Autophosphorylation Assays Research Articles

Genetic Burden of TNNI3K in Diagnostic Testing of Patients With Dilated Cardiomyopathy and Supraventricular Arrhythmias.

Genetic variants in TNNI3K (troponin-I interacting kinase) have previously been associated with dilated cardiomyopathy (DCM), cardiac conduction disease, and supraventricular tachycardias. However, the link between TNNI3K variants and these cardiac phenotypes shows a lack of consensus concerning phenotype and protein function. We describe a systematic retrospective study of a cohort of patients undergoing genetic testing for cardiac arrhythmias and cardiomyopathy including TNNI3K. We further performed burden testing of TNNI3K in the UK Biobank. For 2 novel TNNI3K variants, we tested cosegregation. TNNI3K kinase function was estimated by TNNI3K autophosphorylation assays. We demonstrate enrichment of rare coding TNNI3K variants in DCM patients in the Amsterdam cohort. In the UK Biobank, we observed an association between TNNI3K missense (but not loss-of-function) variants and DCM and atrial fibrillation. Furthermore, we demonstrate genetic segregation for 2 rare variants, TNNI3K-p.Ile512Thr and TNNI3K-p.His592Tyr, with phenotypes consisting of DCM, cardiac conduction disease, and supraventricular tachycardia, together with increased autophosphorylation. In contrast, TNNI3K-p.Arg556_Asn590del, a likely benign variant, demonstrated depleted autophosphorylation. Our findings demonstrate an increased burden of rare coding TNNI3K variants in cardiac patients with DCM. Furthermore, we present 2 novel likely pathogenic TNNI3K variants with increased autophosphorylation, suggesting that enhanced autophosphorylation is likely to drive pathogenicity.

CDK19-related disorder results from both loss-of-function and gain-of-function de novo missense variants

PurposeTo expand the recent description of a new neurodevelopmental syndrome related to alterations in CDK19. MethodsIndividuals were identified through international collaboration. Functional studies included autophosphorylation assays for CDK19 Gly28Arg and Tyr32His variants and in vivo zebrafish assays of the CDK19G28R and CDK19Y32H. ResultsWe describe 11 unrelated individuals (age range: 9 months to 14 years) with de novo missense variants mapped to the kinase domain of CDK19, including two recurrent changes at residues Tyr32 and Gly28. In vitro autophosphorylation and substrate phosphorylation assays revealed that kinase activity of protein was lower for p.Gly28Arg and higher for p.Tyr32His substitutions compared with that of the wild-type protein. Injection of CDK19 messenger RNA (mRNA) with either the Tyr32His or the Gly28Arg variants using in vivo zebrafish model significantly increased fraction of embryos with morphological abnormalities. Overall, the phenotype of the now 14 individuals with CDK19-related disorder includes universal developmental delay and facial dysmorphism, hypotonia (79%), seizures (64%), ophthalmologic anomalies (64%), and autism/autistic traits (56%). ConclusionCDK19 de novo missense variants are responsible for a novel neurodevelopmental disorder. Both kinase assay and zebrafish experiments showed that the pathogenetic mechanism may be more diverse than previously thought.

A high-throughput integrated microfluidics method enables tyrosine autophosphorylation discovery

Autophosphorylation of receptor and non-receptor tyrosine kinases is a common molecular switch with broad implications for pathogeneses and therapy of cancer and other human diseases. Technologies for large-scale discovery and analysis of autophosphorylation are limited by the inherent difficulty to distinguish between phosphorylation and autophosphorylation in vivo and by the complexity associated with functional assays of receptors kinases in vitro. Here, we report a method for the direct detection and analysis of tyrosine autophosphorylation using integrated microfluidics and freshly synthesized protein arrays. We demonstrate the efficacy of our platform in detecting autophosphorylation activity of soluble and transmembrane tyrosine kinases, and the dependency of in vitro autophosphorylation assays on membranes. Our method, Integrated Microfluidics for Autophosphorylation Discovery (IMAD), is high-throughput, requires low reaction volumes and can be applied in basic and translational research settings. To our knowledge, it is the first demonstration of posttranslational modification analysis of membrane protein arrays.

Expression and Characterization of the Receptor Tyrosine Kinase Tie2

Nearly one‐third of human kinases are implicated in disease, and this enzyme class has proved to be an enormously successful drug target for combating many diseases. Kinases are key regulators in signaling pathways responsible for such processes as growth, proliferation, and angiogenesis. Tie2 is an endothelium‐specific receptor tyrosine kinase (RTK) responsible for angiogenesis and vasculature maintenance. Mutations in this enzyme can cause venous malformations for which no current therapies are available, and Tie2 has been proposed to be a target for anticancer therapies. However, the molecular mechanisms of Tie2 activity are not well understood. Here, we developed a robust purification strategy using heterologous expression in insect cells, optimizing expression and purification with a post‐infection incubation time of 72 hours and utilization of affinity chromatography. We have created autophosphorylation assays to establish catalytic rates of wild‐type Tie2 and mutants implicated in disease. We hypothesize that mutant Tie2 will have faster rates of autophosphorylation, likely through stabilization of the active conformation of the protein. These studies in kinase activity can improve our understanding of the catalytic features of wild‐type and mutant Tie2, and can inform the development of therapeutic targets for vascular disease.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fluorescence Measurement of Kinetics of CheY Autophosphorylation with Small Molecule Phosphodonors.

The Escherichia coli chemotaxis protein CheY is a model receiver domain containing a native tryptophan residue that serves as a fluorescent probe for CheY autophosphorylation with small molecule phosphodonors. Here we describe fluorescence measurement of apparent bimolecular rate constants for reaction of wild type and mutant CheY with phosphodonors acetyl phosphate, phosphoramidate, or monophosphoimidazole. Step-by-step protocols to synthesize phosphoramidate (K+ salt) and monophosphoimidazole (Na+ salt), which are not commercially available, are provided. Key factors to consider in developing autophosphorylation assays for other response regulators are also discussed.

Heme-independent Redox Sensing by the Heme-Nitric Oxide/Oxygen-binding Protein (H-NOX) from Vibrio cholerae

Heme nitric oxide/oxygen (H-NOX)-binding proteins act as nitric oxide (NO) sensors among various bacterial species. In several cases, they act to mediate communal behavior such as biofilm formation, quorum sensing, and motility by influencing the activity of downstream signaling proteins such as histidine kinases (HisKa) in a NO-dependent manner. An H-NOX/HisKa regulatory circuit was recently identified in Vibrio cholerae, and the H-NOX protein has been spectroscopically characterized. However, the influence of the H-NOX protein on HisKa autophosphorylation has not been evaluated. This process may be important for persistence and pathogenicity in this organism. Here, we have expressed and purified the V. cholerae HisKa (HnoK) and H-NOX in its heme-bound (holo) and heme-free (apo) forms. Autophosphorylation assays of HnoK in the presence of H-NOX show that the holoprotein in the Fe(II)-NO and Fe(III) forms is a potent inhibitor of HnoK. Activity of the Fe(III) form and aerobic instability of the Fe(II) form suggested that Vibrio cholerae H-NOX may act as a sensor of the redox state as well as NO. Remarkably, the apoprotein also showed robust HnoK inhibition that was dependent on the oxidation of cysteine residues to form disulfide bonds at a highly conserved zinc site. The importance of cysteine in this process was confirmed by mutagenesis, which also showed that holo Fe(III), but not Fe(II)-NO, H-NOX relied heavily upon cysteine for activation. These results highlight a heme-independent mechanism for activation of V. cholerae H-NOX that implicates this protein as a dual redox/NO sensor.

Distal Hydrogen-bonding Interactions in Ligand Sensing and Signaling by Mycobacterium tuberculosis DosS

Mycobacterium tuberculosis DosS is critical for the induction of M. tuberculosis dormancy genes in response to nitric oxide (NO), carbon monoxide (CO), or hypoxia. These environmental stimuli, which are sensed by the DosS heme group, result in autophosphorylation of a DosS His residue, followed by phosphotransfer to an Asp residue of the response regulator DosR. To clarify the mechanism of gaseous ligand recognition and signaling, we investigated the hydrogen-bonding interactions of the iron-bound CO and NO ligands by site-directed mutagenesis of Glu-87 and His-89. Autophosphorylation assays and molecular dynamics simulations suggest that Glu-87 has an important role in ligand recognition, whereas His-89 is essential for signal transduction to the kinase domain, a process for which Arg-204 is important. Mutation of Glu-87 to Ala or Gly rendered the protein constitutively active as a kinase, but with lower autophosphorylation activity than the wild-type in the Fe(II) and the Fe(II)-CO states, whereas the E87D mutant had little kinase activity except for the Fe(II)-NO complex. The H89R mutant exhibited attenuated autophosphorylation activity, although the H89A and R204A mutants were inactive as kinases, emphasizing the importance of these residues in communication to the kinase core. Resonance Raman spectroscopy of the wild-type and H89A mutant indicates the mutation does not alter the heme coordination number, spin state, or porphyrin deformation state, but it suggests that interdomain interactions are disrupted by the mutation. Overall, these results confirm the importance of the distal hydrogen-bonding network in ligand recognition and communication to the kinase domain and reveal the sensitivity of the system to subtle differences in the binding of gaseous ligands.

Activation of PKR by short stem–loop RNAs containing single-stranded arms

Protein kinase R (PKR) is a central component of the innate immunity antiviral pathway and is activated by dsRNA. PKR contains a C-terminal kinase domain and two tandem dsRNA binding domains. In the canonical activation model, binding of multiple PKR monomers to dsRNA enhances dimerization of the kinase domain, leading to enzymatic activation. A minimal dsRNA of 30 bp is required for activation. However, short (∼15 bp) stem–loop RNAs containing flanking single-stranded tails (ss-dsRNAs) are capable of activating PKR. Activation was reported to require a 5′-triphosphate. Here, we characterize the structural features of ss-dsRNAs that contribute to activation. We have designed a model ss-dsRNA containing 15-nt single-stranded tails and a 15-bp stem and made systematic truncations of the tail and stem regions. Autophosphorylation assays and analytical ultracentrifugation experiments were used to correlate activation and binding affinity. PKR activation requires both 5′- and 3′-single-stranded tails but the triphosphate is dispensable. Activation potency and binding affinity decrease as the ssRNA tails are truncated and activation is abolished in cases where the binding affinity is strongly reduced. These results indicate that the single-stranded regions bind to PKR and support a model where ss-dsRNA induced dimerization is required but not sufficient to activate the kinase. The length of the duplex regions in several natural RNA activators of PKR is below the minimum of 30 bp required for activation and similar interactions with single-stranded regions may contribute to PKR activation in these cases.

Abstract 4797: VS-6063 (defactinib) targets cancer stem cells directly and through inhibition of tumor-associated macrophages and cytokine production

Abstract Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase which mediates integrin and growth factor receptor signaling to regulate tumor growth, metastasis and angiogenesis. FAK is also required for the proliferation/survival of cancer stem cells (CSCs). Protein tyrosine kinase 2 (PYK2) is the most closely related protein kinase to FAK, sharing 60% homology in the kinase domain and a similar role in cellular signaling pathways. Several lines of evidence suggest that dual targeting of FAK and PYK2 should confer greater antitumor efficacy than an agent which only inhibits FAK. Inhibition of mammary stem cells or tumor angiogenesis by FAK inhibition can be bypassed by PYK2 activity, suggesting advantages of dual FAK/PYK2 inhibition. Results of PYK2 knockout in mice suggest that PYK2 inhibition is likely to be well tolerated. Based on the observation of reduced macrophage infiltration in PYK2 knockout mice, we investigated effects of PYK2 inhibition on tumor-associated macrophages (TAMs). Abundance of TAMs has been correlated with poor prognosis in multiple cancer types including mesothelioma and breast cancer. VS-6063 is a potent and orally active inhibitor of FAK kinase activity. Preliminary clinical activity has been observed in a Phase 1 single agent trial and in an ongoing Phase 1/1b trial in combination with paclitaxel in patients with ovarian cancer. We find that in addition to potent FAK inhibition, VS-6063 also inhibits PYK2 kinase activity with nanomolar potency in both biochemical enzymatic and cellular autophosphorylation assays. Using both pharmacological FAK inhibitors and FAK siRNA, we have previously demonstrated that FAK inhibition effectively reduces CSCs. We report here that dual targeting of FAK and PYK2 by VS-6063 may more effectively suppress CSCs than inhibition of FAK alone. We find that human monocyte-derived macrophages release IL-6 and IL-8, and these cytokines increase the proportion of ALDH-positive CSCs in mesothelioma and breast cancer cell lines. VS-6063 dose-dependently inhibited production of IL-6 and IL-8, whereas a FAK-only reference inhibitor had no effect on these cytokines. Accordingly, PYK2 siRNA decreased IL-6 release from human macrophages, while FAK siRNA did not. In the MDA-MB-231 breast cancer xenograft model in vivo, VS-6063 reduced serum IL-6 while the FAK-only reference compound did not. Furthermore, VS-6063 substantially reduced the number of F4/80-positive macrophages in the tumors. These data support a model in which TAMs release IL-6 and IL-8 in a PYK2-dependent manner. Dual inhibition of FAK and PYK2 by VS-6063 effectively decreases both the presence of TAMs in tumors and the ability of TAMs to release cytokines that stimulate CSC proliferation and survival, and may therefore provide the opportunity for more durable clinical response than inhibition of FAK alone. Citation Format: Jonathan A. Pachter, Jennifer E. Ring, Vihren N. Kolev, Irina M. Shapiro, Christian M. Vidal, Mahesh V. Padval, Qunli Xu. VS-6063 (defactinib) targets cancer stem cells directly and through inhibition of tumor-associated macrophages and cytokine production. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4797. doi:10.1158/1538-7445.AM2014-4797

Expression profiling and functional characterization of a CBL-interacting protein kinase gene from Populus euphratica

This is the first report on the function of a member of the CIPK family in Populus euphratica. The Ca(2+)-dependent salt overly sensitive (SOS) pathway has been shown to play an essential role in maintaining ion homeostasis and conferring salt tolerance. One component of the SOS pathway, SOS1, was identified in the salt-resistant tree P. euphratica. In this study, we identified and functionally characterized another component of the SOS pathway in this tree called PeSOS2 or PeCIPK26. On the basis of protein sequence similarity and complementation studies in Arabidopsis, PeCIPK26 was concluded to be the functional homolog of Arabidopsis AtSOS2. Yeast two-hybrid assays revealed that PeCIPK26 can interact with four calcineurin B-like (CBL) genes, i.e., PeCBL1, PeCBL4/PeSOS3, PeCBL9 and PeCBL10. Autophosphorylation assays showed that PeCIPK26 is an active protein kinase. Expression profile analysis demonstrated that PeCIPK26 is expressed in root, stem and leaf, and throughout the cell including cell membrane, cytoplasm and nucleus; in addition, it can be induced under salt-stress treatment. Functions of PeCIPK26 in salt tolerance were evaluated by gene overexpression in Arabidopsis cipk24 mutants. The better salt tolerance of transgenic plants relative to mutants was shown by their higher germination rate, lower Na(+) accumulation and higher capacity to discharge Na(+) when grown with NaCl. These results suggest the involvement of PeCIPK26 in the salt-stress response of P. euphratica.

Activation of PKR by Stem-Loop RNAs with Flanking SsRNA Tails

Protein Kinase R (PKR) is a central component of the innate immunity antiviral pathway and is activated by double stranded RNA (dsRNA). PKR contains a C-terminal kinase domain and two tandem dsRNA binding motifs. In the accepted model for activation, binding of multiple PKR monomers to dsRNA enhances dimerization of the kinase domain. A minimum dsRNA length of 30 bp is required for binding two PKR monomers and eliciting strong enzymatic activation. However, short (15 bp) stem-loop RNAs containing flanking single stranded tails (ss-dsRNAs) are capable of activating PKR. Activation requires a 5'-triphosphate and the presence of both 5' and 3' ssRNA tails. The mechanism of PKR activation by ss-dsRNAs is not understood. Here, we have characterized the structural features of ss-dsRNAs that contribute to activation. We have designed a model ss-dsRNA PKR activator containing two single stranded tails of 15 nt and a 15 bp stem (5'-15-15-15-3') and made systematic truncations of the tail and stem regions. Analytical ultracentrifugation experiments combined with autophosphorylation assays were used to correlate RNA binding affinity with the ability to activate the kinase. Both tails are required for PKR activation; however, they can be truncated to produce either 10-15-5 or 5-15-10 without abolishing activation. Activity is retained upon reducing the stem to 10 bp but is lost upon further reduction to 5 bp. All of the ss-dsRNAs bind two PKR monomers in 75 mM NaCl. The loss of PKR activation is correlated with weaker PKR binding, consistent with a model where the autophosphorylation rates are proportional to the concentration of RNA species containing two PKR monomers. A 5'-triphosphate is required for activation. Although the binding affinity decreases upon removal of the 5'-triphosphate, the reduction is not sufficient to explain the loss of activation.

Abstract B283: Defactinib (VS-6063) targets cancer stem cells directly and through inhibition of tumor-associated macrophages and cytokine production.

Abstract Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase which mediates integrin and growth factor receptor signaling to regulate tumor growth, metastasis and angiogenesis. FAK is also required for the proliferation/survival of cancer stem cells (CSCs). Protein tyrosine kinase 2 (PYK2) is the most closely related protein kinase to FAK, sharing 60% amino acid identity in the kinase domain and a similar role in cellular signaling pathways. Several lines of evidence suggest that dual targeting of FAK and PYK2 should confer greater antitumor efficacy than an agent which only inhibits FAK. Inhibition of tumor angiogenesis or mammary stem cells by FAK inhibition can be bypassed by PYK2 activity, suggesting advantages of dual FAK/PYK2 inhibition. Accordingly, a critical role of PYK2 in CSC regulation and NSCLC patients’ prognosis has recently been reported (Kuang et al. 2013, Br J Cancer). Furthermore, preclinical data suggest that PYK2 inhibition can prevent growth of metastatic tumor cells in bone. Results of PYK2 knockout in mice suggest that PYK2 inhibition is likely to be well tolerated. Based on the observation of reduced macrophage infiltration in PYK2 knockout mice, we investigated effects of PYK2 inhibition on tumor-associated macrophages (TAMs). Abundance of TAMs has been correlated with poor prognosis in multiple cancer types including mesothelioma and breast cancer. Defactinib is a potent and orally active inhibitor of FAK kinase activity. Preliminary clinical activity has been observed in a Phase 1 single agent trial and in an ongoing trial in combination with paclitaxel in patients with ovarian cancer. We find that in addition to potent FAK inhibition, defactinib also inhibits PYK2 kinase activity with nanomolar potency in both biochemical enzymatic and cellular autophosphorylation assays. Using both pharmacological FAK inhibitors and FAK siRNA, we have previously demonstrated that FAK inhibition effectively reduces CSCs. We report here that dual targeting of FAK and PYK2 by defactinib may more effectively suppress CSCs than inhibition of FAK alone. We find that differentiated THP-1 macrophages release IL-6 and IL-8, and these cytokines increase the proportion of ALDH-positive CSCs in mesothelioma and breast cancer cell lines. Defactinib dose-dependently inhibited production of IL-6 and IL-8, whereas a FAK-only reference inhibitor had no effect on these cytokines. Furthermore, in MM87 mesothelioma and MDA-MB-231 breast cancer xenograft models, defactinib substantially reduced the number of F4/80-positive TAMs in the tumors. These data support a model in which TAMs release IL-6 and IL-8 in a PYK2-dependent manner. Dual inhibition of FAK and PYK2 by defactinib effectively decreases both the presence of TAMs in tumors and the ability of TAMs to release cytokines that stimulate CSC proliferation and survival, and may therefore provide the opportunity for more durable clinical response than inhibition of FAK alone. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B283. Citation Format: Jennifer E. Ring, Vihren N. Kolev, Irina M. Shapiro, Quentin G. Wright, Joseph R. Testa, Mahesh V. Padval, Qunli Xu, Jonathan A. Pachter. Defactinib (VS-6063) targets cancer stem cells directly and through inhibition of tumor-associated macrophages and cytokine production. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B283.

Identification of a redox-sensitive switch within the JAK2 catalytic domain

Four cysteine residues (Cys866, Cys917, Cys1094, and Cys1105) have direct roles in cooperatively regulating Janus kinase 2 (JAK2) catalytic activity. Additional site-directed mutagenesis experiments now provide evidence that two of these residues (Cys866 and Cys917) act together as a redox-sensitive switch, allowing JAK2's catalytic activity to be directly regulated by the redox state of the cell. We created several variants of the truncated JAK2 (GST/(NΔ661)rJAK2), which incorporated cysteine-to-serine or cysteine-to-alanine mutations. The catalytic activities of these mutant enzymes were evaluated by in vitro autokinase assays and by in situ autophosphorylation and transphosphorylation assays. Cysteine-to-alanine mutagenesis revealed that the mechanistic role of Cys866 and Cys917 is functionally distinct from that of Cys1094 and Cys1105. Most notable is the observation that the robust activity of the CC866,917AA mutant is unaltered by pretreatment with dithiothreitol or o-iodosobenzoate, unlike all other JAK2 variants previously examined. This work provides the first direct evidence for a cysteine-based redox-sensitive switch that regulates JAK2 catalytic activity. The presence of this redox-sensitive switch predicts that reactive oxygen species can impair the cell's response to JAK-coupled cytokines under conditions of oxidative stress, which we confirm in a murine pancreatic β-islet cell line.

Abstract B238: Novel imidazo[1,2-a]pyridines as inhibitors of the IGF-1R tyrosine kinase.

Abstract The Insulin-like Growth Factor-1 Receptor (IGF-1R) is a receptor tyrosine kinase which plays an important role in cancer by promoting proliferation and survival through activation of the Ras/Raf/MAPK and PI3K/Akt signaling pathways. Deregulation of the IGF-1R axis has been reported in a variety of solid cancers and occurs by overexpression of either the receptor or its ligands (IGF-1 and IGF-2) or by down-regulation of IGF binding proteins (in particular IGFBP3). Therapeutic approaches to inhibit IGF-1R signaling include antibodies targeting the receptor and small molecule inhibitors of the tyrosine kinase. The latter strategy offers a potential advantage by simultaneously inhibiting the closely related Insulin Receptor A (IR-A) tyrosine kinase. Indeed, IR-A can drive tumor growth in some cancer cell lines and has been suggested has a potential escape mechanism for IGF-1R inhibition. Following a cell based high throughput screening, we have identified a series of imidazo[1,2-a]pyridines inhibiting the IGF-1R kinase. Although hit compounds carried a significant activity against CDK2, modifications of the aniline part has led to more potent and selective inhibitors. We selected 1-[4-[4-[(5-chloro-4-imidazo[1,2-a]pyridin-3-yl-pyrimidin-2-yl)amino]-3-methoxy-phenyl]piperazin-1-yl]ethanone (5) as a lead compound having good pharmacokinetics in pre-clinical species. Compound (5) displays a dual inhibition of IGF-1R and the insulin receptor tyrosine kinase as measured in biochemical assays as well as in cellular auto-phosphorylation assays. Structure-activity relationship around (5) is described for IGF-1R inhibition as well as for the hERG ion channel. Compound (5) has been assessed in an in vivo efficacy model based on the NIH 3T3 fibroblast cell line transfected to express human IGF-1R and IGF1 ligand. In contrast to the non-transfected parent line, this cell line rapidly grows as a sub-cutaneous xenograft in nude mouse, thus providing a suitable model to measure anti-IGF-1R pharmacology by tumor growth inhibition. The high expression of IGF-1R also enables the generation of good quality PD data using receptor phosphorylation as an endpoint. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B238.

Motesanib inhibits Kit mutations associated with gastrointestinal stromal tumors

BackgroundActivating mutations in Kit receptor tyrosine kinase or the related platelet-derived growth factor receptor (PDGFR) play an important role in the pathogenesis of gastrointestinal stromal tumors (GIST).MethodsThis study investigated the activity of motesanib, an inhibitor of vascular endothelial growth factor receptors (VEGFR) 1, 2, and 3; PDGFR; and Kit, against primary activating Kit mutants and mutants associated with secondary resistance to imatinib. Single- and double-mutant isoforms of Kit were evaluated for their sensitivity to motesanib or imatinib in autophosphorylation assays and in Ba/F3 cell proliferation assays.ResultsMotesanib inhibited Kit autophosphorylation in CHO cell lines expressing primary activating mutations in exon 9 (AYins503-504, IC50 = 18 nM) and exon 11 (V560 D, IC50 = 5 nM; Δ552-559, IC50 = 1 nM). Motesanib also demonstrated activity against kinase domain mutations conferring imatinib resistance (V560D/V654A, IC50 = 77 nM; V560D/T670I, IC50 = 277 nM; Y823 D, IC50 = 64 nM) but failed to inhibit the imatinib-resistant D816V mutant (IC50 > 3000 nM). Motesanib suppressed the proliferation of Ba/F3 cells expressing Kit mutants with IC50 values in good agreement with those observed in the autophosphorylation assays.ConclusionsIn conclusion, our data suggest that motesanib possesses inhibitory activity against primary Kit mutations and some imatinib-resistant secondary mutations.

Abstract 3624: A novel, selective FGFR inhibitor that causes blockade of FGFR autophosphorylation shows a broad-spectrum of anti-tumor activity in preclinical xenograft tumor models

Abstract Anti-angiogenic agents targeting VEGF/VEGFR including Bevacizumab, Sorafenib, and Sunitinib that target blood vessel formation in rapidly growing cancer cells are very effective cancer therapies in the clinic. However, these anti-angiogenic agents also have debilitating side effects, such as hypertension and bleeding, which thereby limit their use in many patients in which the anti-VEGF/VEGFR based therapies are contraindicated. Furthermore, the emergence of resistance to the anti-VEGF/VEGFR based therapies in the clinic due to over-expression of FGF2 or other growth factors could pose a serious challenge to their current use in the clinic. The FGFRs are receptor tyrosine kinases that are present in many types of tumor cells as well as endothelial cells, and play an important role in tumor cell growth, survival, and migration, and also in maintaining tumor angiogenesis. Over-expression of FGFRs or aberrant regulation of their activities has been implicated in many forms of human malignancies. Therefore, targeting FGFRs for cancer treatment represents an attractive strategy that has the potential to treat a variety of cancers by simultaneously inhibiting tumor cell growth, survival, and migration, and also tumor angiogenesis without the side effects associated with the VEGF/VEGFR based therapies. Here, we describe a selective small molecule inhibitor of FGFRs. The FGFR inhibitor is active against all four FGF receptors (IC50 = 2.6 to 7.7 nM) as revealed by biochemical autophosphorylation assays. It also exhibits a potent activity against FGFR or FGFR-mediated Erk phosphorylation in several cancer cell lines (IC50 = 0.6 to 1.5 nM). In addition, in vitro cell based assays have shown that this inhibitor is much more active at inhibiting the proliferation of FGFR-dependent than FGFR-independent cancer cell lines (IC50s = 3-2,600 nM). Furthermore, in vitro selectivity assays have established that this inhibitor exhibits a robust preferential activity in inhibiting FGF- over VEGF-mediated cord formation (IC50s = 0.6 nM for FGF and 3.6 nM for VEGF). Consistent with these in vitro selectivity data, in vivo target inhibition assays have also demonstrated that the FGFR inhibitor has a potent, yet much higher activity against FGF- than VEGF-mediated target phosphorylation in mice (TEC50s and TEC90s of 6 and 29 nM for FGFR, and 35 and 252 nM for VEGFR, respectively). Finally, the FGFR inhibitor shows an excellent broad-spectrum anti-tumor activity in xenograft tumor models representing several major cancer histologies including bladder cancer when dosed at TED50 (1.3 mg/kg) and especially at TED90 (3.2 mg/kg). Thus, the FGFR inhibitor exhibits a very well behaved PK/PD relationship. Currently, the FGFR inhibitor is being further evaluated for its potential utility in the clinic. 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 3624.

Myelosuppression and kinase selectivity of multikinase angiogenesis inhibitors

Background:Myelosuppression has been observed with several multikinase angiogenesis inhibitors in clinical studies, although the frequency and severity varies among the different agents. Inhibitors targeting vascular endothelial growth factor receptor (VEGFR) often inhibit other kinases, which may contribute to their adverse-event profiles.Methods:Kinase selectivity of pazopanib, sorafenib, and sunitinib was evaluated in a panel of 242 kinases. Cellular potency was measured using autophosphorylation assays. Effect on human bone marrow progenitor growth in the presence of multiple growth factors was evaluated and correlated with the kinase selectivity.Results:Sunitinib inhibited more kinases than pazopanib and sorafenib, at potencies within 10-fold of VEGFR-2. All three compounds potently inhibited VEGFR-2, platelet-derived growth factor receptor-β and c-Kit, However, pazopanib was less active against Flt-3 in both kinase and cellular assays. The inhibitory properties of pazopanib, sorafenib, and sunitinib were dependent on the growth factor used to initiate bone marrow colony formation. Addition of stem cell factor and/or Flt-3 ligand with granulocyte-macrophage colony stimulating factor resulted in significant shifts in potency for sorafenib and sunitinib but less so for pazopanib.Conclusion:Activity against c-kit and Flt-3 by multikinase angiogenesis inhibitors provide a potential explanation for the differences in myelosuppression observed with these agents in patients.

The Role of Phosphorylation in Redox Regulation of Photosynthesis Genes psaA and psbA during Photosynthetic Acclimation of Mustard

The long-term response (LTR) to light-quality gradients improves performance and survival of plants in dense stands. It involves redox-controlled transcriptional regulation of the plastome-encoded genes psaAB (encoding the P700 apoproteins of photosystem I) and psbA (encoding the D1 protein of photosystem II) and requires the action of plastid-localized kinases. To study the potential impact of phosphorylation events on plastid gene expression during the LTR, we analyzed mustard seedlings acclimated to light sources favoring either photosystem I or photosystem II. Primer extension analyses of psaA transcripts indicate that the redox regulation occurs at the principal bacterial promoters, suggesting that the plastid encoded RNA polymerase (PEP) is the target for redox signals. Chloroplast protein fractions containing PEP and other DNA-binding proteins were purified from mustard via heparin-Sepharose chromatography. The biochemical properties of these fractions were analyzed with special emphasis on promoter recognition and specificity, phosphorylation state, and kinase activity. The results demonstrate that the LTR involves the action of small DNA-binding proteins; three of them exhibit specific changes in the phosphorylation state. Auto-phosphorylation assays, in addition, exhibit large differences in the activity of endogenous kinase activities. Chloroplast run-on transcription experiments with the kinase inhibitor H7 and the reductant DTT indicate that phosphorylation events are essential for the mediation of redox signals toward psaA and psbA transcription initiation, but require the synergistic action of a thiol redox signal. The data support the idea that redox signals from the thylakoid membrane are linked to gene expression via phosphorylation events; however, this mediation appears to require a complex network of interacting proteins rather than a simple phosphorelay.

Casein kinase activity in etiolated Cucumis sativus cotyledons

Two calcium- and light-dependent protein kinases have been reported in etiolated Cucumis sativus cotyledons (Vidal et al. 2007). In the present work, we studied casein kinase (CK) activity in etiolated cucumber cotyledons of in-gel and in vitro kinase assays, using specific CK inhibitors, and ATP and GTP as phosphate donors. Two proteins with CK activity were detected in both casein gels and autophosphorylation assays. One of them, with a molecular mass of approximately 36 kDa, showed biochemical CK1 characteristics: it was inhibited by specific CK1 inhibitors and only used ATP as phosphate donor. The second, with a molecular mass of approximately 38 kDa, had CK2 characteristics; it used both ATP and GTP as phosphate donors, was inhibited by all specific CK2 inhibitors, and was recognized by a polyclonal antibody directed against the alpha catalytic subunit of a CK2 from tobacco. The kinase activity of the CK2 detected in etiolated cucumber cotyledons showed circadian rhythmicity in both in vitro and in-gel casein phosphorylation and in autophosphorylation assays. Thus, our results suggest that the CK2 of approximately 38 kDa could be related to the circadian oscillator of C. sativus cotyledons.

Synthesis and biological evaluation of 1,2,4-trisubstituted imidazoles and 1,3,5-trisubstituted pyrazoles as inhibitors of transforming growth factor β type 1 receptor (ALK5)

Two series of nitrogenous heterocycle compounds—1,2,4-trisubstituted imidazoles and 1,3,5-trisubstituted pyrazoles have been synthesized and evaluated for their ALK5 inhibitory activity and cytotoxicity in TGFβ-Smad2 assay and MTT assay, respectively. The ALK4/5/7 inhibitory activity of some compound was also evaluated in ALK4/5/7 autophosphorylation assays. Compounds 6c and 14c showed relatively potent ALK5 inhibition while weak cytotoxicity. At the same time, compounds 6c and 14c display relatively better ALK5 selectivity versus ALK4/ALK7 (nearly 10-fold) compared with SB431542, a well known ALK5 inhibitor. Compound 6g2 proved to be a moderately selective ALK4 inhibitor versus ALK5 and ALK7 (>10-fold). The binding mode of 14c generated by flexible docking study shows that 14c fits well into the site cavity of ALK5 by forming several tight interactions.