|
| 1. |
Galkin, A.V., Melnick, J.S., Kim, S., Hood, T.L., Li, N., Li, L., Xia, G., Steensma, R., Chopiuk, G., Jiang, J., Wan, Y., Ding, P., Liu, Y., Sun, F., Schultz, P.G., Gray, N.S. and Warmuth, M.
(2007)
Identification of NVP-TAE684, a potent, selective and efficacious inhibitor of NPM-ALK
Proc. Natl. Acad. Sci. USA
104
,
270-275
.
|
| |
Notes:
NVP-TAE684 was identified as an inhibitor of the constitutive anaplastic lymphoma kinase (ALK) activity associated with the NPM-ALK fusion. NPM-ALK fusion protein is created by translocation event characteristic of anaplastic large-cell lymophomas. NVP-TAE684 was screened against a panel of 35 Ba/F3 cell lines expressing a variety of tyrosine kinases constitutively activated by fusion to TEL. The CellTiter-Glo® Luminescent Cell Viability Assay was used to detect any decrease in viability as a result of treatment with NVP-TAE684. The inhibitory effect of NVP-TAE684 was specific for ALK-associated cell proliferation. In a secondary screen to determine potency, TAE684 was screened against two human anaplastic large-cell lymphoma cell lines. Inhibition of proliferation correlated with dosage and was assessed using the Bright-Glo® Luciferase Assay System.
(0003738) |
| |
 |
| |
Products: Bright-Glo™ Luciferase Assay System | CellTiter-Glo® Luminescent Cell Viability Assay |
| 2. |
Rose, S.D., Kim, D.H., Amarzguioui, M., Heidel, J.D., Collingwood, M.A., Davis, M.E., Rossi, J.J. and Behlke, M.A.
(2005)
Functional polarity is introduced by Dicer processing of short substrate RNAs.
Nucleic Acids Res.
33
,
4140–56
.
|
| |
Notes:
Having observed that blunt 27mers had increased potency in RNAi compared to 21mers or 27mers with 3' or 5' overhangs, these authors investigated what differences may account for these changes in gene silencing activity using the same target sequence in enhanced green fluorescent protein (EGFP). For one experiment, a PCR-generated fragment of the EGFP coding region spanning sites EGFPS1 and EGFPS2 was cloned into psiCHECK™-2 Vector in both sense and antisense orientations. Also, a PCR-generated fragment of the human heterogeneous nuclear ribonucleoprotein H (hnRNPH) coding region spanning sites H1 and H3 was similarly cloned in sense and antisense orientations. HEK293 cells were transfected with 150ng EGFP sense and antisense vectors plus EGFPS2 or control duplex RNAs. HCT116 cells were transfected with 100ng sense and antisense hnRNPH vectors with H3 or control duplex RNAs. The Dual-Luciferase® assay was used to evaluate luciferase expression 24 hours post-transfection. In a separate EGFP RNAi experiment, the Steady-Glo® Luciferase Assay System was used to monitor firefly luciferase activity to normalize transfection of HEK 293 cells.
A further RNAi experiment targeted the firefly luciferase gene in the pGL3-Control Vector cotransfected with 20, 2 or 0.4 nM siRNA duplexes into HeLa cells. After 48 hours, the cells were lysed and 10µl tested using the Luciferase Assay System. To test the level of expression of human La antigen targeted for gene silencing, total RNA was harvested from HeLa cells using the SV 96 Total RNA Isolation System, reverse transcribed and used in real-time PCR.
(0003289) |
| |
|
| |
Products: Luciferase Assay System | pGL3-Control Vector | psiCHECK™-2 Vector | Steady-Glo® Luciferase Assay System | SV 96 Total RNA Isolation System |
| 3. |
Wang, Y., Klock,H., Yin, H., Wolff, K., Bieza, K., Niswonger, K., Matzen, J., Gunderson, D., Hale, J, Lesley, S., Kuhen, K., Caldwell, J, Brinker, A.
(2005)
Homogeneous high-throughput screening assays for HIV-1 integrase 3' processing and strand transfer activities
J. Biomol. Screen.
10
,
456-462
.
|
| |
Notes:
HIV-1 integrase (HIV-IN) catalyzes a two-step reaction that results in HIV-1 provirus incorporation into the host cell genome. The steps involve an endonucleolytic 3’-processing (3P) followed by a strand transfer (ST) reaction. In past research it has been demonstrated that small molecule inhibitors of the in vitro activity of the 3P reaction only, lack antiviral activity, while inhibition of the HIV-IN ST reaction has been shown to be the key to effective suppression of viral replication in vivo. In the ST inhibitor realm, two series of diketo acids and naphthridine carboxamides have demonstrated antiviral activity in cell culture-based models. However, no compounds have completed clinical trials so far, due to either limited potency or high toxicity. Traditional integrase assays have been low-throughput, gel-based assays involving radiolabeled oligonucleotides. More recently high-throughput assays have been developed, but these microtiter-based assays, while amenable to automation, are complicated and labor intensive due to the need for plate coating and washing. The authors describe development of two robust, homogeneous time-resolved fluorescent energy transfer (TR-FRET)-based assays for HIV-IN 3P and ST reactions that are optimized for 384-well amd 1536-well plate formats. A screen for HIV-IN inhibitors was performed on a 1.36 x 106 compound library, resulting in a series of novel HIV-IN inhibitors that preferentially block integrase ST activity and show potential for further development as new antiviral drugs.
(0003776) |
| |
|
| |
Products: Bright-Glo™ Luciferase Assay System |