A chemoproteomic method for identifying cellular targets of covalent kinase inhibitors

Protein kinases are attractive drug targets for numerous human diseases including cancers, diabetes and neurodegeneration. A number of kinase inhibitors that covalently target a cysteine residue in their target kinases have recently entered use in the cancer clinic. Despite the advantages of covalent kinases inhibitors, their inherent reactivity can lead to non-specific binding to other cellular proteins and cause off- target effects in cells. It is thus essential to determine the identity of these off targets in order to fully account for the phenotype and to improve the selectivity and efficacy of covalent inhibitors. Herein we present a detailed protocol for a chemoproteomic method to enrich and identify cellular targets of covalent kinase inhibitors.


INTRODUCTION
Protein kinases are a large family of enzymes that transfer the γ-phosphate group of ATP to the tyrosine, serine or threonine residues of substrate proteins thus modulating numerous biological processes in eukaryotes.
There are approximately 530 protein kinases in human, which constitute about 1.7% of all human genes [1]. Kinases play important roles in signal transduction and thus regulate a variety of cellular processes including metabolism, transcription, cell cycle progression, cytoskeletal rearrangement, cell movement, apoptosis and differentiation [2,3]. Mutations and dysregulation of protein kinases have been implicated in numerous human diseases including cancers, diabetes and neurodegeneration [4,5]. Frequent occurrence of the disease-causing mutations in protein kinases make them attractive targets for therapeutic discovery.
Numerous small molecules have been tested for inhibition against protein kinases and evaluated as targeted cancer therapies. Approximately 30 kinase inhibitors have been approved by the FDA for treating various types of cancer in the clinic [6]. Imatinib (Gleevec), a small-molecule inhibitor of the oncogenic fusion kinase BCR-ABL, was first approved by the FDA in 2001 for the treatment of chronic myeloid leukemia (CML) [7]. Subsequently, numerous other kinase inhibitors, such as gefitinib, erlotinib, sorafenib, sunitinib, lapatinib, dasatinib, crizotinib, and vemurafenib, have been approved by the FDA for the treatment of a variety of cancers including non-small cell lung carcinoma, breast cancer, hepatocellular carcinoma, renal cell carcinoma and melanoma [6].
The majority of clinically approved kinase inhibitors rely on non-covalent forces such as hydrogen bonds, ionic bonds and van der waals interactions to bind to the kinase active site [6]. A small number of kinase inhibitors can form covalent interactions with the sulfurhydryl group of cysteine in protein kinases [8]. Such covalent interactions provide a number of advantages including high selectivity and potency against the target of interest, as well as prolonged and tunable pharmacodynamics [9,10]. Highly specific inhibitors have been identified for individual kinases by covalently targeting non-conserved, rare cysteine in or near the kinase active site [8][9][10][11][12][13].
A number of covalent kinase inhibitors have entered clinic use. Afatinib, a covalent inhibitor of the epidermal growth factor receptors (EGFR), was approved by the FDA for the treatment of EGFR-driven non-small cell lung carcinoma (NSCLC) in 2013 ( Figure 1A). EGFR receptor tyrosine kinase (RTK) subfamily includes four members in mammals: EGFR (ErbB1), ErbB2, ErbB3, and ErbB4, which play essential roles in cell proliferation, survival and differentiation [14]. Mutations and overexpression of EGFR are observed in various cancer cell types [15,16]. In addition to wild-type EGFR, afatinib irreversibly binds and inhibits ErbB2, ErbB4, and certain EGFR mutants, including those caused by EGFR exon 19 deletion mutations or exon 21 (L858R) substitution mutations, as well as EGFR T790M gatekeeper mutation. The inhibition of these RTKs can result in the inhibition of tumor growth and angiogenesis in tumor cells overexpressing these RTKs. Afatinib carries an electrophilic acrylamide group for targeting Cys797 near the end of the EGFR kinase hinge region, which was confirmed by co-crystal structure [13]. Shortly after the FDA approval of afatinib, ibrutinib, a covalent inhibitor of Bruton's tyrosine kinase (BTK), was first approved by the FDA in 2013 for the treatment of mantle cell lymphoma (MCL) and later approved for the treatment of chronic lymphocytic leukemia (CLL) and Waldenström macroglobulinemia ( Figure 1A) [17]. A member of the TEC family of non-receptor tyrosine kinases, BTK is a key regulator for B cell receptor (BCR) signaling and was found overexpressed in a number of B-cell malignancies [12]. Ibrutinib contains an acrylamide group that forms covalent interaction with Cys481 in BTK (at a homologous position to Cys797 in EGFR) and inhibits BTK kinase activity thus preventing BCR signaling [17]. Inspired by the clinical success of afatinib and ibrutinib, there are currently extensive efforts focusing on the development of irreversible kinase inhibitors [9,18]. For example, osimertinib, a selective covalent inhibitor for the drug-resistant mutant (T790M) of EGFR, has been recently approved by the FDA for treating metastatic NSCLC ( Figure 1A) [11]. As compared to previous FDA-approved EGFR inhibitors that also inhibit wildtype EGFR, osimertinib demonstrated great selectivity for EGFR T970M that is only harbored in tumors thus reducing toxicity on normal cells [19].
Despite the advantages of covalent kinases inhibitors described above, the inherent reactivity of covalent inhibitors can lead to non-specific binding to other cellular proteins and cause off-target effects in cells [10]. The covalent modification of off-targets may complicate the analysis of signaling transduction in cells as well as increase the risk of hapten formation (triggering an immune response to the adducted protein) and could lead to high cytotoxicity due to the sustained off-target engagement [20,21]. It is thus essential to determine the identity of these off targets in order to fully account for the phenotype and to improve the selectivity and efficacy of covalent inhibitors [22].
Target identification of covalent kinase inhibitors can be achieved by using a method that involves selective pull-down of target proteins and mass spectrometry for protein identification [23]. First, the covalent kinase inhibitor is derivatized with a terminal alkyne group to generate a probe compound ( Figure 1B). Cells will be treated with the probe resulting in all cellular targets covalently modified with an alkyne tag ( Figure 2). After cell lysis, the lysate is subjected to the copper(I)catalyzed alkyne-azide cycloaddition (CuAAC) click chemistry to conjugate those target proteins with a biotin tag. The biotin-tagged proteins can then be pulled down on streptavidin resin before the target proteins is selectively eluted by cleaving an azo-linker in the tag with sodium dithionite (Figure 2). The proteins enriched in the eluent can be visualized by SDS-PAGE gel analysis and identified by mass spectrometry analysis. The confidence of target relevance can be evaluated by including negative control of no probe treatment or pretreatment of the original covalent kinase inhibitor to compete off probe labeling (Figure 2).
Cravatt and co-workers have applied a similar method to determine the protein targets of two clinically used covalent kinase inhibitor drugs, afatinib and ibrutinib, in the entire human proteome [24]. Notably, they discovered both kinases and non-kinase proteins among the identified cellular targets of both covalent drugs ( Figure 1C).
Herein we present a detailed protocol for this chemoproteomic method. It can serve as a general method for enriching and identifying targets of not only covalent kinase inhibitors but also covalent probes targeting nonkinase proteins.   Add resuspended proteins to streptavidin beads in 15 mL centrifuge tube and incubate on a rotator for 2 h at RT. 5. Collect beads by centrifugation at 2,000 x g for 3 min at RT. Wash beads sequentially with resuspension buffer (2 x 10 mL), PBS (2 x 10 mL), and 1% SDS in PBS (2 x 10 mL). Transfer beads to a 2 mL dolphin-nose tube using widebore pipette tips. 6. Add 200 μL sodium dithionite buffer to beads and incubate at RT for 30 min with gentle rocking. Collect supernatant by centrifugation at 2,000 x g for 3 min at RT. Repeat the elution once more and combine supernatant. 7. Add 1.6 mL ice-cold MeOH to the combined supernatant and incubate at -20 o C overnight. Centrifuge at 10,000 x g for 10 min at 4 o C to pellet protein before decanting supernatant. Place tube up side down with an angle of 45 degree to air-dry the pellet. Proceed to SDS-PAGE gel analysis.

SDS-PAGE gel analysis
1. Resuspend protein pellet in 15 μL of 4% SDS buffer, bath sonicate briefly, and then add 15 μL of 2X SDS-free loading buffer. 2. Boil the sample at 98 o C for 5 min and centrifuge at 10,000 x g for 3 min at RT before loading all 30 μL of the sample into SDS-PAGE gel. 20 μg of whole cell lysate can be used as input for comparison. 3. The gel is stained with Colloidal Blue for 3 h and then destained with water overnight according to manufacture's manual. 4. Cut out the desired gel slices and send out for mass spectrometry analysis to identify the proteins.