scholarly journals Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

PLoS ONE ◽  
2017 ◽  
Vol 12 (9) ◽  
pp. e0184843 ◽  
Author(s):  
Justin Hall ◽  
Amy Brault ◽  
Fabien Vincent ◽  
Shawn Weng ◽  
Hong Wang ◽  
...  
2004 ◽  
Vol 9 (5) ◽  
pp. 375-381 ◽  
Author(s):  
Joungnam Kim ◽  
Sara Felts ◽  
Laura Llauger ◽  
Huazhong He ◽  
Henri Huezo ◽  
...  

Heat shock protein 90 (Hsp90) is a molecular chaperone with essential functions in maintaining transformation, and there is increasing interest in developing Hsp90 inhibitors as cancer therapeutics. In this study, the authors describe the development and optimization of a novel assay for the identification of Hsp90 inhibitors using fluorescence polarization. The assay is based on the competition of fluorescently (BODIPY) labeled geldanamycin (GM) for binding to purified recombinant Hsp90α (GM is a natural product that binds to the ATP/ADP pocket in the amino terminal of Hsp90). The authors show that GM-BODIPY binds Hsp90α with high affinity. Even at low Hsp90α concentrations (30 nM), the measured polarization value is close to the maximum assay range of 160 mP, making measurements very sensitive. Its performance, as judged by signal-to-noise ratios (> 10) and Z and Z′ values (> 0.5), suggests that this is a robust and reliable assay. GM, PU24FCl, ADP, and ATP, all known to bind to the Hsp90 pocket, compete with GM-BODIPY for binding to Hsp90α with EC50s in agreement with reported values. These data demonstrate that the Hsp90-FP-based assay can be used for high-throughput screening in aiding the identification of novel Hsp90 inhibitors.


2000 ◽  
Vol 5 (1) ◽  
pp. 23-30 ◽  
Author(s):  
Jinzi J. Wu ◽  
Donna R. Yarwood ◽  
Quynhchi Pham ◽  
Matthew A. Sills

In the last few years, fluorescence polarization (FP) has been applied to the development of robust, homogeneous, high throughput assays in molecular recognition research, such as ligand-protein interactions. Recently, this technology has been applied to the development of homogeneous tyrosine kinase assays, since there are high-affinity anti-phosphotyrosine antibodies available. Unlike tyrosine kinases, application of FP to assay development for serine/threonine kinases has been impeded because of lack of high-affinity anti-phosphoserine/threonine antibodies. In the present study, we report the discovery of a high-affinity, monoclonal anti-phosphoserine antibody, 2B9, with a Kd of 250 ± 34 pM for a phosphoserine-containing peptide tracer, fluorescein-RFARKGS(PO4)LRQKNV. Our data suggest that 2B9 is selective for fluorescein-RFARKGS(PO4)LRQKNV. The antibody and tracer have been used for the development of a competitive FP assay for protein kinase C (PKC) in 384-well plates. Phosphatidylserine, which enhances the kinase activity of PKC in a Ca2+-dependent manner and has a structure similar to that of phosphoserine, did not interfere with binding of the peptide tracer to the antibody in the FP assay. The data indicate that the FP assay is more sensitive and robust than the scintillation proximity assay for PKC. The FP assay developed here can be used for rapid screening of hundreds of thousands of compounds for discovery of therapeutic leads for PKC-related diseases.


2003 ◽  
Vol 323 (2) ◽  
pp. 224-233 ◽  
Author(s):  
Cynthia Hess Kenny ◽  
Weidong Ding ◽  
Kerry Kelleher ◽  
Susan Benard ◽  
Elizabeth Glasfeld Dushin ◽  
...  

2002 ◽  
Vol 7 (3) ◽  
pp. 215-222 ◽  
Author(s):  
Wei Huang ◽  
Yan Zhang ◽  
J. Richard Sportsman

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of the 3′-ester bond of cyclic AMP (cAMP) and cyclic GMP (cGMP), important second messengers in the transduction of a variety of extracellular signals. There is growing interest in the study of PDEs as drug targets for novel therapeutics. We describe the development of a homogeneous fluorescence polarization assay for PDEs based on the strong binding of PDE reaction products (i.e., AMP or GMP) onto modified nanoparticles through interactions with immobilized trivalent metal cations. This assay technology (IMAP) is applicable to both cAMP- and cGMP-specific PDEs. Results of the assay in 384- and 1536-well microplates are presented.


2018 ◽  
Vol 62 (11) ◽  
pp. 694-701 ◽  
Author(s):  
Triveni Kalleshamurthy ◽  
Ranjitha Shekar ◽  
Harish Heballi Niranjanamurthy ◽  
Krithiga Natesan ◽  
Bibek Ranjan Shome ◽  
...  

2015 ◽  
Vol 63 (41) ◽  
pp. 9159-9164 ◽  
Author(s):  
Antonio Varriale ◽  
Anna Pennacchio ◽  
Gabriella Pinto ◽  
Giorgia Oliviero ◽  
Stefano D’Errico ◽  
...  

2007 ◽  
Vol 14 (3) ◽  
pp. 299-303 ◽  
Author(s):  
C. Ramírez-Pfeiffer ◽  
K. Nielsen ◽  
P. Smith ◽  
F. Marín-Ricalde ◽  
C. Rodríguez-Padilla ◽  
...  

ABSTRACT The screening Rose Bengal test (RBT), the buffered plate agglutination test (BPAT), and the confirmatory complement fixation test (CFT) are currently approved by the World Organization for Animal Health (OIE) for diagnosis of goat brucellosis. However, RBT (at 3% or 8% cell concentration) is known to be affected by vaccinal antibodies. In the present study, Mexican and Canadian OIE tests were compared with the fluorescence polarization assay (FPA), alone or in combination, using indirect and competitive enzyme-linked immunosorbent assays as classification variables for goat sera obtained from an area of high prevalence and widespread vaccination. The relative sensitivities and specificities were, respectively, 99.7% and 32.5% for RBT3, 92.8% and 68.8% for RBT8, 98.4% and 84.8% for Canadian CFT, 83.7% and 65.5% for Mexican CFT, and 78.1% and 89.3% for FPA. The use of FPA as the confirmatory test in combination with other tests significantly increased the final specificities of the screening tests alone; BPAT, RBT3, and RBT8 plus FPA resulted in final specificities of 90%, 91.2%, and 91.3%, respectively, whereas for the combinations RBT3 plus Mexican CFT, RBT8 plus Mexican CFT, and BPAT plus Canadian CFT, specificities were 65.5%, 63.2%, and 91.7%, respectively. We suggest that FPA may be routinely applied as an adaptable screening test for diagnosis of goat brucellosis and as a confirmatory test for screening test series. Some advantages of FPA are that its cutoff can be adjusted to improve its sensitivity or specificity, it is a low-cost and easy-to-perform test of choice when specificity is relevant or when an alternative confirmatory test is not available, and it is not affected by vaccination, thus reducing the number of misdiagnosed and killed goats.


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