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Published in final edited form as: Curr Opin Chem Biol. 2021 Jul 23;65:101–108. doi: 10.1016/j.cbpa.2021.06.007

Advances in chemical proteomic evaluation of lipid kinases – DAG kinases as a case study

Timothy B Ware 1, Ku-Lung Hsu 1,2,3,4,*
PMCID: PMC8671151  NIHMSID: NIHMS1720655  PMID: 34311404

Abstract

Advancements in chemical proteomics and mass spectrometry lipidomics are providing new opportunities to understand lipid kinase activity, specificity and regulation on a global cellular scale. Here, we describe recent developments in chemical biology of lipid kinases with a focus on those members that phosphorylate diacylglycerols. We further discuss future implications of how these mass spectrometry-based approaches can be adapted for studies of additional lipid kinase members with the aim of bridging the gap between protein and lipid kinase-focused investigations.

Introduction

Kinases are among the largest and most diverse class of proteins with 500+ members that constitute ~2% of the human genome [1,2]. Biochemically, kinases catalyze the transfer of a phosphate group to protein [3] and small molecule [4] substrates using adenosine triphosphate (ATP) as the preferred substrate [5]. The reversible phosphorylation of proteins by kinases – and complementary dephosphorylation by phosphatases – on serine/threonine [6] and tyrosine [7,8] residues can alter protein conformation and activation, subcellular localization, and protein-protein interactions [9,10]. Thus, kinases function as molecular switches [11] to regulate cell biology through post-translational modification of signaling proteins [10]. Given their role in cancer, inflammatory, and neurodegenerative diseases, kinases are also prominent drug targets [12].

Historically, kinases that phosphorylate proteins have been the focus of basic and therapeutic investigations, with a few notable exceptions. Consequently, the available biochemical and pharmacological tools have been tailored for analysis of protein kinases to probe their function in cell biology. The methods to study kinases that phosphorylate small molecule substrates has lagged in development by comparison. The diversity of small molecules that can serve as kinase substrates is vast and not easily predicted from genomic analyses. Several metabolic kinases catalyze phosphate transfer in membrane environments that are not easily assayed by conventional methods. Advancements in chemical proteomics [13–15] and mass spectrometry lipidomics [16] are providing exciting opportunities to understand lipid kinase activity, specificity and regulation on a global cellular scale. Here, we describe recent developments in chemical biology of lipid kinases with a particular focus on members that phosphorylate diacylglycerols.

The mammalian lipid kinome

Lipid kinases catalyze the ATP-dependent phosphorylation of hydrophobic or amphipathic small molecules. Lipids in nature are chemically diverse [16], which suggests that lipid kinases would be different in protein topology based on substrate preference. A phylogenetic analysis of lipid kinases could provide a visual tool to compare molecular features based on primary protein sequence. A phylogenetic ‘tree’ of protein kinases has served as an important tool for visualizing functional relationships and inhibitor binding across the kinome [13,14,17]. Here, we provide a complementary phylogenetic dendrogram of lipid kinases as shown in Figure 1 and Table 1.

Figure 1. A phylogenetic tree of the lipid kinome.

Figure 1.

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Phylogenetic analysis of human lipid kinases was performed by first identifying members using the GeneOntology (GO) term for ‘lipid phosphorylation’(GO:0046834) followed by inclusion of entries found on UniProtKB/Swiss-Prot (i.e. reviewed entries). After manual exclusion of members devoid of lipid kinase activity (either from a published report or lack of the lipid kinase domain), multiple sequence alignment of whole protein sequences was performed to generate the lipid kinase phylogenetic tree. Additional information on the individual members is provided in Table 1. In total, 37 lipid kinases that regulate phosphorylation of glycerolipid-, inositol-, and sphingosine-containing metabolites were identified. Numbers in parentheses for DGKs denote the five groups for organizing isozymes in this family based on structural features. Multiple sequence alignments were performed using MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle/) on whole protein FASTA sequences available from https://www.uniprot.org/. Inference of evolutionary history used to generate the phylogenetic tree was performed using the MEGA-X program (https://www.megasoftware.net/) following implementation of the maximum likelihood (ML) method and Whelan and Goldman (WAG) model. All positions with less than 95% site coverage were eliminated, that is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. The scale bar represents the branch length measured in the number of substitutions per site. The current version is updated from a previous phylogenetic analysis of lipid kinases [17].

Table 1. Compendium of the lipid kinase superfamily.

Proteins from the lipid kinases tree in Figure 1 are listed with information on topology, structure and probe-binding detection by chemical proteomics. Probe binding was determined from the following references: ATP acyl phosphate probe [17,42–44,46–48]; TH211 sulfonyl-triazole probe [15]; XO44 sulfonyl-fluoride probe [14]; kinobeads and Omipalisib beads [13]. Asterisk denotes recombinant protein was analyzed.

Gene UniProtKB NCBI_ID Domains/Motifs Protein Data Bank IDs Probe Detection
AGK Q53H12 AGK_HUMAN DAGKc 7CGP ATP acyl phosphate, TH211
CERK Q8TCT0 CERK1_HUMAN DAGKc
DGKA P23743 DGKA_HUMAN DAGK N-termnus, EF-hand, C1, DAGKc, DAGKa 6IIE, 1TUZ ATP acyl phosphate, TH211
DGKB Q9Y6T7 DGKB_HUMAN DAGK N-termnus, EF-hand, C1, DAGKc, DAGKa ATP acyl phosphate*
DGKD Q16760 DGKD_HUMAN PH, C1, DAGKc, DAGKa, SAM 1R79, 3BQ7
DGKE P52429 DGKE_HUMAN C1, DAGKc, DAGKa ATP acyl phosphate*
DGKG P49619 DGKG_HUMAN DAGK N-termnus, EF-hand, C1, DAGKc, DAGKa ATP acyl phosphate*
DGKH Q86XP1 DGKH_HUMAN PH, C1, DAGKc, DAGKa, SAM ATP acyl phosphate
DGKI O75912 DGKI_HUMAN MARCKS homology, Ankyrin, C1, DAGKc, DAGKa, PDZ-binding
DGKK Q5KSL6 DGKK_HUMAN PH, C1, DAGKc, DAGKa ATP acyl phosphate*
DGKQ P52824 DGKQ_HUMAN C1, Ras-associating domain, DAGKc, DAGKa, LXXLL motif ATP acyl phosphate*
DGKZ Q13574 DGKZ_HUMAN MARCKS homology, Ankyrin, C1, DAGKc, DAGKa, Nuclear export signal, PDZ-binding 5ELQ ATP acyl phosphate*
IPMK Q8NFU5 IPMK_HUMAN IPK, Nuclear localization signal 5W2G, 5W2H, 5W2I, 6E7F, 6M88, 6M89, 6M8A, 6M8B, 6M8C, 6M8D, 6M8E ATP acyl phosphate
PI4K2A Q9BTU6 P4K2A_HUMAN PI3_PI4_kinase 4HND, 4HNE, 4PLA, 4YC4, 5EUT, 5I0N TH211
PI4K2B Q8TCG2 P4K2B_HUMAN PI3_PI4_kinase 4WTV
PI4KA P42356 PI4KA_HUMAN PI3_PI4_kinase, PI3Ka, ARM-type fold 6BQ1 ATP acyl phosphate, Omipalisib beads
PI4KAP2 A4QPH2 PI4P2_HUMAN PH, PI3_PI4_kinase ATP acyl phosphate
PI4KB Q9UBF8 PI4KB_HUMAN PI3_PI4_kinase, PI3Ka, ARM-type fold 2N73, 4D0L, 4D0M, 4WAE, 4WAG, 5C46, 5C4G, 5EUQ, 5FBL, 5FBQ, 5FBR, 5FBV, 5FBW, 5LX2, 5NAS, 6GL3 ATP acyl phosphate, Omipalisib beads
PIK3C2A O00443 P3C2A_HUMAN C2, PI3K Ras-binding domain, PI3_PI4_kinase, PI3Ka, PX domain, ARM-type fold 2AR5, 2IWL, 2REA, 2RED, 6BTY, 6BTZ, 6BU0, 6BUB Omipalisib beads
PIK3C2B O00750 P3C2B_HUMAN C2, PI3K Ras-binding domain, PI3_PI4_kinase, PI3Ka, PX domain, ARM-type fold Kinobeads, Omipalisib beads
PIK3C2G O75747 P3C2G_HUMAN C2, PI3K Ras-binding domain, PI3_PI4_kinase, PI3Ka, PX domain, ARM-type fold 2WWE
PIK3C3 Q8NEB9 PK3C3_HUMAN C2, PI3_PI4_kinase, PI3Ka, ARM-type fold 3IHY, 3LS8, 4OYS, 4PH4, 4UWF, 4UWG, 4UWH, 4UWK, 4UWL, 5ANL, 5ENN, 6HOG, 6HOH, 6I3U ATP acyl phosphate, XO44, Omipalisib beads
PIK3CA P42336 PK3CA_HUMAN C2, PI3K Ras-binding domain, PI3K adaptor-binding domain, PI3_PI4_kinase, PI3Ka, ARM-type fold 2ENQ, 2RD0, 3HHM, 3HIZ, 3ZIM, 4JPS, 4L1B, 4L23, 4L2Y, 4OVU, 40VV, 4TUU, 4TV3, 4WAF, 4YKN, 4ZOP, 5DXH, 5DXT, 5FI4, 5ITD, 5SW8, 5SWG, 5SWO, 5SWP, 5SWR, 5SWT, 5SX8, 5SX9, 5SXA, 5SXB, 5SXC, 5SXD, 5SXE, 5SXF, 5SXI, 5SXJ, 5SXK, 5UBR, 5UK8, 5UKJ, 5UL1, 5XGH, 5XGI, 5XGJ, 6GVF, 6GVG, 6GVH, 6GVI, 6NCT, 6OAC, 6PYS, 7K6M, 7K6N, 7K6O, 7K71 ATP acyl phosphate, Omipalisib beads
PIK3CB P42338 PK3CB_HUMAN C2, PI3K Ras-binding domain, PI3K adaptor-binding domain, PI3_PI4_kinase, PI3Ka, ARM-type fold ATP acyl phosphate
PIK3CD O00329 PK3CD_HUMAN C2, PI3K Ras-binding domain, PI3K adaptor-binding domain, PI3_PI4_kinase, PI3Ka, ARM-type fold 5DXU, 5M6U, 5T8F, 5UBT, 5VLR, 6G6W, 6OCO, 6OCU, 6PYR, 6PYU, 7JIS ATP acyl phosphate
PIK3CG P48736 PK3CG_HUMAN C2, PI3K Ras-binding domain, PI3K adaptor-binding domain, PI3_PI4_kinase, PI3Ka, ARM-type fold 1E8Y, 1E8Z, 1HE8, 2A4Z, 2A5U, 2CHW, 2CHX, 2CHZ, 2V4L, 3APC, 3APD, 3APF, 3CSF, 3CST, 3DBS, 3DPD, 3ENE, 3IBE, 3L08, 3L13, 3L16, 3L17, 3L54, 3LJ3, 3MJW, 3ML8, 3ML9, 3NZS, 3NZU, 3OAW, 3P2B, 3PRE, 3PRZ, 3PS6, 3QAQ, 3QAR, 3QJZ, 3QK0, 3R7Q, 3R7R, 3S2A, 3SD5, 3T8M, 3TJP, 3TL5, 3ZVV, 3ZW3, 4ANU, 4ANV, 4ANW, 4ANX, 4AOF, 4DK5, 4EZJ, 4EZK, 4EZL, 4F1S, 4FA6, 4FAD, 4FHJ, 4FHK, 4FJY, 4FJZ, 4FLH, 4FUL, 4G11, 4GB9, 4HLE, 4HVB, 4J6I, 4KZ0, 4KZC, 4PS3, 4PS7, 4PS8, 4URK, 4WWN, 4WWO, 4WWP, 4XX5, 4XZ4, 5EDS, 5G2N, 5G55, 5JHA, 5JHB, 5KAE, 5OQ4, 5T23, 6AUD, 6C1S, 6FH5, 6GQ7, 6T3B, 6T3C, 6XRL, 6XRM ATP acyl phosphate, Omipalisib beads
PIKFYVE Q9Y2I7 FYV1_HUMAN DEP domain, FYVE-type zinc finger, Chaperonin-like domain, PIPKc 7K2V ATP acyl phosphate
PIP4K2A P48426 PI42A_HUMAN PIP5K 2YBX, 6OSP, 6UX9 ATP acyl phosphate, Kinobeads
PIP4K2B P78356 PI42B_HUMAN PIP5K 1BO1, 3WZZ, 3X01, 3X02, 3X03, 3X04, 3X05, 3X06, 3X07, 3X08, 3X09, 3X0A, 3X0B, 3X0C, 6K4G, 6K4H ATP acyl phosphate, Kinobeads
PIP4K2C Q8TBX8 PI42C_HUMAN PIP5K 2GK9 ATP acyl phosphate, TH211, Kinobeads
PIP5K1A Q99755 PI51A_HUMAN PIP5K TH211
PIP5K1B O14986 PI51B_HUMAN PIP5K
PIP5K1C O60331 PI51C_HUMAN PIP5K 2G35, 3H1Z, 3H85 TH211
PIP5KL1 Q5T9C9 PI5L1_HUMAN PIP5K
PIPSL A2A3N6 PIPSL_HUMAN PIP5K, VWF_A, UIM
SPHK1 Q9NYA1 SPHK1_HUMAN DAGKc, Nuclear export signal 3VZB, 3VZC, 3VZD, 4L02, 4V24
SPHK2 Q9NRA0 SPHK2_HUMAN DAGKc, Nuclear export signal, Nuclear localization signal
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Our lipid kinase dendrogram can be divided into several groups based on the preferred lipid substrate of sequence-related kinases. The phosphoinositide-3-kinases (PI3Ks) phosphorylate phosphatidylinositol (PI) and its phosphorylated derivatives (e.g. PI(4,5)P2) at position 3 of the inositol ring to produce 3-phosphoinositides (e.g. PI(3,4,5)P3) [18]. The eight PI3K members are divided into class IA (p110α, PIK3CA; p110β, PIK3CB; p110δ, PIK3CD [19]), class IB (p110γ, PIK3CG), class II (PI3K-C2α, PIK3C2A; PI3K-C2β, PIK3C2B; PI3K-C2γ, PIK3C2G), and class III (VPS34, PIK3C3). Related to the PI3Ks are the phosphatidylinositol 4-kinases (PI4Kα, PI4KA [20]; PI4Kβ, PI4KB[21]; PI4K type II-α, PI4K2A [22]; PI4K type II-β, PI4K2B [22]) that phosphorylate PI to produce PI(4)P, the immediate precursor of PI(4,5)P2. PI4KAP2 is a potential pseudogene that functions as a dominant-negative inhibitor of PI4KA signaling [23]. The PIP(5)K/PIP(4)K family members consist of type 1 (PIP5K1A, PIP5K1B, PIP5K1C) and type 2 (PIP4K2A, PIP4K2B, PIP4K2C) enzymes that phosphorylate PI(4)P or PI(5)P, respectively, to produce PI(4,5)P2 [24]. The type 3 member PIKFYVE phosphorylates PI(3)P to produce PI(3,5)P2 [24]. Other sequence-related members include PIP5KL1 [25], PIPSL [26], and IPMK [27].

The dendrogram also includes sphingosine kinases (SPHK1/SPHK2) that catalyze the phosphorylation of sphingosine to produce sphingosine 1-phosphate [28,29]. Ceramide kinase (CERK) produces ceramide 1-phosphate through phosphorylation of ceramide [30]. Acylglycerol kinase (AGK) catalyzes the phosphorylation of both monoacylglycerol (MAG) and diacylglycerol (DAG) to form lysophosphatidic acid (LPA) and phosphatidic acid (PA), respectively [31]. A subset of lipid kinases phosphorylate DAG lipids (diacylglycerol kinases, DGKs), which serve as the focus for the remainder of this review.

Diacylglycerol kinases are key regulators of DAG metabolism and signaling

Diacylglycerol kinases (DGKs) catalyze the ATP-dependent phosphorylation of DAG to generate PA lipid messengers. In mammals, ten distinct isoforms have been identified; the individual members are classified into five subtypes based on similarity in primary protein sequence (Figure 1). All DGKs contain a shared lipid kinase catalytic domain that contains a DAGKc and DAGKa region, and at least two C1 domains (tandem C1A and C1B). DGKs are differentiated by regulatory domains that are implicated in controlling DGK activation, membrane localization, and protein-protein interactions (Table 1). We recommend the following reviews for additional information on the DGK family [32,33] and DGK function in immunology [34] and neurobiology [35].

Regulation of DGK lipid metabolism and signaling can be achieved through cell- and tissue- specific expression of DGK isoforms (Figure 2A). For example, the expression of DGKα and DGKζ isoforms is increased in T cells compared with other cell types . Genetic knockout of DGKα or DGKζ in mice produces T cells that exhibit enhanced Ras and mitogen-activated protein kinase (MAPK) activation in response to T cell receptor engagement [36,37]. These findings support a role for DGKs in negative regulation of TCR-induced Ras-MAPK signaling by, for example, limiting cellular DAGs required for membrane recruitment of the guanine nucleotide–exchange factor RasGRP1 [38] (Figure 2B). Although DGKα and DGKζ share overlapping functions, DGKζ was found to have a more dominant role over DGKα in regulatory T cell development and TCR-mediated signaling in primary T cells despite a lower protein abundance [39].

Figure 2. Regulation of DGK lipid metabolism and signaling.

Figure 2.

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(A) RNA expression data for individual DGK isoforms across tissues were downloaded from Human Protein Atlas (consensus dataset, http://www.proteinatlas.org). Hierarchical clustering of the log2 of the consensus normalized expression levels, Spearman correlation distance, and complete linkage clustering was performed using Heatmapper (http://www2.heatmapper.ca/). (B) DGKα and DGKζ play overlapping and distinct roles in regulation of T cell metabolism and signaling. (C) Structures of the observed DAG lipid biomarkers for recombinant type 1 rat DGK isoforms; C18:0_18:0 DAG for DGKα, C18:0_18:1 DAG for DGKβ, C16:0_20:3 DAG for DGKγ. DAG substrate specificity tracked with the identity of the host tandem C1 domains when protein engineered into recipient type 1 DGK isoforms as determined by LC-MS lipidomics [48].

Chemical proteomics of diacylglycerol kinases – a functional perspective of the active site

Recently applications of chemical proteomics to the DGKs have identified new ligandable sites for developing DGK small molecule inhibitors with improved selectivity. Chemical proteomics combines small molecule probes and quantitative liquid chromatography-mass spectrometry (LC-MS) for global analysis of hundreds to thousands of protein-ligand interactions in native biological systems (Figure 3A). Activity-based probes range from protein class-targeted agents (e.g. fluorophosphonate probes of serine hydrolases [40]) to electrophilic probes that generally react with nucleophilic groups on protein residues [41]. Chemical probes can be designed to interact with residues located in catalytic, regulatory, or allosteric sites of proteins important for biochemical function. These modified sites and/or proteins can be identified using quantitative LC-MS approaches to assess activity state as well as competition with ligands for medicinal chemistry of potent and selective inhibitors [40].

Figure 3. Chemical proteomic evaluation of DGK ligandability.

Figure 3.

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(A) Schematic of quantitative LC-MS chemical proteomics workflow to identify ligand-binding sites of proteins using activity-based probes. Stable isotope labeling of amino acids in cell culture (SILAC) technology (or other isotopic labeling strategies) is employed to create isotopically distinct proteome pools for semi-quantitative capabilities in multiplexed LC-MS analyses. MS1 extracted ion chromatograms (EIC) are used for quantitation of probe-modified peptide abundances. MS2 spectra are used for identification of the probe-modified sequence including the site of probe binding. (B) Proposed interdomain active site of DGKα highlighting substrate/inhibitor-sensitive probe-modified sites in the C1A and catalytic domain (DAGKc/DAGKa). ATP acyl phosphate probe studies were performed in vitro against recombinant rat DGKα lysate (modified lysine sites are shown). TH211 sulfonyl-triazole probe studies were carried out in live cells for activity-based profiling of endogenous human DGKα (modified tyrosine sites are shown).

ATP acyl phosphate probes are valuable chemical tools used to globally profile the ATP-binding regions of kinases and other ATP-utilizing proteins. This probe class is directed to kinase active sites by ATP recognition followed by covalent modification of catalytic lysines via the acyl phosphate electrophile [42–45] (Figure 3B). Franks et al. demonstrated the ability to adapt these probes for discovery of DGK sites important for substrate and inhibitor binding in native lysates [46]. Competition studies with free ATP identified probe-modified sites in the catalytic domain of members of all 5 DGK subtypes involved in ATP substrate recognition [46]. Furthermore, this study implicated the cysteine rich (C1) domain of DGKα (K237 on the rat protein), DGKζ (K323 on human isoform 2) and DGKθ (K202 on the human protein) in recognition of the ATP acyl phosphate probe (Figure 3B). Unlike the typical C1 domains used by protein kinase C (PKC) for DAG-mediated translocation, DGKs, with the exception of DGKβ and DGKγ, express atypical variants that have poorly-defined function [33]. Chemical proteomics was used to discover that the DGKα C1A domain is a (target) site for inhibitor binding [46]. The near- equivalent sensitivity of the C1A, DAGKc, and DAGKa probe-modified sites to fragment inhibitor binding suggested a putative interdomain architecture for the DGKα active site [47] (Figure 3B).

Lipidomics discovery of DGK C1 domain regulation of substrate specificity

Lipidomics was utilized to test the importance of C1 domains for biochemical and metabolic function of DGKs. In a report by Ware et al., all 10 mammalian DGKs were recombinantly expressed in mammalian cells and cellular alterations in DAG and PA in the larger lipidome were analyzed by LC-MS lipidomics [48]. The outcome of these global studies was identification of key lipid biomarkers of individual DGK isoforms (e.g. DGKα, C18:0_C18:0 DAG; DGKβ, C18:0_C18:1 DAG; DGKγ, C16:0_C20:3 DAG; Figure 2C). Several of the DAG substrates identified in the recombinant gain-of-function approach were validate as authentic substrates through knockdown studies of the corresponding endogenous DGK. The identification of lipid biomarkers for tracking activity of individual DGK isoforms in cells enabled the assessment of C1 domain function in DGK lipid metabolism. Site-directed mutagenesis of reported ATP acyl phosphate probe-modified sites [46] in the C1A (K237A) or catalytic domain (K377A, K539A; Figure 3B) disrupted metabolic function of DGKα [48]. Treatment of recombinant DGKα-expressing cells with an inhibitor that engages DGKα C1A also blocked metabolic function [48].

The identification of isoform-specific lipid biomarkers also permitted evaluation of our hypothesis that DGK C1 domains regulate fatty acyl specificity of DAG substrates. We developed C1 chimera protein constructs that exchanged the tandem C1 regions (~150 amino acids) between DGK isoforms while preserving the backbone regulatory and catalytic domains of host protein. The activity of DGK wild-type and C1 chimeric proteins were evaluated by LC-MS lipidomics to demonstrate that the DAG fatty acyl specificity of DGKs could be programmed by exchanging C1 domains in live cells [48]. The cellular lipidomics findings combined with previous chemical proteomic investigation support C1 domains as a component of the DGK active site that help select lipid substrates and thereby regulate DGK specificity in biological systems.

Covalent probes for activity-based profiling of lipid kinases in live cells

The ATP acyl phosphate probe has proven effective for chemical proteomic profiling of DGKs and other lipid kinases but is limited generally to lysate studies because of poor cell permeability due to its polyanionic nature [17]. The ability to probe kinase activity in live cells is important because protein conformation is dynamically regulated in the cellular milieu [14]. This is particularly important for lipid kinases that require interfacial activation at membranes in order to catalyze biochemical functions [33]. In a recent report, a cell-permeable sulfonyl-triazole probe containing a DGKα fragment ligand recognition element was used to covalently target kinase active sites in lysates and live cells [15] (TH211, Figure 3B). Sulfonyl-triazoles represent a new class of electrophiles that preferentially modify tyrosines through sulfur-triazole exchange (SuTEx) chemistry [49].

Huang et al. demonstrated that TH211 was capable of modifying tyrosine residues in the C1A (Y240) and catalytic domain (Y544, Y623) of native human DGKα detected in live Jurkat T cells [15] (Figure 3B). Mutation of the C1A probe-modified site resulted in a DGKα mutant protein (Y240F) that was catalytically impaired compared with wild-type counterpart. The TH211 probe also enabled competitive studies to confirm target engagement of DGK and general ATP-competitive kinase inhibitors at TH211-modified sites in live cells. The consistent identification of ligandable sites in the C1A and catalytic domains of DGKα using distinct activity-based probes further supports these regions as ‘hotspots’ for inhibitor development (Figure 3B). TH211 also identified modified tyrosine and lysine sites in additional lipid kinases including AGK, PIP4K2C, PIP5K1A, PIP5K1C, and PI4K2A from live cell chemical proteomics [15] (Figure 1 and Table 1). The ability to identify ligandable sites in less conserved regions of kinases (e.g. allosteric sites) will be important for guiding development of selective lipid kinase inhibitors as chemical probes and potential therapeutic agents.

Conclusions and future outlook

The distinct immunological and biochemical effects from perturbing DGKα versus DGKζ in T cells offers a glimpse of potential therapeutic opportunities afforded by targeting individual DGK isoforms [50]. A next step is to develop isoform-selective inhibitors of DGKs to understand how acute perturbations of individual isoforms affects lipid biology in a physiological setting devoid of potential network-wide compensation. Chemical proteomics is poised to aid in these efforts by providing a global method for assessing ligand binding that has already transformed our molecular perspective of the DGK active site. The availability of complementary lipidomics methods for assessing the effects of DGK inhibitors on cellular lipid metabolism will be key for target validation and understanding of compound mode of action. The lessons learned from DGKs will help guide future efforts to tackle additional targets found in the lipid kinome.

Acknowledgements

This work was supported by the National Institutes of Health Grants (DA053107 to T.B.W. and DA043571 to K.-L.H.), National Science Foundation (CHE-1942467 to K.-L.H.), the Robbins Family-MRA Young Investigator Award from the Melanoma Research Alliance (K.-L. H.), and the University of Virginia Cancer Center (NCI Cancer Center Support Grant no. 5P30CA044579-27 to K.-L.H).

Footnotes

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Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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