The measurements of cytokine levels in serum and tissues are important to study the immune response, inflammatory disease status and drug responses. Radioimmunoassay (RIA) was first developed to measure the concentration of insulin in plasma [18
]. RIA is a sensitive in vitro
method that is also applied to measure other antigen and cytokines. Although this assay is sensitive and specific, it requires the use of radioisotope and thus produces the radioactive waste. The RIA assay involves a specific antibody that recognizes the radioactive labeled cytokine tracer and multiple assay steps including extensive wash cycles. In the 1970s, ELISA [19
], also known as an enzyme immunoassay (EIA), was introduced, where the specific antigen (cytokine)-antibody reaction was measured using colorimetric readout instead of a radioactive signal. In the ELISA method, two specific antibodies are usually used with one antibody linked to a reporter enzyme. After several extensive reagent additions and plate washes, the substrate is added and a color change results from an enzyme reaction due to the binding of cytokine to enzyme linked antibody. The intensity of the color produced is proportional to the levels of the cytokine such as TNF-α. The ELISA method is widely utilized in biomedical research because it doesn’t use radioactivity and is a standard method used in immunological experiments to detect cytokines.
For the large scale compound library screening, the throughput using ELISA is low and the assay procedure is complicated because the ELISA assay requires extensive wash cycles and antibody pre-coated plates. Therefore, the homogenous and simple assay method for measurement of cytokine level is needed for compound library screens. The assay should be miniaturized into high plate density formats such as 384-well and 1536-well plates as well as being sensitive to compounds. We have optimized the HTRF-based and AlphaLISA-based TNF-α assay into the homogenous format. The detection reagents are directly added into the wells of the assay plate treated with stimulant and compounds without involving the medium transferring that is needed in the original assay protocol. We found that the activities of TNF-α inhibitors identified in these homogenous assays were well correlated with those obtained from the ELISA-based assay.
Compared to the traditional ELISA method, these assays can be easily miniaturized into a 1536-well plate format and are homogeneous, and no plate wash or supernatant transfer steps. The frequent plate incubations needed in the ELISA assay was reduced significantly. Furthermore, the ratiometric readout from the dual emissions (665 and 615 nm) used in the HTRF-based TNF-α assay minimizes the well-to-well and plate-to-plate variations that are caused by subtle differences in cell numbers and dispensing error.
We also found that the potencies of compounds obtained in the HTRF-based TNF-α assay were slightly lower than those measured in the ELISA method. Among these compounds tested, a 2- to 7-fold shift of activities was observed. This could be due to the fact that ELISA can have increased sensitivity because it is a plate based, and fixed assay system which increases the capture ability of the antibody. Also, the many wash steps in the ELISA protocol will remove any non-specific binding of the antibody. Finally, the antibodies used in the different assays may have different affinities for the antigen, resulting in different sensitivity of the assay. However, the potency ranking orders of compounds tested in ELISA and homogenous assay formats were similar. Therefore, both the HTRF-based and AlphaLISA-based TNF-α assays are still preferable for high throughput screening of large compound collection. The ELISA based TNF-α assay can be used as a confirmation assay to validate the active compounds identified from the primary screens.
Beta-adrenergic agonists are known to suppress LPS-induced TNF-α production [21
]. After a beta-adrenergic agonist binds to its receptor, adenylyl cyclase (AC) is activated by GS
α protein which leads to an increase of intracellular cAMP. It has been shown that the increased cAMP stimulated by beta-adrenergic agonists can reduce the TNF-α mRNA levels [22
]. Consistent with previous reports, we found more than a dozen beta adrenergic agonists including norepinephrine, epinephrine, dobutamine and fenoterol from the LOPAC library with an inhibitory effect on TNF-α production. In addition, we also found that histamine inhibited LPS-induced TNF-α production in THP-1 cells, which is consistent with a previous report that histamine suppressed the LPS-induced synthesis of TNF-α in peripheral blood mononuclear cells [23
]. The inhibition of histamine on TNF-α production is caused by a similar mechanism as the beta adrenergic agonists. Histamine binds to and activates the histamine type 2 (H2) receptor that resulting in activation of GS
α protein, and AC and thus increasing intracellular cAMP [23
The binding of TNF-α to its receptor initiates the signaling cascade resulting in activation of the NF-κB and MAP kinase signaling pathways [7
]. Previous studies reported that Bay 11-7082 [24
], Bay 11-7085 [25
], N-p-tosyl-L-phenylalanine chloromethyl ketone [26
], IRAK-1/4 inhibitor I [27
], and parthenolide [28
] had inhibitory effects on the NF-κB signaling pathway. PD 169316 [29
] was reported to inhibit the p38 MAP kinase. These compounds were found in our LOPAC library screen to inhibit the LPS induced TNF-α production in THP-1 cells, suggesting that the inhibition of NF-κB and MAP kinase signaling pathways by these compounds could be a result of the inhibition of TNF-α production.
BTO-1, CCG-2046, and ellipticine were identified as inhibitors of LPS induced TNF-α production from this LOPAC library screen. BTO-1 is a polo-like kinase I inhibitor, which inhibits spindle assembly, mitotic entry and chromosome segregation [30
]. Ellipticine, an alkaloid derived from the leaves of the evergreen tree, is known as a DNA intercalating agent [31
] and an inhibitor of the enzyme topoisomerase II [32
]. However, the roles of these compounds and the mechanism of action on LPS-induced TNF-α production still need further investigation. These compounds might be useful as research tools in the fields of inflammation and immunology.
In summary, we have optimized and validated two homogenous TNF-α assays using HTRF and AlphaLISA assay formats. Both assays were miniaturized into 1536-well plates in a qHTS format. From a test screen of the LOPAC library, we have identified twenty-five TNF-α inhibitors. All these compounds showed reproducible activities in these two assays. Eight out of the 25 compounds were further evaluated in the traditional ELISA based TNF-α assay. The IC50 values of these compounds measured from the homogenous HTRF-based TNF-α assay correlated very well with those determined in the TNF-α ELISA assay. Out of the 25 compounds, 15 are beta adrenergic receptor agonists, five are known inhibitors of the NF-κB pathway, and one compound is a histamine H2 receptor agonist. The identification of these 21 known inhibitors further demonstrated that these two homogenous TNF-α assays reported here are valid for compound screening. We also found four compounds including BTO-1, CCG-2046, ellipticine, and PD 169316 as new inhibitors of LPS induced TNF-α production. Taken together, our results indicated that these homogenous TNF-α assays can be used to quickly and efficiently screen large compound collections and to identify compounds that can potentially inhibit TNF-α production.