The hindrance effect of amine-AZD4547 (3) on HeLa and HeLa-FGFR3 cells is in accor-dance with the MTT assay results for the RT112 cell line with overexpressed FGFR3. m. Abbreviations: QD, quantum dot; FGFR3, fibroblast growth factor receptor 3. ijn-12-5345s3.tif (811K) GUID:?FB681542-08D8-4789-A984-92D61E96C97A Figure S4: Pearsons correlation coefficient analysis of QD-GFP colocalization arising from QD-FGFR3 binding.Notes: HeLa-FGFR3 cells were treated Bepridil hydrochloride with methoxy-QD and AZD4547 probes for 2 h and excess AZD4547 were treated for 1 h. Hundreds of cells were analyzed. Abbreviations: QD, quantum dot; GFP, green fluorescent protein; FGFR3, fibroblast growth factor receptor 3. ijn-12-5345s4.tif (157K) GUID:?6542F179-18D5-4AD8-B0B1-79E90E46AD21 Abstract Fibroblast growth factor receptors (FGFRs) play an important role in determining cell proliferation, differentiation, migration, and survival. Although a variety of small-molecule FGFR inhibitors have been developed for cancer therapeutics, the interaction between FGFRs and FGFR inhibitors has not been well characterized. The FGFRCinhibitor interaction can be characterized using a new imaging probe that has strong, stable signal properties for in situ cellular imaging of the interaction without quenching. We developed a kinaseCinhibitor-modified quantum dot (QD) probe to investigate the interaction between FGFR and potential inhibitors. Especially, turbo-green fluorescent protein-FGFR3s were overexpressed in HeLa cells to investigate the colocalization of FGFR3 and AZD4547 using the QD-AZD4547 probe. The result indicates that this probe is useful for investigating the binding behaviors of FGFR3 with the FGFR inhibitor. Thus, this new inhibitor-modified QD probe is a promising tool for understanding the interaction between FGFR and inhibitors and for creating future high-content, cell-based drug screening strategies. strong class=”kwd-title” Keywords: quantum dot, fibroblast growth factor 3, AZD4547, kinaseCinhibitor, in situ imaging Introduction In situ cellular imaging has attracted significant attention in the drug-discovery field for its possible applications in mechanism studies, target identification, and cell-based drug screening.1C3 Although several promising studies have suggested using in situ cellular imaging as a tool,4,5 investigating the interaction between target proteins and drugs using this tool remains difficult due to a lack of resolution and throughput. Hence, a new probe must be introduced for in situ cellular imaging to become a major process in new drug development. Therefore, this new probe must have high resolution, high stability, and a strong imaging signal that does not decrease over time. One Bepridil hydrochloride strategy for developing an imaging probe that can Rabbit Polyclonal to Cyclin H investigate mechanisms and support drug discovery is to conjugate a fluorescent chemical with a novel drug that binds to the target protein in cells. Small-molecule fluorescent probes were introduced to study the interaction between a target protein and a drug.6,7 These probes provide a method for screening the activity of potential drug for target enzyme inhibition or for evaluating the drug efficacy associated with biological phenomena. Bepridil hydrochloride However, no technique is available to image inhibitorCkinase binding in situ while tracking the inhibitorCkinase interaction in cells due to the lack of potent imaging probes that do not quench while tracking inhibitors. Therefore, a probe with a long-lasting, highly stable signal is needed to assess the distribution of potential drug movement in cells. Quantum dots (QDs) were selected as fluorescent probes to conjugate with potential drugs (ie, inhibitors in our study) because of their high fluorescence intensity,8 photostability,9 and facile surface chemistry.10 We proposed kinaseCinhibitorCQD conjugates as probes for in situ cellular imaging. The receptor tyrosine kinases (RTKs), which are transmembrane proteins, were selected as the target proteins for the selected inhibitor since the interaction occurs at the cell membrane and thus can be easily detected.11 Recently, fibroblast growth factor receptor (FGFR) signaling in cancer has received attention for its ability to cause tumor angiogenesis12 by deregulating fibroblast growth factors (FGFs). This may lead to tumor cell proliferation, survival, and chemoresistance.13 The FGFR inhibitor AZD4547 is a frontrunner with its enhanced therapeutic results against a variety of FGFR-deregulated cancer models.14 Although many studies on the efficacy of AZD4547 in various cancer cells have been reported,15C17 the physical interaction between AZD4547 and FGFR has not been addressed. In this study, we prepared the known FGFR inhibitor (AZD4547)-modified QD (QD-AZD4547) probe to image FGFRs in situ to investigate the interaction between FGFR and AZD4547 in cells. QDs were conjugated with AZD4547 using an amide-bond-forming reaction between the carboxylic acid groups of QDs and the amine-AZD4547 in the presence of ethylcarbodiimidehydrochloride (EDC)/sulfo-N-hydroxysulfosuccinimide (NHS). This simple conjugation method has been.