Molecular imaging of glycogen synthase kinase-3β and casein kinase-1α kinases

Nyati, Shyam; Ranga, Rajesh; Ross, Brian D.; Rehemtulla, Alnawaz; Bhojani, Mahaveer S.

doi:10.1016/j.ab.2010.06.020

Cited by 15 publications

(15 citation statements)

References 53 publications

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“…The BTR-WT was constructed using the appropriate primers (CTA GAG GAG GAA GTG GAG GGT TAA CTC AGA TGG GAT CCC CTT CAG TGC GTT GCT CAA GCA TGT CAC and CCG GGT GAC ATG CTT GAG CAA CGC ACT GAA GGG GAT CCC ATC TGA GTT AAC CCT CCA CTT CCT CCT). The mutant reporter, BTR-MUT, was generated by substituting both serines of the SXS motif with alanine using a single primer mutagenesis protocol, with minor modifications as described earlier (15), using the following primer: CCT TCA GTG CGT TGC TCA GCC ATG GCA CCC GGG GGA GGA GCT GGA GGA TCC GGT TAT GT. All the clones were sequence verified.…”

Section: Methodsmentioning

confidence: 99%

Molecular Imaging of TGFβ-Induced Smad2/3 Phosphorylation Reveals a Role for Receptor Tyrosine Kinases in Modulating TGFβ Signaling

Nyati

Schinske

Ray

et al. 2011

Clinical Cancer Research

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Purpose: The dual modality of TGFb, both as a potent tumor suppressor and a stimulator of tumor progression, invasion, and metastasis, make it a critical target for therapeutic intervention in human cancers. The ability to carry out real-time, noninvasive imaging of TGFb-activated Smad signaling in live cells and animal models would significantly improve our understanding of the regulation of this unique signaling cascade. To advance these efforts, we developed a highly sensitive molecular imaging tool that repetitively, noninvasively, and dynamically reports on TGFBR1 kinase activity.Experimental Design: The bioluminescent TGFbR1 reporter construct was developed using a split firefly luciferase gene containing a functional sensor of Smad2 phosphorylation, wherein inhibition of TGFb receptor1 kinase activity leads to an increase in reporter signaling. The reporter was stably transfected into mammalian cells and used to image in vivo and in vitro bioluminescent activity as a surrogate for monitoring TGFBR1 kinase activity.Results: The reporter was successfully used to monitor direct and indirect inhibition of TGFb-induced Smad2 and SMAD3 phosphorylation in live cells and tumor xenografts and adapted for high-throughput screening, to identify a role for receptor tyrosine kinase inhibitors as modulators of TGFb signaling.Conclusion: The reporter is a dynamic, noninvasive imaging modality for monitoring TGFb-induced Smad2 signaling in live cells and tumor xenografts. It has immense potential for identifying novel effectors of R-Smad phosphorylation, for validating drug-target interaction, and for studying TGFb signaling in different metastasis models. Clin Cancer Res; 17(23); 7424-39. Ó2011 AACR.

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Section: Methodsmentioning

confidence: 99%

Molecular Imaging of TGFβ-Induced Smad2/3 Phosphorylation Reveals a Role for Receptor Tyrosine Kinases in Modulating TGFβ Signaling

Nyati

Schinske

Ray

et al. 2011

Clinical Cancer Research

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“…No vial of cells was cultured for more than 1–2 months. A549-FKR and SW620-BGCR cells have been previously described (18–19). A549-FKR findings were validated using freshly obtained A549 cultures from the ATCC.…”

Section: Methodsmentioning

confidence: 99%

“…(19). In brief, SW620-BGCR cells were plated in 12-well plates and treated with 250 μM CKI7 and 3 μM NSC 47147 for 3 hours.…”

Section: Methodsmentioning

confidence: 99%

A Novel Kinase Inhibitor of FADD Phosphorylation Chemosensitizes through the Inhibition of NF-κB

Schinske

Nyati²,

Khan

et al. 2011

Molecular Cancer Therapeutics

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FADD (Fas-associated protein with death domain) is a cytosolic adapter protein essential for mediating death receptor-induced apoptosis. It has also been implicated in a number of non-apoptotic activities including embryogenesis, cell-cycle progression, cell proliferation, and tumorigenesis. Our recent studies have demonstrated that high levels of phosphorylated FADD in tumor cells correlates with increased activation of the anti-apoptotic transcription factor NF-κB and is a biomarker for aggressive disease and poor clinical outcome. These findings suggest that inhibition of FADD phosphorylation is a viable target for cancer therapy. A high throughput screen using a cell-based assay for monitoring FADD-kinase activity identified NSC 47147 as a small molecule inhibitor of FADD phosphorylation. The compound was evaluated in live cells and mouse tumors for its efficacy as an inhibitor of FADD-kinase activity through the inhibition of CK1α. NSC 47147 was shown to decrease levels of phosphorylated FADD and NF-κB activity such that combination therapy lead to greater induction of apoptosis and enhanced tumor control as compared to either agent alone. The studies described here demonstrate the utility of bioluminescent cell based assays for the identification of active compounds and the validation of drug target interaction in a living subject. In addition, the presented results provide proof of principle studies as to the validity of targeting FADD-kinase activity as a novel cancer therapy strategy.

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“…We have successfully adapted this platform for monitoring GSK3β/CK1α kinase activities using a β-catenin substrate sequence [77]. Towards a comprehensive understanding of oncological signaling and accelerating the drug discovery process, we are currently developing imaging tools for several high priority oncological targets such as EGFR, Her2, c-Met, Ras-Raf-MEK-ERK (MAPK), mTor, and TGFβ receptor.…”

Section: Imaging Of Kinasesmentioning

confidence: 99%

Novel molecular imaging platform for monitoring oncological kinases

et al. 2010

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Recent advances in oncology have lead to identification of a plethora of alterations in signaling pathways that are critical to oncogenesis and propagation of malignancy. Among the biomarkers identified, dysregulated kinases and associated changes in signaling cascade received the lion's share of scientific attention and have been under extensive investigations with goal of targeting them for anti-cancer therapy. Discovery of new drugs is immensely facilitated by molecular imaging technology which enables non-invasive, real time, dynamic imaging and quantification of kinase activity. Here, we review recent development of novel kinase reporters based on conformation dependent complementation of firefly luciferase to monitor kinase activity. Such reporter system provides unique insights into the pharmacokinetics and pharmacodynamics of drugs that modulate kinase signaling and have a huge potential in drug discovery, validation, and drug-target interactions.

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Molecular imaging of glycogen synthase kinase-3β and casein kinase-1α kinases

Cited by 15 publications

References 53 publications

Molecular Imaging of TGFβ-Induced Smad2/3 Phosphorylation Reveals a Role for Receptor Tyrosine Kinases in Modulating TGFβ Signaling

Molecular Imaging of TGFβ-Induced Smad2/3 Phosphorylation Reveals a Role for Receptor Tyrosine Kinases in Modulating TGFβ Signaling

A Novel Kinase Inhibitor of FADD Phosphorylation Chemosensitizes through the Inhibition of NF-κB

Novel molecular imaging platform for monitoring oncological kinases

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