RESUMEN
Stimulator of interferon genes (STING) is a dimeric transmembrane adapter protein that plays a key role in the human innate immune response to infection and has been therapeutically exploited for its antitumor activity. The activation of STING requires its high-order oligomerization, which could be induced by binding of the endogenous ligand, cGAMP, to the cytosolic ligand-binding domain. Here we report the discovery through functional screens of a class of compounds, named NVS-STGs, that activate human STING. Our cryo-EM structures show that NVS-STG2 induces the high-order oligomerization of human STING by binding to a pocket between the transmembrane domains of the neighboring STING dimers, effectively acting as a molecular glue. Our functional assays showed that NVS-STG2 could elicit potent STING-mediated immune responses in cells and antitumor activities in animal models.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas de la Membrana , Animales , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Bioensayo , Citosol , Inmunidad Innata , Ligandos , Proteínas de la Membrana/metabolismoRESUMEN
[This corrects the article DOI: 10.1021/acscentsci.2c01317.].
RESUMEN
Targeted protein degradation with molecular glue degraders has arisen as a powerful therapeutic modality for eliminating classically undruggable disease-causing proteins through proteasome-mediated degradation. However, we currently lack rational chemical design principles for converting protein-targeting ligands into molecular glue degraders. To overcome this challenge, we sought to identify a transposable chemical handle that would convert protein-targeting ligands into molecular degraders of their corresponding targets. Using the CDK4/6 inhibitor ribociclib as a prototype, we identified a covalent handle that, when appended to the exit vector of ribociclib, induced the proteasome-mediated degradation of CDK4 in cancer cells. Further modification of our initial covalent scaffold led to an improved CDK4 degrader with the development of a but-2-ene-1,4-dione ("fumarate") handle that showed improved interactions with RNF126. Subsequent chemoproteomic profiling revealed interactions of the CDK4 degrader and the optimized fumarate handle with RNF126 as well as additional RING-family E3 ligases. We then transplanted this covalent handle onto a diverse set of protein-targeting ligands to induce the degradation of BRD4, BCR-ABL and c-ABL, PDE5, AR and AR-V7, BTK, LRRK2, HDAC1/3, and SMARCA2/4. Our study undercovers a design strategy for converting protein-targeting ligands into covalent molecular glue degraders.
RESUMEN
Photoaffinity labeling (PAL) is a powerful tool for the identification of non-covalent small molecule-protein interactions that are critical to drug discovery and medicinal chemistry, but this approach is limited to only a small subset of robust photocrosslinkers. The identification of new photoreactive motifs capable of covalent target capture is therefore highly desirable. Herein, we report the design, synthesis, and evaluation of a new class of PAL warheads based on the UV-triggered 1,2-photo-Brook rearrangement of acyl silanes, which hitherto have not been explored for PAL workflows. Irradiation of a series of probes in cell lysate revealed an iPr-substituted acyl silane with superior photolabeling and minimal thermal background labeling compared to other substituted acyl silanes. Further, small molecule (+)-JQ1- and rapamycin-derived iPr acyl silanes were shown to selectively label recombinant BRD4-BD1 and FKBP12, respectively, with minimal background. Together, these data highlight the untapped potential of acyl silanes as a novel, tunable scaffold for photoaffinity labeling.
RESUMEN
Many diseases are driven by proteins that are aberrantly ubiquitinated and degraded. These diseases would be therapeutically benefited by targeted protein stabilization (TPS). Here we present deubiquitinase-targeting chimeras (DUBTACs), heterobifunctional small molecules consisting of a deubiquitinase recruiter linked to a protein-targeting ligand, to stabilize the levels of specific proteins degraded in a ubiquitin-dependent manner. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48-ubiquitin-specific deubiquitinase OTUB1. We showed that a DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR), robustly stabilized ΔF508-CFTR protein levels, leading to improved chloride channel conductance in human cystic fibrosis bronchial epithelial cells. We also demonstrated stabilization of the tumor suppressor kinase WEE1 in hepatoma cells. Our study showcases covalent chemoproteomic approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS.
Asunto(s)
Fibrosis Quística , Quimera/metabolismo , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Enzimas Desubicuitinizantes/metabolismo , Enzimas Desubicuitinizantes/uso terapéutico , Humanos , Ligandos , Ubiquitina/metabolismoRESUMEN
PHY34 is a synthetic small molecule, inspired by a compound naturally occurring in tropical plants of the Phyllanthus genus. PHY34 was developed to have potent in vitro and in vivo anticancer activity against high grade serous ovarian cancer (HGSOC) cells. Mechanistically, PHY34 induced apoptosis in ovarian cancer cells by late-stage autophagy inhibition. Furthermore, PHY34 significantly reduced tumor burden in a xenograft model of ovarian cancer. In order to identify its molecular target/s, we undertook an unbiased approach utilizing mass spectrometry-based chemoproteomics. Protein targets from the nucleocytoplasmic transport pathway were identified from the pulldown assay with the cellular apoptosis susceptibility (CAS) protein, also known as CSE1L, representing a likely candidate protein. A tumor microarray confirmed data from mRNA expression data in public databases that CAS expression was elevated in HGSOC and correlated with worse clinical outcomes. Overexpression of CAS reduced PHY34 induced apoptosis in ovarian cancer cells based on PARP cleavage and Annexin V staining. Compounds with a diphyllin structure similar to PHY34 have been shown to inhibit the ATP6V0A2 subunit of V(vacuolar)-ATPase. Therefore, ATP6V0A2 wild-type and ATP6V0A2 V823 mutant cell lines were tested with PHY34, and it was able to induce cell death in the wild-type at 246 pM while the mutant cells were resistant up to 55.46 nM. Overall, our data demonstrate that PHY34 is a promising small molecule for cancer therapy that targets the ATP6V0A2 subunit to induce autophagy inhibition while interacting with CAS and altering nuclear localization of proteins.
Asunto(s)
Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Núcleo Celular/metabolismo , Proteína de Susceptibilidad a Apoptosis Celular/metabolismo , Cistadenocarcinoma Seroso/metabolismo , Neoplasias Ováricas/metabolismo , ATPasas de Translocación de Protón/antagonistas & inhibidores , Transporte Activo de Núcleo Celular/efectos de los fármacos , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proteína de Susceptibilidad a Apoptosis Celular/genética , Cistadenocarcinoma Seroso/tratamiento farmacológico , Cistadenocarcinoma Seroso/patología , Femenino , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Phyllanthus/química , PronósticoRESUMEN
Proteolysis-targeting chimeras (PROTACs), heterobifunctional compounds that consist of protein-targeting ligands linked to an E3 ligase recruiter, have arisen as a powerful therapeutic modality for targeted protein degradation (TPD). Despite the popularity of TPD approaches in drug discovery, only a small number of E3 ligase recruiters are available for the >600 E3 ligases that exist in human cells. Here, we have discovered a cysteine-reactive covalent ligand, EN106, that targets FEM1B, an E3 ligase recently discovered as the critical component of the cellular response to reductive stress. By targeting C186 in FEM1B, EN106 disrupts recognition of the key reductive stress substrate of FEM1B, FNIP1. We further establish that EN106 can be used as a covalent recruiter for FEM1B in TPD applications by demonstrating that a PROTAC linking EN106 to the BET bromodomain inhibitor JQ1 or the kinase inhibitor dasatinib leads to the degradation of BRD4 and BCR-ABL, respectively. Our study showcases a covalent ligand that targets a natural E3 ligase-substrate binding site and highlights the utility of covalent ligand screening in expanding the arsenal of E3 ligase recruiters suitable for TPD applications.
Asunto(s)
Acetamidas/química , Proteínas de Ciclo Celular/metabolismo , Proteolisis , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Animales , Azepinas/química , Sitios de Unión , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular , Cisteína/química , Dasatinib/química , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Ratones , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Triazoles/química , Complejos de Ubiquitina-Proteína Ligasa/antagonistas & inhibidores , Complejos de Ubiquitina-Proteína Ligasa/genéticaRESUMEN
While there are hundreds of predicted E3 ligases, characterizing their applications for targeted protein degradation has proved challenging. Here, we report a chemical biology approach to evaluate the ability of modified recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for specific E3 ligase binders, we use maleimide-thiol chemistry for covalent functionalization followed by E3 electroporation (COFFEE) in live cells. We demonstrate that electroporated recombinant von Hippel-Lindau (VHL) protein, covalently functionalized at its ligandable cysteine with JQ1 or dasatinib, induces degradation of BRD4 or tyrosine kinases, respectively. Furthermore, by applying COFFEE to SPSB2, a Cullin-RING ligase 5 receptor, as well as to SKP1, the adaptor protein for Cullin-RING ligase 1 F box (SCF) complexes, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligase components, and provide further evidence that not only E3 ligase receptors but also adaptors can be directly hijacked for neo-substrate degradation.
Asunto(s)
Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular , Femenino , Humanos , Masculino , Proteínas Recombinantes/metabolismoRESUMEN
The translation of functionally active natural products into fully synthetic small-molecule mimetics has remained an important process in medicinal chemistry. We recently discovered that the terpene natural product nimbolide can be utilized as a covalent recruiter of the E3 ubiquitin ligase RNF114 for use in targeted protein degradation-a powerful therapeutic modality within modern-day drug discovery. Using activity-based protein profiling-enabled covalent ligand-screening approaches, here we report the discovery of fully synthetic RNF114-based recruiter molecules that can also be exploited for PROTAC applications, and demonstrate their utility in degrading therapeutically relevant targets, such as BRD4 and BCR-ABL, in cells. The identification of simple and easily manipulated drug-like scaffolds that can mimic the function of a complex natural product is beneficial in further expanding the toolbox of E3 ligase recruiters, an area of great importance in drug discovery and chemical biology.
Asunto(s)
Productos Biológicos/metabolismo , Proteómica , Ubiquitina-Proteína Ligasas/metabolismo , Productos Biológicos/química , Humanos , Estructura Molecular , UbiquitinaciónRESUMEN
Molecular glues are an intriguing therapeutic modality that harness small molecules to induce interactions between proteins that typically do not interact. However, such molecules are rare and have been discovered fortuitously, thus limiting their potential as a general strategy for therapeutic intervention. We postulated that natural products bearing one or more electrophilic sites may be an unexplored source of new molecular glues, potentially acting through multicovalent attachment. Using chemoproteomic platforms, we show that members of the manumycin family of polyketides, which bear multiple potentially reactive sites, target C374 of the putative E3 ligase UBR7 in breast cancer cells, and engage in molecular glue interactions with the neosubstrate tumor-suppressor TP53, leading to p53 transcriptional activation and cell death. Our results reveal an anticancer mechanism of this natural product family, and highlight the potential for combining chemoproteomics and multicovalent natural products for the discovery of new molecular glues.
Asunto(s)
Antineoplásicos/química , Neoplasias de la Mama/tratamiento farmacológico , Polienos/química , Policétidos/química , Alcamidas Poliinsaturadas/química , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral , Reactivos de Enlaces Cruzados/química , Descubrimiento de Drogas , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Conformación Molecular , Estructura Molecular , Polienos/farmacología , Alcamidas Poliinsaturadas/farmacología , Electricidad Estática , Relación Estructura-Actividad , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Targeted protein degradation (TPD) and proteolysis-targeting chimeras (PROTACs) have arisen as powerful therapeutic modalities for degrading specific proteins in a proteasome-dependent manner. However, a major limitation of TPD is the lack of E3 ligase recruiters. Recently, we discovered the natural product nimbolide as a covalent recruiter for the E3 ligase RNF114. Here, we show the broader utility of nimbolide as an E3 ligase recruiter for TPD applications. We demonstrate that a PROTAC linking nimbolide to the kinase and BCR-ABL fusion oncogene inhibitor dasatinib, BT1, selectively degrades BCR-ABL over c-ABL in leukemia cancer cells, compared to previously reported cereblon or VHL-recruiting BCR-ABL degraders that show opposite selectivity or, in some cases, inactivity. Thus, we further establish nimbolide as an additional general E3 ligase recruiter for PROTACs, and we demonstrate the importance of expanding upon the arsenal of E3 ligase recruiters, as such molecules confer differing selectivity for the degradation of neo-substrate proteins.
Asunto(s)
Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Limoninas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteolisis/efectos de los fármacos , Tiazoles/farmacología , Proteínas de Fusión bcr-abl/química , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Células K562 , Limoninas/química , Inhibidores de Proteínas Quinasas/química , Tiazoles/química , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we combine phenotypic screening with chemical genetics to identify pre-messenger RNA endonuclease cleavage and polyadenylation specificity factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a synthetic lethal node in a subset of acute myeloid leukemia (AML) and Ewing's sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing's sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing an avenue to therapeutic intervention in cancer.
Asunto(s)
Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Leucemia Mieloide Aguda/metabolismo , Precursores del ARN/metabolismo , Sarcoma de Ewing/metabolismo , Animales , Apoptosis/efectos de los fármacos , Sitios de Unión , Hidrolasas de Éster Carboxílico/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Células HEK293 , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Fenotipo , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , Piperazinas/farmacología , Unión Proteica , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Sarcoma de Ewing/tratamiento farmacológicoRESUMEN
Nimbolide, a terpenoid natural product derived from the Neem tree, impairs cancer pathogenicity; however, the direct targets and mechanisms by which nimbolide exerts its effects are poorly understood. Here, we used activity-based protein profiling (ABPP) chemoproteomic platforms to discover that nimbolide reacts with a novel functional cysteine crucial for substrate recognition in the E3 ubiquitin ligase RNF114. Nimbolide impairs breast cancer cell proliferation in-part by disrupting RNF114-substrate recognition, leading to inhibition of ubiquitination and degradation of tumor suppressors such as p21, resulting in their rapid stabilization. We further demonstrate that nimbolide can be harnessed to recruit RNF114 as an E3 ligase in targeted protein degradation applications and show that synthetically simpler scaffolds are also capable of accessing this unique reactive site. Our study highlights the use of ABPP platforms in uncovering unique druggable modalities accessed by natural products for cancer therapy and targeted protein degradation applications.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Productos Biológicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Proteínas Portadoras/metabolismo , Limoninas/farmacología , Proteolisis/efectos de los fármacos , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Limoninas/química , Limoninas/aislamiento & purificación , Ubiquitina-Proteína LigasasRESUMEN
Targeted protein degradation has arisen as a powerful strategy for drug discovery allowing the targeting of undruggable proteins for proteasomal degradation. This approach most often employs heterobifunctional degraders consisting of a protein-targeting ligand linked to an E3 ligase recruiter to ubiquitinate and mark proteins of interest for proteasomal degradation. One challenge with this approach, however, is that only a few E3 ligase recruiters currently exist for targeted protein degradation applications, despite the hundreds of known E3 ligases in the human genome. Here, we utilized activity-based protein profiling (ABPP)-based covalent ligand screening approaches to identify cysteine-reactive small-molecules that react with the E3 ubiquitin ligase RNF4 and provide chemical starting points for the design of RNF4-based degraders. The hit covalent ligand from this screen reacted with either of two zinc-coordinating cysteines in the RING domain, C132 and C135, with no effect on RNF4 activity. We further optimized the potency of this hit and incorporated this potential RNF4 recruiter into a bifunctional degrader linked to JQ1, an inhibitor of the BET family of bromodomain proteins. We demonstrate that the resulting compound CCW 28-3 is capable of degrading BRD4 in a proteasome- and RNF4-dependent manner. In this study, we have shown the feasibility of using chemoproteomics-enabled covalent ligand screening platforms to expand the scope of E3 ligase recruiters that can be exploited for targeted protein degradation applications.
Asunto(s)
Complejos de Coordinación/química , Proteínas Nucleares/metabolismo , Proteolisis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complejos de Coordinación/metabolismo , Cisteína/química , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Dominios Proteicos , Bibliotecas de Moléculas Pequeñas/metabolismo , Relación Estructura-Actividad , Ubiquitinación , Zinc/químicaRESUMEN
The identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the pathway. A phenotypic screen identified compounds that interfere with trafficking of Notch and induce apoptosis via an endoplasmic reticulum (ER) stress mechanism. Target identification approaches revealed a role for SLC39A7 (ZIP7), a zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound-resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, and an analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and investigate ZIP7 as a novel druggable node in the Notch pathway.
Asunto(s)
Proteínas de Transporte de Catión/genética , Estrés del Retículo Endoplásmico/fisiología , Receptor Notch1/genética , Animales , Apoptosis , Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión/metabolismo , Proteínas de Transporte de Catión/fisiología , Línea Celular , Transformación Celular Neoplásica , Retículo Endoplásmico/fisiología , Humanos , Mutación , Transporte de Proteínas , Receptor Notch1/fisiología , Transducción de Señal , Zinc/metabolismoRESUMEN
Using a comprehensive chemical genetics approach, we identified a member of the lignan natural product family, HTP-013, which exhibited significant cytotoxicity across various cancer cell lines. Correlation of compound activity across a panel of reporter gene assays suggested the vacuolar-type ATPase (v-ATPase) as a potential target for this compound. Additional cellular studies and a yeast haploinsufficiency screen strongly supported this finding. Competitive photoaffinity labeling experiments demonstrated that the ATP6V0A2 subunit of the v-ATPase complex binds directly to HTP-013, and further mutagenesis library screening identified resistance-conferring mutations in ATP6V0A2. The positions of these mutations suggest the molecule binds a novel pocket within the domain of the v-ATPase complex responsible for proton translocation. While other mechanisms of v-ATPase regulation have been described, such as dissociation of the complex or inhibition by natural products including bafilomycin A1 and concanamycin, this work provides detailed insight into a distinct binding pocket within the v-ATPase complex.
Asunto(s)
Productos Biológicos/metabolismo , Productos Biológicos/farmacología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Productos Biológicos/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Células HCT116 , Células HEK293 , Humanos , Estructura Molecular , Neurospora crassa/metabolismo , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/químicaRESUMEN
The combination of photoaffinity labeling (PAL) and quantitative chemoproteomics enables the comprehensive, unbiased determination of protein interaction profiles to support target identification of bioactive small molecules. This approach is amenable to cells in culture and compatible with pharmacologically relevant transmembrane target classes like G-protein coupled receptors and ions channels which have been notoriously hard to access by conventional chemoproteomics approaches. Here, we describe a strategy that combines PAL probe titration and competition with excess parental compounds with the goal of enabling the identification of specific interactors as well as assessing the functional relevance of a binding event for the phenotype under investigation.
Asunto(s)
Etiquetas de Fotoafinidad/química , Proteómica/métodos , Bibliotecas de Moléculas Pequeñas/análisis , Química Clic , Conductometría , Diseño de Fármacos , Proteínas de Unión al GTP/análisis , Células HEK293 , Humanos , Espectrometría de Masas , Receptores Acoplados a Proteínas G/análisisRESUMEN
Quantitative chemoproteomics has recently emerged as an experimental approach to determine protein interaction profiles of small molecules in a given cell line or tissue. In contrast to standard biochemical and biophysical kinase assays, application of this method to kinase inhibitors determines compound binding to endogenously expressed kinases under conditions approximating the physiological situation with regard to the molecular state of the kinase and presence of required cofactors and regulatory proteins. Using a dose-dependent, competition-based experimental design in combination with quantitative mass spectrometry approaches, such as the use of tandem mass tags (TMT) for isobaric labeling described here, allows to rank-order interactions of inhibitors to kinase by binding affinity.