlled in a temporal and spatial manner by kinases and phosphatases, deregulation of which leads to disease. The phosphatidylinositol 5-phosphate 4-kinases (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 PI5P4KA/B/C. 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. PIKfyve, a related understudied lipid kinase, has also been validated as a target in Non-Hodgkin lymphoma and Ebola viral infection. We observed that THZ-P1-2 was also capable of covalently binding to PIKfyve, albeit at a much lower preference than PI5P4K. We reengineered compound selectivity towards PIKfyve and identified a class of compounds from the THZ-P1-2 scaffold exhibiting picomolar-to-nanomolar IC50s. These inhibitors exhibit cellular on-target engagement and vacuolar enlargement, a well-established PIKfyve inhibitory phenotype. Docking/modeling studies and mass spectrometry revealed a similar distant cysteine, Cys1970, to be covalently labeled by these compounds. Lastly, this THZ-family of inhibitors, particularly two top compounds MFH-5-3 and MFH-5-4, exhibit potent antiproliferative activity in lymphoma cell lines and inhibition of Ebola viral infectivity. Taken together, our studies demonstrate that the PI5P4Ks and PIKfyve are tractable targets, with inhibitors serving as useful tools to further interrogate the therapeutic potential of these noncanonical lipid kinases and inform drug discovery campaigns in the context of cancer, Ebola, and potentially other autophagy-dependent diseases.