scholarly journals A Microfluidics-Based Mobility Shift Assay to Discover New Tyrosine Phosphatase Inhibitors

2006 ◽  
Vol 11 (8) ◽  
pp. 996-1004 ◽  
Author(s):  
Dominique Perrin ◽  
Christèle Frémaux ◽  
Dominique Besson ◽  
Wolfgang HB Sauer ◽  
Alexander Scheer
Keyword(s):  
Drug Target ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Mobility Shift ◽  
Phosphatase Inhibitors ◽  
Mobility Shift Assay ◽  
Diabetes And Obesity ◽  
Major Drug ◽  
Specific Peptide

Protein tyrosine phosphatases (PTPs) play key roles in regulating tyrosine phosphorylation levels in cells. Since the discovery of PTP1B as a major drug target for diabetes and obesity, PTPs have emerged as a new and promising class of signaling targets for drug development in a variety of therapeutic areas. The routine use of generic substrate 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) in our hands led to the discovery of very similar and often not very selective molecules. Therefore, to increase the chances to discover novel chemical scaffolds, a side-by-side comparison between the DiFMUP assay and a chip-based mobility shift assay with a specific phosphopeptide was performed, on 1 PTP, using a focused set of compounds. Assay robustness and sensitivity were comparable for both the DiFMUP and mobility shift assays. The off-chip mobility shift assay required a longer development time because of identification, synthesis, and characterization of a specific peptide, and its cost per point was higher. However, although most potent scaffolds found with the DiFMUP assay were confirmed in the mobility shift format, the off-chip mobility shift assay led to the identification of previously unidentified chemical scaffolds with improved druglike properties.

Design and Biological Evaluation of Novel Imidazolyl Flavonoids as Potent and Selective Protein Tyrosine Phosphatase Inhibitors

2020 ◽  
Vol 16 (4) ◽  
pp. 563-574 ◽  
Author(s):  
Rong Y. Han ◽  
Yu Ge ◽  
Ling Zhang ◽  
Qing M. Wang
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Structural Features ◽  
Biological Evaluation ◽  
Cell Protein ◽  
Phosphatase Inhibitors ◽  
Protein Tyrosine ◽  
Therapeutic Development ◽  
Chemical Studies

Background: Protein tyrosine phosphatases 1B are considered to be a desirable validated target for therapeutic development of type II diabetes and obesity. Methods: A new series of imidazolyl flavonoids as potential protein tyrosine phosphatase inhibitors were synthesized and evaluated. Results: Bioactive results indicated that some synthesized compounds exhibited potent protein phosphatase 1B (PTP1B) inhibitory activities at the micromolar range. Especially, compound 8b showed the best inhibitory activity (IC50=1.0 µM) with 15-fold selectivity for PTP1B over the closely related T-cell protein tyrosine phosphatase (TCPTP). Cell viability assays indicated that 8b is cell permeable with lower cytotoxicity. Molecular modeling and dynamics studies revealed the reason for selectivity of PTP1B over TCPTP. Quantum chemical studies were carried out on these compounds to understand the structural features essential for activity. Conclusion: Compound 8b should be a potential selective PTP1B inhibitor.

Olf-1-binding site: characterization of an olfactory neuron-specific promoter motif

1993 ◽  
Vol 13 (5) ◽  
pp. 3002-3014
Author(s):  
K Kudrycki ◽  
C Stein-Izsak ◽  
C Behn ◽  
M Grillo ◽  
R Akeson ◽  
...  
Keyword(s):  
Nuclear Protein ◽  
Consensus Sequence ◽  
Binding Motif ◽  
Sequence Motif ◽  
Olfactory Neuron ◽  
Sequence Motifs ◽  
Mobility Shift ◽  
Specific Expression ◽  
Mobility Shift Assay

We report characterization of several domains within the 5' flanking region of the olfactory marker protein (OMP) gene that may participate in regulating transcription of this and other olfactory neuron-specific genes. Analysis by electrophoretic mobility shift assay and DNase I footprinting identifies two regions that contain a novel sequence motif. Interactions between this motif and nuclear proteins were detected only with nuclear protein extracts derived from olfactory neuroepithelium, and this activity is more abundant in olfactory epithelium enriched in immature neurons. We have designated a factor(s) involved in this binding as Olf-1. The Olf-1-binding motif consensus sequence was defined as TCCCC(A/T)NGGAG. Studies with transgenic mice indicate that a 0.3-kb fragment of the OMP gene containing one Olf-1 motif is sufficient for olfactory tissue-specific expression of the reporter gene. Some of the other identified sequence motifs also interact specifically with olfactory nuclear protein extracts. We propose that Olf-1 is a novel, olfactory neuron-specific trans-acting factor involved in the cell-specific expression of OMP.

scholarly journals Olf-1-binding site: characterization of an olfactory neuron-specific promoter motif.

1993 ◽  
Vol 13 (5) ◽  
pp. 3002-3014 ◽  
Author(s):  
K Kudrycki ◽  
C Stein-Izsak ◽  
C Behn ◽  
M Grillo ◽  
R Akeson ◽  
...  
Keyword(s):  
Nuclear Protein ◽  
Consensus Sequence ◽  
Binding Motif ◽  
Sequence Motif ◽  
Olfactory Neuron ◽  
Sequence Motifs ◽  
Mobility Shift ◽  
Specific Expression ◽  
Mobility Shift Assay

We report characterization of several domains within the 5' flanking region of the olfactory marker protein (OMP) gene that may participate in regulating transcription of this and other olfactory neuron-specific genes. Analysis by electrophoretic mobility shift assay and DNase I footprinting identifies two regions that contain a novel sequence motif. Interactions between this motif and nuclear proteins were detected only with nuclear protein extracts derived from olfactory neuroepithelium, and this activity is more abundant in olfactory epithelium enriched in immature neurons. We have designated a factor(s) involved in this binding as Olf-1. The Olf-1-binding motif consensus sequence was defined as TCCCC(A/T)NGGAG. Studies with transgenic mice indicate that a 0.3-kb fragment of the OMP gene containing one Olf-1 motif is sufficient for olfactory tissue-specific expression of the reporter gene. Some of the other identified sequence motifs also interact specifically with olfactory nuclear protein extracts. We propose that Olf-1 is a novel, olfactory neuron-specific trans-acting factor involved in the cell-specific expression of OMP.

scholarly journals Characterization of the Epstein–Barr virus BALF2 promoter

1999 ◽  
Vol 80 (10) ◽  
pp. 2747-2750 ◽  
Author(s):  
Chien-Hui Hung ◽  
Shih-Tung Liu
Keyword(s):  
Transcription Factors ◽  
Epstein Barr Virus ◽  
Early Stage ◽  
Lytic Cycle ◽  
Mobility Shift ◽  
Response Elements ◽  
Barr Virus ◽  
Epstein Barr ◽  
Mobility Shift Assay

BALF2, which encodes the major DNA-binding protein of Epstein–Barr virus (EBV), is expressed during the early stage of the lytic cycle. The location of the BALF2 promoter was identified by primer extension, which indicated that the transcription start is located at nucleotide 164,782 of the EBV genome. Transfection analyses revealed that, similar to other EBV early promoters, the BALF2 promoter is activated by the EBV-encoded transcription factors Rta and Zta. The promoter is also synergistically activated if both transcription factors are present in B lymphocytes and in epithelial cells. Deletion analysis and electrophoretic mobility-shift assay revealed that the region between nucleotides −134 and −64 contains Zta- response elements and the region between nucleotides −287 and −254 contains Rta-response elements. This study demonstrates the importance of Rta and Zta in regulating the transcription of EBV early genes.

Evaluation of electrophoretic mobility shift assay as a method to determine plasma protein binding of siRNA

Bioanalysis ◽  
2019 ◽  
Vol 11 (21) ◽  
pp. 1927-1939 ◽  
Author(s):  
Carrie Rocca ◽  
Sean Dennin ◽  
Yongli Gu ◽  
Joohwan Kim ◽  
Samantha Chigas ◽  
...  
Keyword(s):  
Protein Binding ◽  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Plasma Protein Binding ◽  
Plasma Protein ◽  
Plasma Proteins ◽  
Small Interfering Rnas ◽  
Mobility Shift ◽  
Mobility Shift Assay

Aim: The electrophoretic mobility shift assay (EMSA) was evaluated as an alternative to ultrafiltration (UF) to assess plasma protein binding (PPB) of small interfering RNAs (siRNA) and antisense oligonucleotides (ASO). Results & methodology: EMSA analysis showed that PPB depended on siRNA and plasma concentration. Conversely, when analyzed by ultrafiltration, siRNA bound the filtration device nonspecifically and PPB remained >98% across physiologically relevant siRNA concentrations. Using EMSA, siRNA exhibited charge-based interactions with plasma proteins, while ASO remained highly bound to plasma proteins or albumin in the presence of 500 mM salt. Conclusion: PPB characteristics of siRNA and ASO can be distinguished using EMSA. Characterization of siRNA PPB by EMSA enhances our knowledge of siRNA absorption, distribution, metabolism and excretion and advanced development of RNA interference therapeutics.

Characterization of the human E2F4 promoter region and its response to 12-O-tetradecanoylphorbol-13-acetate

2019 ◽  
Vol 166 (4) ◽  
pp. 363-373 ◽  
Author(s):  
Hiroshi Hamada ◽  
Yuta Goto ◽  
Jun Arakawa ◽  
Erisa Murayama ◽  
Yui Ogawa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Promoter Region ◽  
Expression Vectors ◽  
Reporter Assay ◽  
Rt Pcr ◽  
Mobility Shift ◽  
A Cell ◽  
Mobility Shift Assay ◽  
E2f Transcription Factors

Abstract The E2F transcription factors (TFs), which control the progression of the cell cycle in response to DNA-damage and various stresses, are known to interact with a tumour suppressor, Retinoblastoma 1 (RB1). We previously showed that the response of the human RB1 promoter to a 12-O-tetradecanoylphorbol-13-acetate (TPA) in HL-60 cells is mediated by a duplicated GGAA motif, which is also present in the 5′-upstream of the E2F family genes. The motifs are especially rich in the 5′-upstream of the E2F4 gene. In the present study, we constructed luciferase (Luc) expression vectors containing a 466 bp of the 5′-upstream of the human E2F4 gene. The transfection of this plasmid and deletion/mutation-introduced derivatives into HL-60 cells and a Luc reporter assay showed that duplicated and triplicated GGAA (TTCC) motifs in the E2F4 promoter respond to TPA. As expected, electrophoretic mobility shift assay indicated that SPI1 (PU.1) binds to the GGAA motif-containing element. A quantitative RT-PCR and western blotting showed that the E2F4 transcripts and its encoding proteins accumulate during the differentiation of HL-60 into macrophage-like cells. In contrast, the expression of the E2F1 gene and the protein, which possibly acts as a cell cycle accelerator, was greatly diminished.

scholarly journals Src family kinases-mediated negative regulation of sperm acrosome reaction in chickens (Gallus gallus domesticus)

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0241181
Author(s):  
Chathura Priyadarshana ◽  
Rangga Setiawan ◽  
Atsushi Tajima ◽  
Atsushi Asano
Keyword(s):  
Acrosome Reaction ◽  
Tyrosine Phosphatase ◽  
Functional Characterization ◽  
Src Family Kinases ◽  
Prolonged Storage ◽  
Phosphatase Inhibitors ◽  
Calcium Dependent ◽  
Mammalian Sperm ◽  
Unique Mechanism

The acrosome reaction (AR) is a strictly-regulated, synchronous exocytosis that is required for sperm to penetrate ova. This all-or-nothing process occurs only once in the sperm lifecycle through a sequence of signaling pathways. Spontaneous, premature AR therefore compromises fertilization potential. Although protein kinase A (PKA) pathways play a central role in AR across species, the signaling network used for AR induction is poorly understood in birds. Mechanistic studies of mammalian sperm AR demonstrate that PKA activity is downstreamly regulated by Src family kinases (SFKs). Using SFK inhibitors, our study shows that in chicken sperm, SFKs play a role in the regulation of PKA activity and spontaneous AR without affecting motility. Furthermore, we examined the nature of SFK phosphorylation using PKA and protein tyrosine phosphatase inhibitors, which demonstrated that unlike in mammals, SFK phosphorylation in birds does not occur downstream of PKA and is primarily regulated by calcium-dependent tyrosine phosphatase activity. Functional characterization of SFKs in chicken sperm showed that SFK activation modulates the membrane potential and plays a role in inhibiting spontaneous AR. Employing biochemical isolation, we also found that membrane rafts are involved in the regulation of SFK phosphorylation. This study demonstrates a unique mechanism for regulating AR induction inherent to avian sperm that ensure fertilization potential despite prolonged storage.

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