Chemoproteomic Selectivity Profiling of PIKK and PI3K Kinase Inhibitors

Abstract

Chemical proteomic approaches utilizing immobilized, broad-selective kinase inhibitors (Kinobeads) have proven valuable for the elucidation of a compound's target profile under close-to-physiological conditions and often revealed potentially synergistic or toxic off-targets. Current Kinobeads enrich more than 300 native protein kinases from cell line or tissue lysates but do not systematically cover phosphatidylinositol 3-kinases (PI3Ks) and phosphatidylinositol 3-kinase-related kinases (PIKKs). Some PIKKs and PI3Ks show aberrant activation in many human diseases and are indeed validated drug targets. Here, we report the development of a novel version of Kinobeads that extends kinome coverage to these proteins. This is achieved by inclusion of two affinity probes derived from the clinical PI3K/MTOR inhibitors Omipalisib and BGT226. We demonstrate the utility of the new affinity matrix by the profiling of 13 clinical and preclinical PIKK/PI3K inhibitors. The large discrepancies between the PI3K affinity values obtained and reported results from recombinant assays led us to perform a phosphoproteomic experiment showing that the chemoproteomic assay is the better approximation of PI3K inhibitor action in cellulo. The results further show that NVP-BEZ235 is not a PI3K inhibitor. Surprisingly, the designated ATM inhibitor CP466722 was found to bind strongly to ALK2, identifying a new chemotype for drug discovery to treat f ibrodysplasia ossif icans progressiva.

Drug mechanism‐of‐action discovery through the integration of pharmacological and CRISPR screens

“…Conversely, two pan-PI3K inhibitors (buparlisib and omipalisib) displayed no significant association with any PI3K isoform (Dataset EV5), consistent with its lack of isoform specificity and potential polypharmacology. Interestingly, MTOR and pan-PI3K inhibitor, dactolisib, had significant associations with RPTOR and MTOR but none with PI3K isoforms (Dataset EV5), consistent with recently reported greater specificity for inhibition of the MTOR complex (Reinecke et al, 2019). Similarly, we observed that selective EGFR inhibitors cetuximab, erlotinib and gefitinib (Fig 3-EGFR inhibitors panel) were associated with EGFR but not ERBB2, whereas sapatinib, afatinib and poziotinib (Fig 3 -ERBB2; EGFR inhibitors panel) were all associated with both EGFR and ERBB2.…”

“…While informative, this approach fails to recapitulate the native context of the full‐length protein in cells which could influence true drug activity, nor does it identify potential non‐kinase off‐target effects. Cellular‐based approaches to investigate mechanism‐of‐action include transcriptional profiling following drug treatment of cells, chemical proteomics approaches such as kinobeads (Bantscheff et al , ; Médard et al , ) and cellular thermal shift assay (Savitski et al , ) to measure drug–protein interactions, and multiplexed imaging or flow‐cytometry to measure multiple cellular parameters upon drug treatment (Li et al , ; Subramanian et al , ; Reinecke et al , ). Despite the utility of these different approaches, gaining a full picture of drug mechanism‐of‐action, particularly in cells, remains a challenge and new approaches would be beneficial.…”

Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC–MS/MS

“…Kinobeads, FLAG based APs and BioID pull-downs. Kinobeads selectivity profiling of AT-9283 was performed with the standard protocol 49 . The K-562 (ATCC, CCL-243), COLO-205 (ATCC, CCL-222) and MV-4-11 (ATCC, CRL-9591) cells were cultured in RPMI 1640 medium (Biochrom GmbH) supplemented with 10% (v/v) FBS (Biochrom GmbH) and 1% (v/v) antibiotics.…”

Target deconvolution of HDAC pharmacopoeia highlights MBLAC2 as common off-target

“…For instance, we and others have identi ed novel targets of kinase inhibitors within (e.g. activin receptor ALK2 for the ATM inhibitor CP466722) 60 or outside the target class (e. g. ferrochelatase for the BRAF inhibitor Vemurafenib and others) 61 . The current study, uncovered ve such cases for HDAC inhibitors, notably ALDH2 (n=9 drugs, most potent pK d app = 6.5), GATD3A (n=7 drugs, most potent pK d app = 5.8), ISOC1 (n=7 drugs, most potent pK d app = 6.8), ISOC2 (n=8 drugs, most potent pK d app = 8.2) and MBLAC2 (n=24 drugs, most potent pK d app = 7.6).…”