Limits...
Two High Throughput Screen Assays for Measurement of TNF-α in THP-1 Cells.

Leister KP, Huang R, Goodwin BL, Chen A, Austin CP, Xia M - Curr Chem Genomics (2011)

Bottom Line: These compounds were also confirmed in a traditional ELISA assay.We also identified several novel inhibitors of TNF-α, such as BTO-1, CCG-2046, ellipticine, and PD 169316.The results demonstrated that both homogeneous TNF-α assays are robust and suitable for high throughput screening.

View Article: PubMed Central - PubMed

Affiliation: NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Tumor Necrosis Factor-α (TNF-α), a secreted cytokine, plays an important role in inflammatory diseases and immune disorders, and is a potential target for drug development. The traditional assays for detecting TNF-α, enzyme linked immunosorbent assay (ELISA) and radioimmunoassay, are not suitable for the large size compound screens. Both assays suffer from a complicated protocol, multiple plate wash steps and/or excessive radioactive waste. A simple and quick measurement of TNF-α production in a cell based assay is needed for high throughput screening to identify the lead compounds from the compound library. We have developed and optimized two homogeneous TNF-α assays using the HTRF (homogeneous time resolved fluorescence) and AlphaLISA assay formats. We have validated the HTRF based TNF-α assay in a 1536-well plate format by screening a library of 1280 pharmacologically active compounds. The active compounds identified from the screen were confirmed in the AlphaLISA TNF-α assay using a bead-based technology. These compounds were also confirmed in a traditional ELISA assay. From this study, several beta adrenergic agonists have been identified as TNF-α inhibitors. We also identified several novel inhibitors of TNF-α, such as BTO-1, CCG-2046, ellipticine, and PD 169316. The results demonstrated that both homogeneous TNF-α assays are robust and suitable for high throughput screening.

No MeSH data available.


Related in: MedlinePlus

Concentration response curves of TNF-α inhibitors in a HTRF-based TNF-α assay (▼) and ELISA-based assay (■). TNF-α level was measured after THP-1 cells were incubated with various concentrations of Bay 11-7085 (A), CCG-2046 (B), IRAK-1/4 inhibitor (C) and PD 169316 (D) in the presence of 1 µg/ml LPS for 17 hr
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3106354&req=5

Figure 5: Concentration response curves of TNF-α inhibitors in a HTRF-based TNF-α assay (▼) and ELISA-based assay (■). TNF-α level was measured after THP-1 cells were incubated with various concentrations of Bay 11-7085 (A), CCG-2046 (B), IRAK-1/4 inhibitor (C) and PD 169316 (D) in the presence of 1 µg/ml LPS for 17 hr

Mentions: To further validate the TNF-α inhibitors identified from both the homogenous HTRF-based and the AlphaLISA-based TNF-α assays, eight compounds were selected based on potency and diversity of mechanism of action and purchased from commercial chemical vendors. The inhibitory effect of these compounds on LPS induced TNF-α production in THP-1 cells was determined using a traditional ELISA assay that is the gold standard for cytokine measurement. As shown in Table 2, all eight compounds inhibited LPS induced TNF-α production with IC50s ranging from 10 nM to 1.95 µM. It was notable that the potencies of all the compounds tested in the ELISA-based assay were higher than the potencies obtained from the HTRF-based assay, with IC50 shifts ranging from 2- to 7-fold (Table 2). For example, Bay 11-7085 had higher potency in the ELISA-based assay (IC50, 0.293 µM) than in the HTRF-based assay (IC50, 1.43 µM), with a 4.9-fold IC50 shift (Fig. 5A). The IC50 shifts of CCG-2046 (2.32 µM in the HTRF assay, and 0.66 µM in the ELISA assay, Fig. 5B), IRAK-1/4 inhibitor I (2.76 µM in HTRF assay, and 0.52 µM in ELISA assay, Fig. 5C), and PD 169316 (78.2 nM in HTRF assay, and 10.6 nM in ELISA assay, Fig. 5D) were 4-, 5- and 7-fold, respectively. The other four compounds, BTO-1, dobutamine, ellipticine and histamine, had 2 to 3-fold IC50 shifts (Table 2). However, the ranking orders of the compound potencies were comparable between the two assay formats. The IC50 values of these compounds in both the ELISA-based and HTRF-based assays correlated very well with an R2 of 0.93. In addition, these eight compounds were also tested in a cell viability assay to evaluate the potential cytotoxicity because the cytotoxic compounds could show the false effect on the reduction of TNF-α production in the cell based assays. We found that all these compounds showed none or weak cytotoxic effect (Table 2). Taken together, these data suggest that the homogenous HTRF-based TNF-α assays can be used to identify small molecule compounds that inhibit TNF-α production.


Two High Throughput Screen Assays for Measurement of TNF-α in THP-1 Cells.

Leister KP, Huang R, Goodwin BL, Chen A, Austin CP, Xia M - Curr Chem Genomics (2011)

Concentration response curves of TNF-α inhibitors in a HTRF-based TNF-α assay (▼) and ELISA-based assay (■). TNF-α level was measured after THP-1 cells were incubated with various concentrations of Bay 11-7085 (A), CCG-2046 (B), IRAK-1/4 inhibitor (C) and PD 169316 (D) in the presence of 1 µg/ml LPS for 17 hr
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3106354&req=5

Figure 5: Concentration response curves of TNF-α inhibitors in a HTRF-based TNF-α assay (▼) and ELISA-based assay (■). TNF-α level was measured after THP-1 cells were incubated with various concentrations of Bay 11-7085 (A), CCG-2046 (B), IRAK-1/4 inhibitor (C) and PD 169316 (D) in the presence of 1 µg/ml LPS for 17 hr
Mentions: To further validate the TNF-α inhibitors identified from both the homogenous HTRF-based and the AlphaLISA-based TNF-α assays, eight compounds were selected based on potency and diversity of mechanism of action and purchased from commercial chemical vendors. The inhibitory effect of these compounds on LPS induced TNF-α production in THP-1 cells was determined using a traditional ELISA assay that is the gold standard for cytokine measurement. As shown in Table 2, all eight compounds inhibited LPS induced TNF-α production with IC50s ranging from 10 nM to 1.95 µM. It was notable that the potencies of all the compounds tested in the ELISA-based assay were higher than the potencies obtained from the HTRF-based assay, with IC50 shifts ranging from 2- to 7-fold (Table 2). For example, Bay 11-7085 had higher potency in the ELISA-based assay (IC50, 0.293 µM) than in the HTRF-based assay (IC50, 1.43 µM), with a 4.9-fold IC50 shift (Fig. 5A). The IC50 shifts of CCG-2046 (2.32 µM in the HTRF assay, and 0.66 µM in the ELISA assay, Fig. 5B), IRAK-1/4 inhibitor I (2.76 µM in HTRF assay, and 0.52 µM in ELISA assay, Fig. 5C), and PD 169316 (78.2 nM in HTRF assay, and 10.6 nM in ELISA assay, Fig. 5D) were 4-, 5- and 7-fold, respectively. The other four compounds, BTO-1, dobutamine, ellipticine and histamine, had 2 to 3-fold IC50 shifts (Table 2). However, the ranking orders of the compound potencies were comparable between the two assay formats. The IC50 values of these compounds in both the ELISA-based and HTRF-based assays correlated very well with an R2 of 0.93. In addition, these eight compounds were also tested in a cell viability assay to evaluate the potential cytotoxicity because the cytotoxic compounds could show the false effect on the reduction of TNF-α production in the cell based assays. We found that all these compounds showed none or weak cytotoxic effect (Table 2). Taken together, these data suggest that the homogenous HTRF-based TNF-α assays can be used to identify small molecule compounds that inhibit TNF-α production.

Bottom Line: These compounds were also confirmed in a traditional ELISA assay.We also identified several novel inhibitors of TNF-α, such as BTO-1, CCG-2046, ellipticine, and PD 169316.The results demonstrated that both homogeneous TNF-α assays are robust and suitable for high throughput screening.

View Article: PubMed Central - PubMed

Affiliation: NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Tumor Necrosis Factor-α (TNF-α), a secreted cytokine, plays an important role in inflammatory diseases and immune disorders, and is a potential target for drug development. The traditional assays for detecting TNF-α, enzyme linked immunosorbent assay (ELISA) and radioimmunoassay, are not suitable for the large size compound screens. Both assays suffer from a complicated protocol, multiple plate wash steps and/or excessive radioactive waste. A simple and quick measurement of TNF-α production in a cell based assay is needed for high throughput screening to identify the lead compounds from the compound library. We have developed and optimized two homogeneous TNF-α assays using the HTRF (homogeneous time resolved fluorescence) and AlphaLISA assay formats. We have validated the HTRF based TNF-α assay in a 1536-well plate format by screening a library of 1280 pharmacologically active compounds. The active compounds identified from the screen were confirmed in the AlphaLISA TNF-α assay using a bead-based technology. These compounds were also confirmed in a traditional ELISA assay. From this study, several beta adrenergic agonists have been identified as TNF-α inhibitors. We also identified several novel inhibitors of TNF-α, such as BTO-1, CCG-2046, ellipticine, and PD 169316. The results demonstrated that both homogeneous TNF-α assays are robust and suitable for high throughput screening.

No MeSH data available.


Related in: MedlinePlus