Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

High-affinity complementation of a small fragment of beta-galactosidase to an inactive deletion mutant of the enzyme forms a stable heteromeric enzyme complex capable of hydrolyzing substrates to produce either chemiluminescent or fluorescent signals. This review describes a series of screening assays in which the small beta-galactosidase fragment, Enzyme Donor or ProLabel, is either chemically conjugated or recombinantly fused to small molecules or proteins, respectively. Chemical conjugation forms the basis of several HitHunter HTS assays in which competitive displacement of the ProLabel conjugate from either a binding protein (receptor or antibody) is induced by the analyte in question. In this manner, a calibration curve is generated, to measure cellular analytes including 3',5'-cyclic AMP. Changes in this second messenger, occurring due to G protein-coupled receptor (GPCR) activation, can thus be easily measured in a homogeneous assay. Similar assays have been developed for tyrosine kinases, serine threonine kinases, nuclear hormone receptors, and proteases. A second form of assay technology involves measurement of cellular protein expression, in which the protein is fused to ProLabel. Analysis can be undertaken in crude cell lysates, or with intact cells, using beta-galactosidase complementation in a microtiter plate. This homogeneous technology is highly sensitive and has been developed to measure protein expression changes occurring in response to pathway activation by targets such as GPCRs, tyrosine kinase receptors, and proteases. In summary, the DiscoveRx technology using beta-galactosidase complementation provides a robust and flexible assay technology for use in cell-free and cell-based HTS.
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PMID:Enzyme fragment complementation: a flexible high throughput screening assay technology. 1509 Jan 61

Activation of cells by the tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) cytokines results in activation of the nuclear factor-kappaB (NF-kappaB) via proteasomal degradation of an associated IkappaB molecule. To monitor cellular IkappaB, the protein was recombinantly expressed as a fusion protein with a novel enzymatic tag, ProLabel (PL). ProLabel is a small 5.5-kDa sequence from the amino-terminal amino acids of beta-galactosidase, possesses a simple ribbon structure, and can be fused to many proteins via the amino or carboxyl terminus. Expression of this construct allows quantitative detection of the recombinant protein in crude lysates by using a method based on beta-galactosidase enzyme fragment complementation (EFC). Transient transfection of IkappaB-PL in HeLa cells generated an EFC signal that was highly correlated with a western analysis of the protein construct. ProLabel expressed alone in the cells did not show any EFC activity, due to rapid proteolytic degradation, indicating a very low background signal from the protein tag. TNF-alpha and IL-1 treatment induced a concentration-dependent degradation of IkappaB-PL, with potency values similar to those reported using other methods. IkappaBM-PL (mutant of IkappaB-PL), in contrast, did not undergo degradation for concentrations up to and including 10 ng/ml TNF-alpha or IL-1, demonstrating that degradation of IkappaB-PL was specific to the NF-kappaB pathway activation. TNF-alpha and IL-1 induced maximal IkappaB-PL degradation within 30 min of induction. This was reversed by several agents that ablate this pathway, including anti-TNF-alpha antibodies and the proteasome inhibitor, MG-132. The assay was amenable to HTS systems, with good precision and reproducibility. Z' values and coefficients of variance for IkappaB-PL degradation were 0.6 and <9%, respectively.
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PMID:Homogeneous assays for cellular protein degradation using beta-galactosidase complementation: NF-kappaB/IkappaB pathway signaling. 1509 Feb 28

This article describes the automation of an in vitro cell-based fusion assay for the identification of novel inhibitors of receptor mediated HIV-1 entry. The assay utilises two stable cell lines: one expressing CD4, CCR5 and an LTR-promoter/beta-galactosidase reporter construct, and the other expressing gp160 and tat. Accumulation of beta-galactosidase can only occur following fusion of these two cell lines via the gp160 and receptor mediators, as this event facilitates the transfer of the tat transcription factor between the two cell types. Although similar cell fusion systems have been described previously, they have not met the requirements for HTS due to complexity, throughput and reagent cost. The assay described in this article provides significant advantage, as (a) no transfection/infection events are required prior to the assay, reducing the potential for variability, (b) cells are mixed in solution, enhancing fusion efficiency compared to adherent cells, (c) miniaturization to low volume enables screening in 384-well plates; and (d) online cell dispensing facilitates automated screening. This assay has been employed to screen approximately 650,000 compounds in a singleton format. The data demonstrate that the assay is robust, with a Z' consistently above 0.6, which compares favourably with less complex biochemical assays.
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PMID:Development and automation of a 384-well cell fusion assay to identify inhibitors of CCR5/CD4-mediated HIV virus entry. 1545 38

Chagas disease is a tropical disease caused by the parasite Trypanosoma cruzi, which is endemic in Central and South America. Few treatments are available with effectiveness limited to the early (acute) stage of disease, significant toxicity and widespread drug resistance. In this work we report the outcome of a HTS-ready assay chemical library screen to identify novel, nontoxic, small-molecule inhibitors of T. cruzi. We have selected 50 compounds that possess hydrazone as a common group. The compounds were screened using recombinant T. cruzi (Tulahuen strain) expressing beta-galactosidase. A 3D quantitative structure-activity relationship (QSAR) analysis was performed using descriptors calculated from comparative molecular field analysis (CoMFA). Our findings show that of the fifty selected hydrazones, compounds LpQM-19, 28 and 31 displayed the highest activity against T. cruzi, leading to a selectivity index (SI) of 20-fold. The 3D-QSAR analysis indicates that a particular electrostatic arrangement, where electron-deficient atoms are aligned along the molecule main axis positively correlates with compound biological activity. These results provide new candidate molecules for the development of treatments against Chagas disease.
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PMID:Compound profiling and 3D-QSAR studies of hydrazone derivatives with activity against intracellular Trypanosoma cruzi. 2696 73