The Ligand Binding Landscape of Diacylglycerol Kinases

Abstract

SUMMARY Diacylglycerol kinases (DGKs) are integral components of signal transduction cascades that regulate cell biology through ATP-dependent phosphorylation of the lipid messenger diacylglycerol. Methods for direct evaluation of DGK activity in native biological systems are lacking and needed to study isoform-specific functions of these multidomain lipid kinases. Here, we utilize ATP acyl phosphate activity-based probes and quantitative mass spectrometry to define, for the first time, ATP- and small molecule-binding motifs of representative members from all five DGK subtypes. We use chemical proteomics to discover an unusual binding mode for the DGK-alpha (DGKα) inhibitor ritanserin, including interactions at the atypical C1 domain distinct from the ATP binding region. Unexpectedly, deconstruction of ritanserin yielded a fragment compound that blocks DGKα activity through a conserved binding mode and enhanced selectivity against the kinome. Collectively, our studies illustrate the power of chemical proteomics to profile protein-small molecule interactions of lipid kinases for fragment-based lead discovery.

Structure activity relationship studies on Amb639752: toward the identification of a common pharmacophoric structure for DGKα inhibitors

“…We have demonstrated the efficacy of DGKa inhibitors for XLP-1 treatment 8 , while others have proposed them for cancer treatment 22 and to remove immune-checkpoints promoting immune vigilance against cancer 40 . Commercially available DGKa inhibitors are limited by poor specificity 25,41 and pharmacokinetic 20 . The CU-3 molecule described by other features a noteworthy activity and specificity but its reactive chemical structure make unlikely an in vivo use 30 .…”

Activity‐Based Kinome Profiling Using Chemical Proteomics and ATP Acyl Phosphates

“…Upload .mzXML and .mzIdent files into Skyline-daily software (Schilling et al, 2012) and perform data analysis as previously described (Franks et al, 2017). Upload .mzXML and .mzIdent files into Skyline-daily software (Schilling et al, 2012) and perform data analysis as previously described (Franks et al, 2017).…”

“…47. Upload .mzXML and .mzIdent files into Skyline-daily software (Schilling et al, 2012) and perform data analysis as previously described (Franks et al, 2017).…”

“…Traditional in vitro assays have been implemented to study kinase activity, including, e.g., radiochemical ATP assays (Hastie, McLauchlan, & Cohen, 2006;Karra, Stippec, & Cobb, 2017) and coupled bioluminescent assays (ADP-Glo; Tai, Bojjireddy, & Balla, 2011;Franks et al, 2017). Advantages of these assays include the potential for medium-to highthroughput analyses.…”

“…The probes bind to the kinase active site, where the acyl phosphate reactive group is placed in proximity to the ε-amino group of conserved lysines involved in ATP binding. Probe-labeled kinases can be visualized by SDS-PAGE with a streptavidin-fluorophore (Franks, Campbell, Purow, Harris, & Hsu, 2017;McCloud et al, 2018), or probe-modified active-site peptides can be enriched by avidin affinity chromatography and identified by tandem liquid chromatography-mass spectrometry (LC-MS/MS) analyses (Patricelli et al, 2007(Patricelli et al, , 2011. 1).…”