A High-Throughput Screening Platform Targeting PDLIM5 for Pulmonary Hypertension.

Publisher: Sage Publications Country of Publication: United States NLM ID: 9612112 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1552-454X (Electronic) Linking ISSN: 10870571 NLM ISO Abbreviation: J Biomol Screen Subsets: MEDLINE

Publication: <2004- > : Thousand Oaks, CA : Sage Publications
Original Publication: Larchmont, NY : Mary Ann Liebert, Inc., c1996-

High-Throughput Screening Assays*; Adaptor Proteins, Signal Transducing/*agonists ; Antihypertensive Agents/*pharmacology ; LIM Domain Proteins/*agonists ; Paclitaxel/*pharmacology; A549 Cells ; Adaptor Proteins, Signal Transducing/antagonists & inhibitors ; Adaptor Proteins, Signal Transducing/genetics ; Adaptor Proteins, Signal Transducing/metabolism ; Animals ; Epithelial Cells/drug effects ; Epithelial Cells/metabolism ; Epithelial Cells/pathology ; Gene Expression ; Genes, Reporter ; Genetic Vectors/antagonists & inhibitors ; Genetic Vectors/chemistry ; Genetic Vectors/metabolism ; Humans ; Hypertension, Pulmonary/drug therapy ; LIM Domain Proteins/antagonists & inhibitors ; LIM Domain Proteins/genetics ; LIM Domain Proteins/metabolism ; Lentivirus/genetics ; Lentivirus/metabolism ; Luciferases/genetics ; Luciferases/metabolism ; Lung/drug effects ; Lung/metabolism ; Lung/pathology ; Mink ; Phosphorylation/drug effects ; RNA, Small Interfering/genetics ; RNA, Small Interfering/metabolism ; Smad2 Protein/antagonists & inhibitors ; Smad2 Protein/genetics ; Smad2 Protein/metabolism ; Smad3 Protein/antagonists & inhibitors ; Smad3 Protein/genetics ; Smad3 Protein/metabolism ; Transforming Growth Factor beta/genetics ; Transforming Growth Factor beta/metabolism

Pulmonary arterial hypertension is a complex disease with multiple etiologic factors. PDLIM5, a member of the Enigma subfamily of PDZ and LIM domain protein family, contains an N-terminal PDZ domain and three LIM domains at its C-terminus. We have previously shown that overexpression of PDLIM5 prevents hypoxia-induced pulmonary hypertension (PH), and deletion of PDLIM5 in smooth muscle cells enhances hypoxia-induced PH in vivo. These results suggest that PDLIM5 may be a novel therapeutic target of PH. In this study, we aim to establish a high-throughput screening platform for PDLIM5-targeted drug discovery. We generated a stable mink lung epithelial cell line (MLEC) containing a transforming growth factor-β/Smad luciferase reporter with lentivirus-mediated suppression of PDLIM5 (MLEC-shPDLIM5) and measured levels of Smad2/3 and pSmad2/3. We found that in MLEC, suppression of PDLIM5 decreased Smad-dependent luciferase activity, Smad3, and pSmad3. We used MLEC-shPDLIM5 and a control cell line (MLEC-shCTL) to screen the Prestwick library (1200 compounds) and identified and validated paclitaxel as a PDLIM5 inhibitor in MLEC. Furthermore, we showed that paclitaxel inhibited Smad2 expression and Smad3 phosphorylation in A549 cells. Our study suggests that this system is robust and suitable for PDLIM5-targeted drug discovery.
(© 2016 Society for Laboratory Automation and Screening.)

R01HL123804 United States HL NHLBI NIH HHS

Keywords: PDLIM5; TGF/Smad; high-throughput screening; luciferase reporter assay

0 (Adaptor Proteins, Signal Transducing)
0 (Antihypertensive Agents)
0 (LIM Domain Proteins)
0 (PDLIM5 protein, human)
0 (RNA, Small Interfering)
0 (SMAD2 protein, human)
0 (SMAD3 protein, human)
0 (Smad2 Protein)
0 (Smad3 Protein)
0 (Transforming Growth Factor beta)
EC 1.13.12.- (Luciferases)
P88XT4IS4D (Paclitaxel)

Date Created: 20160115 Date Completed: 20161213 Latest Revision: 20161230

20221213

10.1177/1087057115625924

26762503