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Background Based on their potential to analyze aberrant cellular signaling in relation to biological function, kinase activity profiling in tumor biopsies by peptide microarrays and mass spectrometry-based phosphoproteomics may guide selection of protein kinase inhibitors in patients with cancer. Variable tissue handling procedures in clinical practice may influence protein phosphorylation status and kinase activity and therewith may hamper biomarker discovery. Here, the effect of cold ischemia time (CIT) on the stability of kinase activity and protein phosphorylation status in fresh-frozen clinical tissue samples was studied using peptide microarrays and mass spectrometry-based phosphoproteomics. Methods Biopsies of colorectal cancer resection specimens from five patients were collected and snap frozen immediately after surgery and at 6 additional time points between 0 and 180 min of CIT. Kinase activity profiling was performed for all samples using a peptide microarray. MS-based global phosphoproteomics was performed in tumors from 3 patients at 4 time points. Statistical and cluster analyses were performed to analyze changes in kinase activity and phosphoproteome resulting from CIT. Results Unsupervised cluster analysis of kinase activity and phosphoproteome data revealed that samples from the same patients cluster together. Continuous ANOVA analysis of all 7 time points for 5 patient samples resulted in 4 peptides out of 210 (2%) with significantly (p 2) altered signal intensity in time. In 4 out of 5 patients tumor kinase activity was stable with CIT. MS-based phosphoproteomics resulted in the detection of 10,488 different phosphopeptides with on average 6044 phosphopeptides per tumor sample. 2715 phosphopeptides were detected in all samples at time point 0, of which 90 (3.3%) phosphopeptides showed significant changes in intensity with CIT (p 2. Conclusions The vast majority of the phosphoproteome as well as the activity of protein kinases in colorectal cancer resection tissue is stable up to 180 min of CIT and reflects tumor characteristics. However, specific changes in kinase activity with increasing CIT were observed. Therefore, stringent tissue collection procedures are advised to minimize changes in kinase activity during CIT.

Chemical tools to monitor drug-target engagement of endogenously expressed protein kinases are highly desirable for preclinical target validation in drug discovery. Here, we describe a chemical genetics strategy to selectively study target engagement of endogenous kinases. By substituting a serine residue into cysteine at the DFG-1 position in the ATP-binding pocket, we sensitize the non-receptor tyrosine kinase FES towards covalent labeling by a complementary fluorescent chemical probe. This mutation is introduced in the endogenous FES gene of HL-60 cells using CRISPR/Cas9 gene editing. Leveraging the temporal and acute control offered by our strategy, we show that FES activity is dispensable for differentiation of HL-60 cells towards macrophages. Instead, FES plays a key role in neutrophil phagocytosis via SYK kinase activation. This chemical genetics strategy holds promise as a target validation method for kinases., Chemical tools to monitor drug-target engagement of endogenous enzymes are essential for preclinical target validation. Here, the authors present a chemical genetics strategy to study target engagement of endogenous kinases, achieving specific labeling and inactivation of FES kinase to provide insights into FES’ role in neutrophil phagocytosis.

In non‐small cell lung cancer, sensitizing mutations in epidermal growth factor receptor (EGFR) or cMET amplification serve as good biomarkers for targeted therapies against EGFR or cMET, respectively. Here we aimed to determine how this different genetic background would affect the interaction between the EGFR‐inhibitor erlotinib and the cMET‐inhibitor crizotinib. To unravel the mechanism of synergy we investigated the effect of the drugs on various parameters, including cell cycle arrest, migration, protein phosphorylation, kinase activity, the expression of drug efflux pumps, intracellular drug concentrations, and live‐cell microscopy. We observed additive effects in EBC‐1, H1975, and HCC827, and a strong synergism in the HCC827GR5 cell line. This cell line is a clone of the HCC827 cells that harbor an EGFR exon 19 deletion and has been made resistant to the EGFR‐inhibitor gefitinib, resulting in cMET amplification. Remarkably, the intracellular concentration of crizotinib was significantly higher in HCC827GR5 compared to the parental HCC827 cell line. Furthermore, live‐cell microscopy with a pH‐sensitive probe showed a differential reaction of the pH in the cytoplasm and the lysosomes after drug treatment in the HCC827GR5 in comparison with the HCC827 cells. This change in pH could influence the process of lysosomal sequestration of drugs. These results led us to the conclusion that lysosomal sequestration is involved in the strong synergistic reaction of the HCC827GR5 cell line to crizotinib–erlotinib combination. This finding warrants future clinical studies to evaluate whether genetic background and lysosomal sequestration could guide tailored therapeutic interventions., In non‐small cell lung cancer, sensitizing mutations in epidermal growth factor receptor (EGFR) or cMET amplification serve as good biomarkers for targeted therapies against EGFR or cMET, respectively. Here we aimed to determine how this different genetic background would affect the interaction between the EGFR‐inhibitor erlotinib and the cMET‐inhibitor crizotinib. Both genetic characteristics and lysosomal function seem to contribute to the synergistic interaction between crizotinib and erlotinib in the HCC827GR5 cell line, and more research is warranted to determine the exact mechanisms linking these cellular properties.

Chemical tools and methods that report on target engagement of endogenously expressed protein kinases by small molecules in human cells are highly desirable. Here, we describe a chemical genetics strategy that allows the study of non-receptor tyrosine kinase FES, a promising therapeutic target for cancer and immune disorders. Precise gene editing was used in combination with a rationally designed, complementary fluorescent probe to visualize endogenous FES kinase in HL-60 cells. We replaced a single oxygen atom by a sulphur in a serine residue at the DFG-1 position of the ATP-binding pocket in an endogenously expressed kinase, thereby sensitizing the engineered protein towards covalent labeling and inactivation by a fluorescent probe. The temporal control offered by this strategy allows acute inactivation of FES activity both during myeloid differentiation and in terminally differentiated neutrophils. Our results show that FES activity is dispensable for differentiation of HL-60 cells towards macrophages. Instead, FES plays a key role in neutrophil phagocytosis by activation of SYK kinase, a central regulator of immune function in neutrophils. This strategy holds promise as a target validation method for kinases.

O-GlcNAc transferase (OGT) attaches a GlcNAc moiety on specific substrate proteins using UDP-GlcNAc as the sugar donor. This modification can alter protein function by regulating cellular signaling and transcription pathways in response to altered nutrient availability and stress. Specific inhibitors of OGT would be valuable tools for biological studies and lead structures for therapeutics. The existing OGT inhibitors are mainly derived from the sugar donor substrate, but poor cell permeability and off-target effects limit their use. Here, we describe our progress on OGT inhibition based on substrate peptides identified by array screening. Subsequently, bisubstrate inhibitors were prepared by conjugating these peptides to uridine in various ways. In parallel, an in silico fragment screening was conducted to obtain small molecules targeting the UDP binding pocket. After evaluation of the initial hits, one of these small molecules was elaborated into a novel OGT hybrid inhibitor, as the replacement of uridine. The novel compounds inhibit OGT activity with IC50 values in the micromolar range. Abstract. Bibliografija: str. 886-887. ARRS China Scholarship Council

O-GlcNAcylation of proteins regulates important cellular processes. A few reports noted that O-GlcNAcylation exhibits cross-talk with tyrosine phosphorylation. With an activity-based microarray analysis of 256 tyrosine kinase peptide substrates, we found that phosphorylation of 6 peptides by Jak2 inhibits their subsequent O-GlcNAcylation. However, O-GlcNAcylation has no detectable effect on their subsequent phosphorylation. A specific peptide (ZO3_357_371), derived from the ZO-3 protein, was studied in detail. Kinetic results show that the presence of a phosphate at Tyr364 of ZO3_357_371 slows the O-GlcNAcylation of nearby Ser369, while the presence of a GlcNAc at Ser369 has no significant effect on the phosphorylation of this peptide at Tyr364. These findings provide a glimpse into the new paradigm for cellular signaling control by cross-talk. Bibliografija: str. 1881-1883. Abstract. ARRS China Scholarship Council

Introduction Lung squamous cell carcinomas (SCC) typically harbor a strong activation of epidermal growth factor receptor (EGFR) pathway. Since one of the most common resistance mechanisms against EGFR inhibition relies on the activation of cMET parallel signaling, we investigated the efficacy of a dual blockade with erlotinib and crizotinib in EGFR and cMET wild-type lung SCC cell lines. Methods Drug sensitivity assays were performed on LUDLU, SKMES-1, H1703, Calu1 and H520 cells. Further studies included analysis of cell cycle, apoptosis, spheroids, migration and Pathscan intracellular signaling array. Expression of emerging proteins was validated by Western blot and evaluated by immunohistochemistry in tissue-microarrays from lung cancer patients. Results Erlotinib and crizotinib showed additive interaction in Calu1, H520 and SKMES-1, and strong synergism in the LUDLU cells (Combination Index: 0.387), associated to G2/M phase arrest, increased apoptosis, spheroid size reduction and inhibition of migration. Remarkably, this combination decreased the phosphorylation of downstream targets of MAPK and PI3K/Akt/mTOR pathways, with the largest decrease observed for PRAS40 Thr246. Moreover, it reduced the expression of both p-Her3 and p-PRAS40 in the synergistic LUDLU cells. Tissue specimens showed a higher expression of both proteins in SCC compared to adenocarcinoma histology. Conclusions Combining erlotinib and crizotinib led to an additive/synergistic interaction in 4 out of 5 SCC cells. By combining both inhibitors, MAPK and PI3K/Akt/mTOR pathways were strongly inhibited, leading to increased cell death. p-Her3 and p-PRAS40 might be used as markers for determining the synergistic effect and for selecting potential candidates for the combination treatment.

Mitogen-activated protein kinase (MAPK) inhibition with the combination of BRAF (Rapidly Accelerated Fibrosarcoma) and MEK (Mitogen-activated protein kinase kinase) inhibitors has become the standard of first-line therapy of metastatic melanoma harbouring BRAF V600 mutations. However, about half of the patients present with primary resistance while the remaining develop secondary resistance under prolonged treatment. Thus, there is a need for predictive biomarkers for sensitivity and/or resistance to further refine the patient population likely to benefit from MAPK inhibitors. In this study, we explored a top-down approach using a multiplex kinase assay, first, to discover a kinome signature predicting sensitivity, intrinsic and acquired resistance to MAPK inhibitors in melanoma, and second, to understand the mechanism of resistance using cell lines. Pre-dose tissues from patients (four responders and three non-responders to BRAFi monotherapy) were profiled for phosphotyrosine kinase (PTK) and serine-threonine kinase (STK) activities on a PamChip® peptide microarray in the presence and absence of ex vivo BRAFi. In addition, molecular studies were conducted on four sensitive parental lines, their offspring with acquired resistance to BRAFi and two lines with intrinsic resistance. PTK and STK activities in cell lysates were measured in the presence and absence of ex vivo BRAFi and/or MEKi. In tissue lysates, concentration-dependent ex vivo inhibition of STK and PTK activities with dabrafenib was stronger in responders than in non-responders. This difference was confirmed in cell lines comparing sensitive and resistant ones. Interestingly, common features of resistance were increased activity of receptor tyrosine kinases, Proto-oncogene tyrosine-protein kinase Src (Src) family kinases and protein kinase B (PKB, AKT) signalling. These latter results were confirmed by Western blots. While dabrafenib alone showed an inhibition of STK and PTK activities in both tissues and cell lines, the combination of dabrafenib and trametinib showed an antagonism on the STK activities and a synergism on PTK activities, resulting in stronger inhibitions of overall tyrosine kinase activities. Altogether; these data reveal that resistance of tumours and cell lines to MAPK inhibitors can be predicted using a multiplex kinase assay and is associated with an increase in specific tyrosine kinase activities and globally to AKT signalling in the patient’s tissue. Thus, such a predictive kinome signature would help to identify patients with innate resistance to MAPK double inhibition in order to propose other therapies.

O-GlcNAc transferase (OGT) attaches a GlcNAc moiety on specific substrate proteins using UDP-GlcNAc as the sugar donor. This modification can alter protein function by regulating cellular signaling and transcription pathways in response to altered nutrient availability and stress. Specific inhibitors of OGT would be valuable tools for biological studies and lead structures for therapeutics. The existing OGT inhibitors are mainly derived from the sugar donor substrate, but poor cell permeability and off-target effects limit their use. Here, we describe our progress on OGT inhibition based on substrate peptides identified by array screening. Subsequently, bisubstrate inhibitors were prepared by conjugating these peptides to uridine in various ways. In parallel, an in silico fragment screening was conducted to obtain small molecules targeting the UDP binding pocket. After evaluation of the initial hits, one of these small molecules was elaborated into a novel OGT hybrid inhibitor, as the replacement of uridine. The novel compounds inhibit OGT activity with IC(50) values in the micromolar range.

Phosphatases and kinases regulate the crucial phosphorylation post-translational modification. In spite of their similarly important role in many diseases and therapeutic potential, phosphatases have received arguably less attention. One reason for this is a scarcity of high-throughput phosphatase assays. Herein, a new real-time, dynamic protein tyrosine phosphatase (PTP) substrate microarray assay measuring product formation is described. PTP substrates comprising a novel 3-nitrophosphotyrosine residue are immobilized in discrete spots. After reaction catalyzed by a PTP a 3-nitrotyrosine residue is formed that can be detected by specific, sequence-independent antibodies. The resulting microarray was successfully evaluated with a panel of recombinant PTPs and cell lysates, which afforded results comparable to data from other assays. Its parallel nature, convenience, and low sample requirements facilitate investigation of the therapeutically relevant PTP enzyme family. Keeping it real: The activity of important protein tyrosine phosphatases has been monitored in real time in parallel with a novel substrate microarray through formation of 3-nitrotyrosine (see figure). Copyright © 2013 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim.

A high-throughput method for the determination of the kinetics of protein tyrosine phosphatase (PTP) activity in a microarray format is presented, allowing real-time monitoring of the dephosphorylation of a 3-nitro-phosphotyrosine residue. The 3-nitro-phosphotyrosine residue is incorporated in potential PTP substrates. The peptide substrates are immobilized onto a porous surface in discrete spots. After dephosphorylation by a PTP, a 3-nitrotyrosine residue is formed that can be detected by a specific, sequence-independent antibody. The rate of dephosphorylation can be measured simultaneously on 12 microarrays, each comprising three concentrations of 48 clinically relevant peptides, using 1.0-5.0 μg of protein from a cell or tissue lysate or 0.1-2.0 μg of purified phosphatase. The data obtained compare well with solution phase assays involving the corresponding unmodified phosphotyrosine substrates. This technology, characterized by high-throughput (12 assays in less than 2 h), multiplexing and low sample requirements, facilitates convenient and unbiased investigation of the enzymatic activity of the PTP enzyme family, for instance by profiling of PTP substrate specificities, evaluation of PTP inhibitors and pinpointing changes in PTP activity in biological samples related to diseases.

The main responses of P2Y(1) ligation are platelet shape change and transient aggregation while P2Y(12) ligation amplifies P2Y(1)-induced aggregation and accelerates aggregation, secretion and thromboxane A(2) production induced by other agonist-receptor complexes. We searched for new targets of P2Y signalling using micro-arrays with 144 peptides representing known phosphosites of protein tyrosine kinases. ADP induced phosphorylation of peptides representing surface receptors, second messenger enzymes and cytoskeletal proteins. Strong phosphorylation was found in peptides representing Ephrin-receptor family members. Blockade of P2Y(1/12) inhibited phosphorylation of EphA4- and EphB1-peptides on micro-arrays. The EphA2/4 inhibitor 2,5-dimethylpyrrolyl benzoic acid derivative interfered with P2Y(1/12)-induced EphA4 phosphorylation, left P2Y(1)-induced aggregation unchanged but inhibited with P2Y(12)-induced secretion, second phase aggregation and thrombus formation on collagen at 1600 s(-1). These results show that platelet EphA4 is an important intermediate in P2Y(12)-induced granule secretion.

Multivalency is an important phenomenon in protein–carbohydrate interactions. In order to evaluate glycodendrimers as multivalent inhibitors of carbohydrate binding proteins, we displayed them on a microarray surface. Valencies were varied from 1 to 8, and corrections were made for the valencies so that all surfaces contained the same amount of the sugar ligand. Five different carbohydrates were attached to the dendrimers. A series of fluorescent lectins was evaluated, and for each of them a binding profile was obtained from a single experiment showing both the specificity of the lectin for a certain sugar and whether it prefers multivalent ligands or not. Very distinct binding patterns were seen for the various lectins. The results were rationalized with respect to the interbinding distances of the lectins.

Nuclear receptors (NRs) are major targets for drug discovery and have key roles in development and homeostasis as well as in many diseases such as obesity, diabetes, and cancer. NRs are ligand-dependent transcription factors that need to work in concert with so-called transcriptional coregulators, including corepressors and coactivators, to regulate transcription. Upon ligand binding, NRs undergo a conformational change, which alters their binding preference for coregulators. Short alpha-helical sequences in the coregulator proteins, LXXLL (in coactivators) or LXXXIXXXL (in corepressors), are essential for the NR-coregulator interactions. However, little is known on how specificity is dictated. To obtain a comprehensive overview of NR-coregulator interactions, we used a microarray approach based on interactions between NRs and peptides derived from known coregulators. Using the peroxisome proliferator-activated receptor gamma (PPARgamma) as a model NR, we were able to generate ligand-specific interaction profiles (agonist rosiglitazone versus antagonist GW9662 versus selective PPARgamma modulator telmisartan) and characterize NR mutants and isotypes (PPARalpha, -beta/delta, and -gamma). Importantly, based on the NR-coregulator interaction profile, we were able to identify TRIP3 as a novel regulator of PPARgamma-mediated adipocyte differentiation. These findings indicate that NR-coregulator interaction profiling may be a useful tool for drug development and biological discovery.

Kinase inhibitors are increasingly important in drug development. Because the majority of current inhibitors target the conserved ATP-binding site, selectivity might become an important issue. This could be particularly problematic for the potential drug target protein kinase C (PKC), of which twelve isoforms with high homology exist in humans. A strategy to increase selectivity is to prepare bisubstrate-based inhibitors that target the more selective peptide-binding site in addition to the ATP-binding site. In this paper a generally applicable, rapid methodology is presented to discover such bisubstrate-based leads. Dynamic peptide microarrays were used to find peptide-binding site inhibitors. These were linked with chemoselective click chemistry to an ATP-binding site inhibitor, and this led to novel bisubstrate structures. The peptide microarrays were used to evaluate the resulting inhibitors. Thus, novel bisubstrate-based inhibitors were obtained that were both more potent and selective compared to their constituent parts. The most promising inhibitor has nanomolar affinity and selectivity towards PKCtheta amongst three isozymes.

A microarray-based mix-and-measure, nonradioactive multiplex method with real-time detection was used for substrate identification, assay development, assay optimisation, and kinetic characterization of protein kinase A (PKA). The peptide arrays included either up to 140 serine/threonine-containing peptides or a concentration series of a smaller number of peptides. In comparison with existing singleplex assays, data quality was high, variation in assay conditions and reagent consumption were reduced considerably, and assay development could be accelerated because phosphorylation kinetics were monitored simultaneously on 4, 12, or 96 arrays. PKA was shown to phosphorylate many peptides containing known PKA phosphorylation sites as well as some new substrates. The kinetic behavior of the enzyme and the mechanism of inhibition by AMP-PNP, staurosporin, and PKA inhibitor peptide on the peptide microarray correlated well with data from homogeneous assays. Using this multiplex setup, we showed that the kinetic parameters of PKA and the potency of PKA inhibitors can be affected by the sequence of the peptide substrate. The technology enables kinetic monitoring of kinase activity in a multiplex setting such as a cell or tissue lysate. Finally, this high-throughput method allows fast identification of peptide substrates for serine/threonine kinases that are still uncharacterized.

In the developing embryo, cell growth, differentiation, and migration are strictly regulated by complex signaling pathways. One of the most important cell signaling mechanisms is protein phosphorylation on tyrosine residues, which is tightly controlled by protein-tyrosine kinases and protein-tyrosine phosphatases. Here we investigated endogenous phosphotyrosine signaling in developing zebrafish embryos. Tyrosine phosphorylated proteins were immunoaffinity-purified from zebrafish embryos at 3 and 5 days postfertilization and identified by multidimensional LC-MS. Among the identified proteins were tyrosine kinases, including Src family kinases, Eph receptor kinases, and focal adhesion kinases, as well as the adaptor proteins paxillin, p130Cas, and Crk. We identified several known and some unknown in vivo tyrosine phosphorylation sites in these proteins. Whereas most immunoaffinity-purified proteins were detected at both developmental stages, significant differences in abundance and/or phosphorylation state were also observed. In addition, multiplex in vitro kinase assays were performed by incubating a microarray of peptide substrates with the lysates of the two developmental stages. Many of the in vivo observations were confirmed by this on-chip in vitro kinase assay. Our experiments are the first to show that global tyrosine phosphorylation-mediated signaling can be studied at endogenous levels in complex multicellular organisms.

Divalent peptidomimetic compounds can bind a signal transduction protein and provide an avenue towards uncovering compounds capable of influencing or interfering with protein–protein interactions in general. High-affinity binding was realized by linking two relatively weakly interacting monophosphorylated peptides with an oligoethylene glycol spacer (see structure) the length of which was tuned by design.

Introduction RAF-MAPK pathway inhibition with BRAF inhibitors or the combination of BRAF and MEK inhibitors has become the mainstay of therapy of metastatic melanoma harboring BRAF V600 mutations with a response rate as high as 50-75 %. A minority of patients present with primary resistance while all patients develop secondary resistance. We explored a reversed translational approach using a novel multiplex kinase assay to first identify individuals not responding to therapy using baseline biopsy samples and second to understand the mechanism of resistance using patient-derived cell lines. Method Pre-dose tissues from 4 non-responders and 3 responders to vemurafenib monotherapy, all harboring the V600E mutation, were profiled for Serine-Threonine Kinase (STK) activities on a PamChip® peptide microarray in the presence and absence of dabrafenib. For molecular studies three melanoma cell lines, all with BRAF V600E mutations, were made resistant to vemurafenib. Protein Tyrosine Kinase (PTK) and STK activities in lysates of sensitive or resistant cells were measured +/- dabrafenib, trametinib or both. Lysates were blotted against a selected panel of (phospho)proteins. Results In tissue lysates, concentration-dependent ex vivo inhibition with dabrafenib was stronger in the patients that were clinical responders than in non-responders. This difference in inhibition of STK activity by dabrafenib was confirmed in the cell lines that were made resistant against vemurafenib. IC50s for the sensitive cell lines were below 0.5 µM and varied from 10-21 µM for cell lines with acquired resistance. PTK activity was increased in resistant cell lines. STK activity was lower, but large differences exist among the cell lines. Common features of resistance were increased activity of receptor tyrosine kinases, Src family kinases and AKT signaling. These results were confirmed by Western blot analysis. Dabrafenib (10 µM) caused inhibition of STK activity while trametinib (10 µM) gave some activation. Dabrafenib (0.5 µM) gave strong inhibition of PTK activity, both in tissue and cell lines. Trametinib had hardly any inhibitory effect. Interestingly, the combination of dabrafenib and trametinib had an antagonistic effect on the STK activity and a synergistic effect on PTK activity, resulting in a stronger inhibition of kinase activity. Conclusion Resistance of tumors and cell lines to vemurafenib can affect kinase activity profiles as detected pre-dose using a multiplex kinase assay. Dabrafenib inhibition of PTK and STK activity is stronger in sensitive cell lines. The overall activation of kinases in a lysate upon MEK inhibition suggests MEK as suppressor of kinase activity. Resistance to MAPK pathway inhibitors could be linked to an increase in tyrosine kinase activity and AKT pathway activity. Biomarkers are needed to identify patients in need of combined MAPK pathway inhibition. Citation Format: Riet Hilhorst, Mohammed Krayem, Adrienne van den Berg, Fabrice Journe, Liesbeth Hovestad-Bijl, Tim van den Hooven, Rik de Wijn, Philippe Aftimos, Ghanem Ghanem, Rob Ruijtenbeek. Discovery of a kinome signature predicting sensitivity and resistance to RAF-MAPK pathway inhibitors in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3163. doi:10.1158/1538-7445.AM2017-3163

The active metabolite (JM118) of the oral platinum analog satraplatin (JM216) was investigated for potential synergism with erlotinib, an epidermal growth factor receptor (EGFR) inhibitor. JM118 sensitivity of 7 cancer cell lines (ovarian: 2008, A2780; colon: Lovo92, WiDr; lung: A549, SW1573; epidermoid: A431), was enhanced most pronounced when JM118 preceded erlotinib, which was associated with increased formation of DNA-platinum adducts. The combination increased G2/M phase accumulation and enhanced apoptosis. JM118 increased the phosphorylation of the cell cycle proteins CDK2 and CHK1 after 24 hr exposure. JM118/erlotinib enhanced Erk and Akt phosphorylation after 2 hr. JM118 significantly decreased the phosphorylation of PTEN, VEGFR, EPHA1, ERBB4, FGF-R, andSTAT3 by 20 (PTEN) to >90% (STAT3). Conclusion: Erlotinib enhanced the effects of JM118, even in cells with mutations in Ras. The mechanism of synergy involved a combination of effects on platinum-DNA adduct formation, cell cycle distribution and signaling.

Peptoid-peptide hybrids are oligomeric peptidomimetics that contain one or more N-substituted glycine residues. In these hybrids, the side chains of one or several amino acids are "shifted" from the alpha-carbon atom to the amide nitrogen atom. A library of phosphorylated peptoid-peptide hybrids derived from the sequence pTyr-Glu-Thr-Leu was synthesized and tested for binding to the tandem SH2 domain of the protein tyrosine kinase Syk. A considerable influence of the side chain position was observed. Compounds 19-21, 24, and 25 comprising a peptoid NpTyr and/or NGlu residue did not show any binding. Compounds 22, 23, and 26 containing an NhThr (hThr=homothreonine) and/or NLeu peptoid residue showed binding with IC(50) values that were only five to eight times higher than that of the tetrapeptide lead compound 18. These data show that side chain shifting is possible with retention of binding capacity, but only at the two C-terminal residues of the tetramer. This method of a peptoid scan using peptoid-peptide hybrids appears to be very useful to explore to what extent a peptide sequence can be transformed into a peptoid while retaining its affinity.

Peptide microarray technology can be used to identify substrates for recombinant kinases, to measure kinase activity and changes thereof in cell lysates and lysates from fresh frozen (tumor) tissue. The effect of kinase inhibitors on the kinase activities in relevant tissues can be investigated as well. The method for performing experiments on dynamic peptide microarrays with real-time readout is described, as well as the influence of assay parameters and suggestions for optimization of experiments.

A number of pro-moieties 8a - • designed for prodrug preparation have been synthesized (chart 2). The pro-moieties, containing a glucuronyl carbamate group linked to a spacer possessing a terminal carboxylic acid group, have been synthesized from isocyanates 6 and anomerically unprotected glucuronic acids I0 (chart 2). The requisite isocyanates had to be prepared using the Curtius rearrangement. Glucuronyl carbamates proved to be excellent substrates for human ~-glucuronidase. The pro-moieties 8a - e can be coupled to hydroxy- or amino group containing dmgs, The resulting prodrugs are designed to be activated by ~-glucuronidase (chart 1) and to be used in ADEPT. Application is demonstrated with the synthesis of daunomycin prodrugs 12a - e (chart 3). The lack of selectivity of cytostatic agents for tumor cells is a serious drawback in conventional cancer chemotherapy. This major problem dictates research in the field of selective chemotherapy. In this context the selectivity of monoclonal antibodies (mAb) for their target tumor cells can be used to selectively deliver cytotoxicity to tumor tissue. In the ADEPT 1 (Antibody Directed Enzyme Prodrug Therapy) approach, specific enzymes are delivered to tumor sites by using mAb's. These enzymes are selected to convert a prodrug, which is administered in a second step after localization of the mAb-enzyme conjugate, to the parent cytotoxic drug 2. We focused our attention on the (3-glucuronidase (GUS) / (3-glucuronide prodrug 3 couple. This system possesses the advantage that GUS is a human lysosomal enzyme present only at minor concentrations in blood 4 and that (3-glucuronides are highly polar compounds which have a poor cell-membrane permeability. In our approach towards glucuronide based prodrugs, a (3-glucuronide is attached to a spacer molecule via a carbamate linkage. A spacer was used in order to couple the 13-glucuronide to a hydroxy- or amino group containing drug as well as to facilitate GUS hydrolysis 5.

A functional detection assay was developed for Escherichia coli secreted shiga-like toxin based on antibody capture and visualization with a multivalent galabiose ligand. It was possible to detect verotoxin in medically relevant E. coli samples in a dose dependent fashion. This method is a new step towards measuring functional protein levels in complex mixtures, which can be used for diagnostic purposes in a clinical setting.

Kinases present an attractive target for drug development, since they are involved in vital cellular processes and are implicated in a variety of diseases, such as cancer and diabetes. However, obtaining selectivity for a specific kinase over others is difficult since many current kinase inhibitors exclusively target the highly conserved kinase ATP binding domain. Previously, a microarray-based strategy to discover so-called bisubstrate-based inhibitors that target the more specific peptide binding groove in addition to the ATP binding site was described. One attractive feature of this strategy is the opportunity to tune the selectivity of these inhibitors by systematically varying components. In an extension to this previous work, this study explores the potential of this guided selectivity modulation, leading to a series of inhibitors with different selectivity profiles against highly homologous protein kinase C (PKC) isozymes. Of the inhibitors studied, most exhibited improved potency and selectivity compared with their constituent parts. Furthermore, the selectivity was found to be tunable either through modification of the pseudosubstrate peptide (peptide binding groove) or the ATP-competitive part (ATP binding site). In a number of cases, the selectivity of the construct could be predicted from the initial peptide substrate profiling experiment. Since this strategy is applicable to all kinase sets, it could be used to rapidly develop uniquely selective inhibitors.

Divalent peptidomimetic compounds can bind a signal transduction protein and provide an avenue towards uncovering compounds capable of influencing or interfering with protein–protein interactions in general. High-affinity binding was realized by linking two relatively weakly interacting monophosphorylated peptides with an oligoethylene glycol spacer (see structure) the length of which was tuned by design.

Dendrimers were fitted out with up to eight mannose moieties by "click" chemistry. They were subsequently attached to aluminum oxide chips via a spacer that was linked to the dendrimer core; this resulted in a microarray of glycodendrimers. Binding of the glycodendrimers to the fluorescent lectins ConA and GNA was observable in real time. In a single experiment it was possible to observe the multivalency enhancement or cluster effect in the binding event. This effect was small for ConA, in agreement with its widely spaced binding sites, whereas it was large for GNA, with its twelve much more closely spaced binding sites. The dendrimer-fitted chip represents a valuable screening tool for multivalency effects. Furthermore kinetic and thermodynamic data on binding events can be deduced. Inhibition experiments are also possible with the system as was shown for ConA with alpha-methyl mannose as the inhibitor.

Background: Most differentiated non medullary thyroid cancers (DTC) are curatively treated by surgery and radio-active iodine ablation therapy. A subset of patients shows recurrence due to a loss of iodine transport. Two main subgroups of recurrent DTC are seen: papillary thyroid cancers (PTC) with somatic BRAF mutations (V600E) and oncocytic follicular cancer. Recurrent DTC are clinically treated by multi-kinase inhibitors such as sorafenib, with a low affinity for BRAF V600E. Vemurafenib and dabrafenib were specifically designed against this mutant. The aim of this study was twofold: - Can benign and malign DTC be classified based on kinase activity profiles? - Do sorafenib (and regorafenib) show different inhibition profiles than dabrafenib? Methods: Tissue cryosections from fresh frozen thyroid tumors were lysed. All tumor specimens were analyzed for BRAF mutations. Serine/threonine kinase (STK) activity profiles of the lysates (0.5 μg protein per array) were generated on PamChip® peptide microarrays, comprising peptide sequences from known human phosphorylation sites. The ex vivo effect of BRAF inhibitors sorafenib, regorafenib and dabrafenib on kinase activity profiles of 14 PTC's was determined as well. Data were analysed with Bionavigator software. Results: A classifier built on the STK kinase activity profiles of 57 thyroid cancer samples was able to classify malignant and benign tumors with a limited error rate. Leave One Out Cross Validation classified 26/35 of malignant and 17/22 of benign samples correctly. Kinase inhibition profiles of PTC's with sorafenib and regorafenib did not discrimate V600E mutants from wild type tumors whereas with dabrafenib 34/144 peptides were identified that potentially differentiated the groups. Conclusions: Serine/threonine kinase activity profiling appears to be able to differentiate benign and malignant thyroid tumors. Ex vivo spiking in of kinase inhibitors shows differential inhibition in tumors with a somatic BRAF mutation. Potentially, an industrial prediction platform can be envisioned for testing of novel drugs in tumor tissue. Whether individual patient responses against registered kinase inhibitors can be predicted must be investigated. Citation Format: Maria H. Hilhorst, Adrienne van den Berg, Tom van Wezel, Tim Kievits, Piet J. Boender, Rik de Wijn, Rob Ruijtenbeek, Wim Corver, Hans Morreau. Kinase activity profiles distinguish papillary thyroid cancers with and without BRAF V600E mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4322. doi:10.1158/1538-7445.AM2015-4322

Nano-electrospray tandem mass spectrometry (nano-ES-MS/MS) was used to record collision-induced dissociation (CID) spectra of a set of peptoid–peptide hybrids and the complete peptoid derived from the phosphopeptide Ac-pTyr-Glu-Thr-Leu-NH2 (1). The presence of B and Y″-type fragment ions in the tandem mass spectra of the protonated molecular ions [M + H]+ allowed confirmation of sequence similar to mass spectrometric sequence analysis in peptides. In the isomeric peptoid compounds studied, one or several amino acid residues were replaced by peptoid residues (N-substituted glycine residues), which resulted in characteristic tandem mass spectra with differently increased relative abundances of Y″-and B-type fragment ions. The increment of a particular Y″-ion was directly correlated to the position of a peptoid residue present. In addition to these increased peak intensities, other characteristic peaks were also observed compared with the spectrum of reference peptide 1. When a peptoid phosphotyrosine was incorporated, the presence of this residue was apparent from the occurrence of a relatively intense peak at m/z 187 representing the positively charged side-chain of phosphotyrosine, which was almost absent in the spectrum of the reference peptide 1. Since the threonine side-chain had to be translated into the homo peptoid analog this substitution was apparent from the presence of [M + H]+ and fragment ions 14 mass units higher than observed in the spectrum of the reference phosphopeptide 1. The presence of an NLeu peptoid residue could be confirmed by the specific fragmentation of the immonium ion showing an intense peak in its tandem mass spectrum at m/z 57, which results from the loss of an neutral imine molecule leading to a positively charged [C4H9]+ ion. By means of these mass spectrometric characteristics, all isomeric peptoid compounds could be distinguished from each other and characterized. The methods used appear to be very useful in future studies of peptoids and peptoid–peptide hybrids. Copyright © 2001 John Wiley & Sons, Ltd.

We explored the use of a newly developed cuvette-based surface plasmon resonance (SPR) instrument (IBIS) to study peptide–protein interactions. We studied the interaction between the SH2 domain of lck and a phosphotyrosine peptide EPQY*EEIPIYL which was immobilized on a sensor chip. No indications for mass transport limitation (MTL) were observed when standard kinetic approaches were used. However, addition of competing peptide during dissociation revealed a high extent of rebinding. A dissociation rate constant ( k d ) of 0.6 ± 0.1 s −1 was obtained in the presence of large amounts of peptide. A simple bimolecular binding model, applying second-order kinetics for the cuvette system, could not adequately describe the data. Fits were improved upon including a step in the model which describes diffusion of the SH2 domain from the bulk to the sensor, especially for a surface with high binding capacity. From experiments in glycerol-containing buffers, it appeared that the diffusion rate decreased with higher viscosity. It is demonstrated that MTL during association and dissociation can be described by the same diffusion rate. A binding constant ( K D ) of 5.9 ± 0.8 nM was obtained from the SPR equilibrium signals by fitting to a Langmuir binding isotherm, with correction for loss of free analyte due to binding. An association rate constant k a of 1.1(±0.2) × 10 8 M −1 · s −1 was obtained from k d / K D . The values for k a and k d obtained in this way were 2–3 orders larger than that from standard kinetic analysis, ignoring MTL. We conclude that in a cuvette the extent of MTL is comparable to that in a flow system.

Tyrosine kinases pose attractive targets for cancer therapy, as their abnormal signaling activity results in enhanced proliferation, migration and angiogenesis. Previously we have been able to generate a comprehensive description of the biological kinase activity present in pediatric brain tumor tissue lysate by the application of a peptide microarray containing 144 different tyrosine kinase peptide substrates. To establish which peptide substrates present on the array are most sensitive and discriminative for VEGFR activity we measured the peptide phosphorylation applying recombinant VEGFR1, 2 and 3. Furthermore, a panel of recombinant kinases with highly diverse VEGFR sequence homology was included to study substrate specificity. An aminopeptidase A (APA) derived peptide proved to be highly phosphorylated by VEGFR 1 and 2. No phosphorylation of this peptide could be observed applying any of the other kinases, arguing that this peptide is discriminative for VEGFR1 and 2 activity. In the peptide array data of pediatric brain tumors, phosphorylation of the APA peptide was observed, suggestive for VEGFR activation. Based on these results we hypothesize that APA can be activated through direct phosphorylation by VEGFR2 and exert functional effects on endothelial cell proliferation and migration. To test this hypothesis we stimulated human umbilical vein endothelial cells (HUVECs) with VEGF and observed phosphorylation of Aminopeptidase A. Literature states that inhibition of APA suppresses VEGF-induced migration and proliferation of endothelial cells and inhibits angiogenesis. Based on our data we hypothesize that these APA-mediated effects of VEGF stimulation are induced through direct activation by VEGFR2 kinase activity. Concluding, peptide microarray technology is able to provide a selective view on tyrosine kinase activation, as has been shown here for VEGFR1 and 2. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2377.

Efficient strategies for the introduction of arginine residues featuring acetylene or azide moieties in their side chains are described. The substituents are introduced in a way that maintains the basicity of the guanidine moiety. The methodology can be used e.g. for non-invasive labeling of arginine-containing peptides. Its applicability is demonstrated by the introduction of 'click' handles into a Protein Kinase C (PKC) pseudosubstrate peptide, and the subsequent preparation and evaluation of a novel bisubstrate-based inhibitor based on such a peptide.

Background Protein-tyrosine kinases (PTKs) regulate virtually all biological processes. PTKs phosphorylate substrates in a sequence-specific manner and relatively short peptide sequences determine selectivity. Here, we developed new technology to determine PTK activity profiles using peptide arrays. The zebrafish is an excellent model system to investigate signaling in the whole organism, given its wealth of genetic tools, including morpholino-mediated knock down technology. We used zebrafish embryo lysates to determine PTK activity profiles, thus providing the unique opportunity to directly compare the effect of protein knock downs on PTK activity profiles on the one hand and phenotypic changes on the other. Methodology We used multiplex arrays of 144 distinct peptides, spotted on a porous substrate, allowing the sample to be pumped up and down, optimizing reaction kinetics. Kinase reactions were performed using complex zebrafish embryo lysates or purified kinases. Peptide phosphorylation was detected by fluorescent anti-phosphotyrosine antibody binding and the porous chips allowed semi-continuous recording of the signal. We used morpholinos to knock down protein expression in the zebrafish embryos and subsequently, we determined the effects on the PTK activity profiles. Results and Conclusion Reproducible PTK activity profiles were derived from one-day-old zebrafiish embryos. Morpholino-mediated knock downs of the Src family kinases, Fyn and Yes, induced characteristic phenotypes and distinct changes in the PTK activity profiles. Interestingly, the peptide substrates that were less phosphorylated upon Fyn and Yes knock down were preferential substrates of purified Fyn and Yes. Previously, we demonstrated that Wnt11 knock down phenocopied Fyn/Yes knock down. Interestingly, Wnt11 knock down induced similar changes in the PTK activity profile as Fyn/Yes knock down. The control Nacre/Mitfa knock down did not affect the PTK activity profile significantly. Our results indicate that the novel peptide chip technology can be used to unravel kinase signaling pathways in vivo.