Optimization of the Lead Compound NVP-BHG712 as a Colorectal Cancer Inhibitor.

The ephrin type-A receptor 2 (EPHA2) kinase belongs to the largest family of receptor tyrosine kinases. There are several indications of an involvement of EPHA2 in the development of infectious diseases and cancer. Despite pharmacological potential, EPHA2 is an under-examined target protein. In this study, we synthesized a series of derivatives of the inhibitor NVP-BHG712 and triazine-based compounds. These compounds were evaluated to determine their potential as kinase inhibitors of EPHA2, including elucidation of their binding mode (X-ray crystallography), affinity (microscale thermophoresis), and selectivity (Kinobeads assay). Eight inhibitors showed affinities in the low-nanomolar regime (KD <10 nM). Testing in up to seven colon cancer cell lines that express EPHA2 reveals that several derivatives feature promising effects for the control of human colon carcinoma. Thus, we have developed a set of powerful tool compounds for fundamental new research on the interplay of EPH receptors in a cellular context.

A novel Cereblon E3 ligase modulator with antitumor activity in gastrointestinal cancer.

Targeted protein degradation offers new opportunities to inactivate cancer drivers and has successfully entered the clinic. Ways to induce selective protein degradation include proteolysis targeting chimera (PROTAC) technology and immunomodulatory (IMiDs) / next-generation Cereblon (CRBN) E3 ligase modulating drugs (CELMoDs). Here, we aimed to develop a MYC PROTAC based on the MYC-MAX dimerization inhibitor 10058-F4 derivative 28RH and Thalidomide, called MDEG-541. We show that a subgroup of gastrointestinal cancer cell lines and primary patient-derived organoids are MDEG-541 sensitive. Although MYC expression was regulated in a CRBN-, proteasome- and ubiquitin-dependent manner, we provide evidence that MDEG-541 induced the degradation of CRBN neosubstrates, including G1 to S phase transition 1/2 (GSPT1/2) and the Polo-like kinase 1 (PLK1). In sum, we have established a CRBN-dependent degrader of relevant cancer targets with activity in gastrointestinal cancers.

Design, Synthesis, and Evaluation of WD-Repeat-Containing Protein 5 (WDR5) Degraders.

Histone H3K4 methylation serves as a post-translational hallmark of actively transcribed genes and is introduced by histone methyltransferase (HMT) and its regulatory scaffolding proteins. One of these is the WD-repeat-containing protein 5 (WDR5) that has also been associated with controlling long noncoding RNAs and transcription factors including MYC. The wide influence of dysfunctional HMT complexes and the typically upregulated MYC levels in diverse tumor types suggested WDR5 as an attractive drug target. Indeed, protein-protein interface inhibitors for two protein interaction interfaces on WDR5 have been developed. While such compounds only inhibit a subset of WDR5 interactions, chemically induced proteasomal degradation of WDR5 might represent an elegant way to target all oncogenic functions. This study presents the design, synthesis, and evaluation of two diverse WDR5 degrader series based on two WIN site binding scaffolds and shows that linker nature and length strongly influence degradation efficacy.

PROTAC-mediated degradation reveals a non-catalytic function of AURORA-A kinase.

The mitotic kinase AURORA-A is essential for cell cycle progression and is considered a priority cancer target. Although the catalytic activity of AURORA-A is essential for its mitotic function, recent reports indicate an additional non-catalytic function, which is difficult to target by conventional small molecules. We therefore developed a series of chemical degraders (PROTACs) by connecting a clinical kinase inhibitor of AURORA-A to E3 ligase-binding molecules (for example, thalidomide). One degrader induced rapid, durable and highly specific degradation of AURORA-A. In addition, we found that the degrader complex was stabilized by cooperative binding between AURORA-A and CEREBLON. Degrader-mediated AURORA-A depletion caused an S-phase defect, which is not the cell cycle effect observed upon kinase inhibition, supporting an important non-catalytic function of AURORA-A during DNA replication. AURORA-A degradation induced rampant apoptosis in cancer cell lines and thus represents a versatile starting point for developing new therapeutics to counter AURORA-A function in cancer.

Radiosensitization by Kinase Inhibition Revealed by Phosphoproteomic Analysis of Pancreatic Cancer Cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers and known for its extensive genetic heterogeneity, high therapeutic resistance, and strong variation in intrinsic radiosensitivity. To understand the molecular mechanisms underlying radioresistance, we screened the phenotypic response of 38 PDAC cell lines to ionizing radiation. Subsequent phosphoproteomic analysis of two representative sensitive and resistant lines led to the reproducible identification of 7,800 proteins and 13,000 phosphorylation sites (p-sites). Approximately 700 p-sites on 400 proteins showed abundance changes after radiation in all cell lines regardless of their phenotypic sensitivity. Apart from recapitulating known radiation response phosphorylation markers such as on proteins involved in DNA damage repair, the analysis uncovered many novel members of a radiation-responsive signaling network that was apparent only at the level of protein phosphorylation. These regulated p-sites were enriched in potential ATM substrates and in vitro kinase assays corroborated 10 of these. Comparing the proteomes and phosphoproteomes of radiosensitive and -resistant cells pointed to additional tractable radioresistance mechanisms involving apoptotic proteins. For instance, elevated NADPH quinine oxidoreductase 1 (NQO1) expression in radioresistant cells may aid in clearing harmful reactive oxygen species. Resistant cells also showed elevated phosphorylation levels of proteins involved in cytoskeleton organization including actin dynamics and focal adhesion kinase (FAK) activity and one resistant cell line showed a strong migration phenotype. Pharmacological inhibition of the kinases FAK by Defactinib and of CHEK1 by Rabusertib showed a statistically significant sensitization to radiation in radioresistant PDAC cells. Together, the presented data map a comprehensive molecular network of radiation-induced signaling, improves the understanding of radioresistance and provides avenues for developing radiotherapeutic strategies.

Proteome activity landscapes of tumor cell lines determine drug responses.

Integrated analysis of genomes, transcriptomes, proteomes and drug responses of cancer cell lines (CCLs) is an emerging approach to uncover molecular mechanisms of drug action. We extend this paradigm to measuring proteome activity landscapes by acquiring and integrating quantitative data for 10,000 proteins and 55,000 phosphorylation sites (p-sites) from 125 CCLs. These data are used to contextualize proteins and p-sites and predict drug sensitivity. For example, we find that Progesterone Receptor (PGR) phosphorylation is associated with sensitivity to drugs modulating estrogen signaling such as Raloxifene. We also demonstrate that Adenylate kinase isoenzyme 1 (AK1) inactivates antimetabolites like Cytarabine. Consequently, high AK1 levels correlate with poor survival of Cytarabine-treated acute myeloid leukemia patients, qualifying AK1 as a patient stratification marker and possibly as a drug target. We provide an interactive web application termed ATLANTiC (http://atlantic.proteomics.wzw.tum.de), which enables the community to explore the thousands of novel functional associations generated by this work.

Identification of molecular targets for the targeted treatment of gastric cancer using dasatinib.

Gastric cancer (GC) remains the third leading cause of cancer-related death despite several improvements in targeted therapy. There is therefore an urgent need to investigate new treatment strategies, including the identification of novel biomarkers for patient stratification. In this study, we evaluated the effect of FDA-approved kinase inhibitors on GC. Through a combination of cell growth, migration and invasion assays, we identified dasatinib as an efficient inhibitor of GC proliferation. Mass-spectrometry-based selectivity profiling and subsequent knockdown experiments identified members of the SRC family of kinases including SRC, FRK, LYN and YES, as well as other kinases such as DDR1, ABL2, SIK2, RIPK2, EPHA2, and EPHB2 as dasatinib targets. The expression levels of the identified kinases were investigated on RNA and protein level in 200 classified tumor samples from patients, who had undergone gastrectomy, but had received no treatment. Levels of FRK, DDR1 and SRC expression on both mRNA and protein level were significantly higher in metastatic patient samples regardless of the tumor stage, while expression levels of SIK2 correlated with tumor size. Collectively, our data suggest dasatinib for treatment of GC based on its unique property, inhibiting a small number of key kinases (SRC, FRK, DDR1 and SIK2), highly expressed in GC patients.

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

Nano-flow liquid chromatography tandem mass spectrometry (nano-flow LC-MS/MS) is the mainstay in proteome research because of its excellent sensitivity but often comes at the expense of robustness. Here we show that micro-flow LC-MS/MS using a 1 × 150 mm column shows excellent reproducibility of chromatographic retention time (<0.3% coefficient of variation, CV) and protein quantification (<7.5% CV) using data from >2000 samples of human cell lines, tissues and body fluids. Deep proteome analysis identifies >9000 proteins and >120,000 peptides in 16 h and sample multiplexing using tandem mass tags increases throughput to 11 proteomes in 16 h. The system identifies >30,000 phosphopeptides in 12 h and protein-protein or protein-drug interaction experiments can be analyzed in 20 min per sample. We show that the same column can be used to analyze >7500 samples without apparent loss of performance. This study demonstrates that micro-flow LC-MS/MS is suitable for a broad range of proteomic applications.

The Formation of a Camalexin Biosynthetic Metabolon.

Arabidopsis (Arabidopsis thaliana) efficiently synthesizes the antifungal phytoalexin camalexin without the apparent release of bioactive intermediates, such as indole-3-acetaldoxime, suggesting that the biosynthetic pathway of this compound is channeled by the formation of an enzyme complex. To identify such protein interactions, we used two independent untargeted coimmunoprecipitation (co-IP) approaches with the biosynthetic enzymes CYP71B15 and CYP71A13 as baits and determined that the camalexin biosynthetic P450 enzymes copurified with these enzymes. These interactions were confirmed by targeted co-IP and Förster resonance energy transfer measurements based on fluorescence lifetime microscopy (FRET-FLIM). Furthermore, the interaction of CYP71A13 and Arabidopsis P450 Reductase1 was observed. We detected increased substrate affinity of CYP79B2 in the presence of CYP71A13, indicating an allosteric interaction. Camalexin biosynthesis involves glutathionylation of the intermediary indole-3-cyanohydrin, which is synthesized by CYP71A12 and especially CYP71A13. FRET-FLIM and co-IP demonstrated that the glutathione transferase GSTU4, which is coexpressed with Trp- and camalexin-specific enzymes, is physically recruited to the complex. Surprisingly, camalexin concentrations were elevated in knockout and reduced in GSTU4-overexpressing plants. This shows that GSTU4 is not directly involved in camalexin biosynthesis but rather plays a role in a competing mechanism.

Chemoproteomic Selectivity Profiling of PIKK and PI3K Kinase Inhibitors.

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 fibrodysplasia ossificans progressiva.

NVP-BHG712: Effects of Regioisomers on the Affinity and Selectivity toward the EPHrin Family.

Erythropoietin-producing hepatocellular (EPH) receptors are transmembrane receptor tyrosine kinases. Their extracellular domains bind specifically to ephrin A/B ligands, and this binding modulates intracellular kinase activity. EPHs are key players in bidirectional intercellular signaling, controlling cell morphology, adhesion, and migration. They are increasingly recognized as cancer drug targets. We analyzed the binding of NVP-BHG712 (NVP) to EPHA2 and EPHB4. Unexpectedly, all tested commercially available NVP samples turned out to be a regioisomer (NVPiso) of the inhibitor, initially described in a Novartis patent application. They only differ by the localization of a single methyl group on either one of two adjacent nitrogen atoms. The two compounds of identical mass revealed different binding modes. Furthermore, both in vitro and in vivo experiments showed that the isomers differ in their kinase affinity and selectivity.

The target landscape of clinical kinase drugs.

Kinase inhibitors are important cancer therapeutics. Polypharmacology is commonly observed, requiring thorough target deconvolution to understand drug mechanism of action. Using chemical proteomics, we analyzed the target spectrum of 243 clinically evaluated kinase drugs. The data revealed previously unknown targets for established drugs, offered a perspective on the "druggable" kinome, highlighted (non)kinase off-targets, and suggested potential therapeutic applications. Integration of phosphoproteomic data refined drug-affected pathways, identified response markers, and strengthened rationale for combination treatments. We exemplify translational value by discovering SIK2 (salt-inducible kinase 2) inhibitors that modulate cytokine production in primary cells, by identifying drugs against the lung cancer survival marker MELK (maternal embryonic leucine zipper kinase), and by repurposing cabozantinib to treat FLT3-ITD-positive acute myeloid leukemia. This resource, available via the ProteomicsDB database, should facilitate basic, clinical, and drug discovery research and aid clinical decision-making.

Pharmacoproteomic characterisation of human colon and rectal cancer.

Most molecular cancer therapies act on protein targets but data on the proteome status of patients and cellular models for proteome-guided pre-clinical drug sensitivity studies are only beginning to emerge. Here, we profiled the proteomes of 65 colorectal cancer (CRC) cell lines to a depth of > 10,000 proteins using mass spectrometry. Integration with proteomes of 90 CRC patients and matched transcriptomics data defined integrated CRC subtypes, highlighting cell lines representative of each tumour subtype. Modelling the responses of 52 CRC cell lines to 577 drugs as a function of proteome profiles enabled predicting drug sensitivity for cell lines and patients. Among many novel associations, MERTK was identified as a predictive marker for resistance towards MEK1/2 inhibitors and immunohistochemistry of 1,074 CRC tumours confirmed MERTK as a prognostic survival marker. We provide the proteomic and pharmacological data as a resource to the community to, for example, facilitate the design of innovative prospective clinical trials.

Chemoproteomics-Aided Medicinal Chemistry for the Discovery of EPHA2 Inhibitors.

The receptor tyrosine kinase EPHA2 has gained attention as a therapeutic drug target for cancer and infectious diseases. However, EPHA2 research and EPHA2-based therapies have been hampered by the lack of selective small-molecule inhibitors. Herein we report the synthesis and evaluation of dedicated EPHA2 inhibitors based on the clinical BCR-ABL/SRC inhibitor dasatinib as a lead structure. We designed hybrid structures of dasatinib and the previously known EPHA2 binders CHEMBL249097, PD-173955, and a known EPHB4 inhibitor in order to exploit both the ATP pocket entrance as well as the ribose pocket as binding epitopes in the kinase EPHA2. Medicinal chemistry and inhibitor design were guided by a chemical proteomics approach, allowing early selectivity profiling of the newly synthesized inhibitor candidates. Concomitant protein crystallography of 17 inhibitor co-crystals delivered detailed insight into the atomic interactions that underlie the structure-affinity relationship. Finally, the anti-proliferative effect of the inhibitor candidates was confirmed in the glioblastoma cell line SF-268. In this work, we thus discovered a novel EPHA2 inhibitor candidate that features an improved selectivity profile while maintaining potency against EPHA2 and anticancer activity in SF-268 cells.

Chemical Proteomics and Structural Biology Define EPHA2 Inhibition by Clinical Kinase Drugs.

The receptor tyrosine kinase EPHA2 (Ephrin type-A receptor 2) plays important roles in oncogenesis, metastasis, and treatment resistance, yet therapeutic targeting, drug discovery, or investigation of EPHA2 biology is hampered by the lack of appropriate inhibitors and structural information. Here, we used chemical proteomics to survey 235 clinical kinase inhibitors for their kinase selectivity and identified 24 drugs with submicromolar affinities for EPHA2. NMR-based conformational dynamics together with nine new cocrystal structures delineated drug-EPHA2 interactions in full detail. The combination of selectivity profiling, structure determination, and kinome wide sequence alignment allowed the development of a classification system in which amino acids in the drug binding site of EPHA2 are categorized into key, scaffold, potency, and selectivity residues. This scheme should be generally applicable in kinase drug discovery, and we anticipate that the provided information will greatly facilitate the development of selective EPHA2 inhibitors in particular and the repurposing of clinical kinase inhibitors in general.

Expression and Purification of EPHA2 Tyrosine Kinase Domain for Crystallographic and NMR Studies.

The receptor tyrosine kinase EPHA2 is overexpressed in several cancers (breast, head and neck, non-small-cell lung cancer). Small-molecule-based inhibition of the EPHA2 kinase domain (KD) is seen as an important strategy for therapeutic intervention. However, obtaining structural information by crystallography or NMR spectroscopy for drug discovery is severely hampered by the lack of pure, homogeneous protein. Here, different fragments of the EPHA2 KD were expressed and purified from both bacterial (Escherichia coli, BL21(DE3) cells) and insect cells (Spodoptera frugiperda, Sf9 cells).1 H,15 N HSQC was used to determine the proper folding and homogeneity of all the constructs. Protein from E. coli was well-folded but unstable, and it did not crystallize. However, a construct (D596-G900) produced in Sf9 cells yielded homogenous, well-folded protein that crystallized readily, thereby resulting in eleven new EPHA2-ligand crystal structures. We have also established a strategy for selective and uniform 15 N-amino acid labeling of EPHA2 KD in Sf9 cells for investigating dynamics and EPHA2-drug interactions by NMR.

Tofacitinib and analogs as inhibitors of the histone kinase PRK1 (PKN1).

AIM: The histone kinase PRK1 has been identified as a potential target to combat prostate cancer but selective PRK1 inhibitors are lacking. The US FDA -approved JAK1-3 inhibitor tofacitinib also potently inhibits PRK1 in vitro. RESULTS: We show that tofacitinib also inhibits PRK1 in a cellular setting. Using tofacitinib as a starting point for structure-activity relationship studies, we identified a more potent and another more selective PRK1 inhibitor compared with tofacitinib. Furthermore, we found two potential PRK1/JAK3-selectivity hotspots. CONCLUSION: The identified inhibitors and the selectivity hotspots lay the basis for the development of selective PRK1 inhibitors. The identification of PRK1, but also of other cellular tofacitinib targets, has implications on its clinical use and on future development of tofacitinib-like JAK inhibitors. [Formula: see text].

Identification of Highly Potent Protein Kinase†C-Related Kinase†1 Inhibitors by Virtual Screening, Binding Free Energy Rescoring, and in†vitro Testing.

Despite the considerable interest in protein kinase C-related kinase 1 (PRK1) as a target in cancer research, there is still a lack of PRK1 inhibitors with suitable selectivity profiles and physicochemical properties. To identify new PRK1 inhibitors we applied a virtual screening approach, which combines ensemble docking, minimization of the protein-ligand complex, binding free energy calculations, and application of quantitative structure-activity relationship (QSAR) models for predicting in vitro activity. The developed approach was then applied in a prospective manner to screen available libraries of kinase inhibitors from Selleck and GlaxoSmithKline (GSK). Compounds that showed favorable prediction were then tested in vitro for PRK1 inhibition. Some of the hits were found to inhibit PRK1 in the low-nanomolar range. Three in vitro hits were additionally tested in a mass-spectrometry-based cellular kinase profiling assay to examine selectivity. Our findings show that nanomolar and drug-like inhibitors can be identified by the virtual screening approach presented herein. The identified inhibitors are valuable tools for gaining a better understanding of PRK1 inhibition, and the identified hits can serve as starting points for further chemical optimization.

Chemical Proteomics Reveals Ferrochelatase as a Common Off-target of Kinase Inhibitors.

Many protein kinases are valid drug targets in oncology because they are key components of signal transduction pathways. The number of clinical kinase inhibitors is on the rise, but these molecules often exhibit polypharmacology, potentially eliciting desired and toxic effects. Therefore, a comprehensive assessment of a compound's target space is desirable for a better understanding of its biological effects. The enzyme ferrochelatase (FECH) catalyzes the conversion of protoporphyrin IX into heme and was recently found to be an off-target of the BRAF inhibitor Vemurafenib, likely explaining the phototoxicity associated with this drug in melanoma patients. This raises the question of whether FECH binding is a more general feature of kinase inhibitors. To address this, we applied a chemical proteomics approach using kinobeads to evaluate 226 clinical kinase inhibitors for their ability to bind FECH. Surprisingly, low or submicromolar FECH binding was detected for 29 of all compounds tested and isothermal dose response measurements confirmed target engagement in cells. We also show that Vemurafenib, Linsitinib, Neratinib, and MK-2461 reduce heme levels in K562 cells, verifying that drug binding leads to a loss of FECH activity. Further biochemical and docking experiments identified the protoporphyrin pocket in FECH as one major drug binding site. Since the genetic loss of FECH activity leads to photosensitivity in humans, our data strongly suggest that FECH inhibition by kinase inhibitors is the molecular mechanism triggering photosensitivity in patients. We therefore suggest that a FECH assay should generally be part of the preclinical molecular toxicology package for the development of kinase inhibitors.