A High-Throughput Assay to Identify Allosteric Inhibitors of the PLC‑γ Isozymes Operating at Membranes
The two phospholipase C-γ (PLC-γ) isozymes are major signaling hubs and emerging therapeutic targets for various diseases, yet there are no selective inhibitors for these enzymes. We have developed a high-throughput, liposome-based assay that features XY-69, a fluorogenic, membrane-associated report...
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| Veröffentlicht in: | Biochemistry (Easton) 2020-10, Vol.59 (41), p.4029-4038 |
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| container_title | Biochemistry (Easton) |
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| creator | Huang, Weigang Carr, Adam J Hajicek, Nicole Sokolovski, Miri Siraliev-Perez, Edhriz Hardy, P. Brian Pearce, Kenneth H Sondek, John Zhang, Qisheng |
| description | The two phospholipase C-γ (PLC-γ) isozymes are major signaling hubs and emerging therapeutic targets for various diseases, yet there are no selective inhibitors for these enzymes. We have developed a high-throughput, liposome-based assay that features XY-69, a fluorogenic, membrane-associated reporter for mammalian PLC isozymes. The assay was validated using a pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) in 384-well format; it is highly reproducible and has the potential to capture both orthosteric and allosteric inhibitors. Selected hit compounds were confirmed with secondary assays, and further profiling led to the interesting discovery that adenosine triphosphate potently inhibits the PLC-γ isozymes through noncompetitive inhibition, raising the intriguing possibility of endogenous, nucleotide-dependent regulation of these phospholipases. These results highlight the merit of the assay platform for large scale screening of chemical libraries to identify allosteric modulators of the PLC-γ isozymes as chemical probes and for drug discovery. |
| doi_str_mv | 10.1021/acs.biochem.0c00511 |
| format | Article |
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Selected hit compounds were confirmed with secondary assays, and further profiling led to the interesting discovery that adenosine triphosphate potently inhibits the PLC-γ isozymes through noncompetitive inhibition, raising the intriguing possibility of endogenous, nucleotide-dependent regulation of these phospholipases. 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Brian</creatorcontrib><creatorcontrib>Pearce, Kenneth H</creatorcontrib><creatorcontrib>Sondek, John</creatorcontrib><creatorcontrib>Zhang, Qisheng</creatorcontrib><title>A High-Throughput Assay to Identify Allosteric Inhibitors of the PLC‑γ Isozymes Operating at Membranes</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>The two phospholipase C-γ (PLC-γ) isozymes are major signaling hubs and emerging therapeutic targets for various diseases, yet there are no selective inhibitors for these enzymes. We have developed a high-throughput, liposome-based assay that features XY-69, a fluorogenic, membrane-associated reporter for mammalian PLC isozymes. The assay was validated using a pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) in 384-well format; it is highly reproducible and has the potential to capture both orthosteric and allosteric inhibitors. Selected hit compounds were confirmed with secondary assays, and further profiling led to the interesting discovery that adenosine triphosphate potently inhibits the PLC-γ isozymes through noncompetitive inhibition, raising the intriguing possibility of endogenous, nucleotide-dependent regulation of these phospholipases. These results highlight the merit of the assay platform for large scale screening of chemical libraries to identify allosteric modulators of the PLC-γ isozymes as chemical probes and for drug discovery.</description><subject>Animals</subject><subject>Cell Membrane - enzymology</subject><subject>Humans</subject><subject>Isoenzymes - chemistry</subject><subject>Isoenzymes - metabolism</subject><subject>Phospholipase C gamma - chemistry</subject><subject>Phospholipase C gamma - metabolism</subject><subject>Signal Transduction - physiology</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kU9OGzEYxS1UBClwAqTKy24m2B7PH2-QoqiFSKnoAtbWN7YnYzQzTm1PpXTVK_Qs3KOH6ElqlBTBpqtP1vfes59_CF1SMqeE0StQYd5YpzozzIkipKD0CM1owUjGhSjeoRkhpMyYKMkpeh_CYzpyUvETdJrnhNWkojNkF_jWbrrsvvNu2nTbKeJFCLDD0eGVNmO07Q4v-t6FaLxVeDV2trHR-YBdi2Nn8Nf18s_PX7-f8Cq4H7vBBHy3NR6iHTcYIv5ihsbDaMI5Om6hD-biMM_Qw-dP98vbbH13s1ou1hlwXsSMt6yivMgh5wp0WdKSAgClFS9ZzWqhal0RoBUrtdalKEBTDXmrG9Ea0XCWn6Hrfe52agajVergoZdbbwfwO-nAyreb0XZy477LmtSU5UUK-HgI8O7bZEKUgw3K9H1q4aYgGeeCFYJWJEnzvVR5F4I37cs1lMhnSDJBkgdI8gApuT68fuGL5x-VJLjaC57dj27yY_qw_0b-BV8Zo5U</recordid><startdate>20201020</startdate><enddate>20201020</enddate><creator>Huang, Weigang</creator><creator>Carr, Adam J</creator><creator>Hajicek, Nicole</creator><creator>Sokolovski, Miri</creator><creator>Siraliev-Perez, Edhriz</creator><creator>Hardy, P. 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The assay was validated using a pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) in 384-well format; it is highly reproducible and has the potential to capture both orthosteric and allosteric inhibitors. Selected hit compounds were confirmed with secondary assays, and further profiling led to the interesting discovery that adenosine triphosphate potently inhibits the PLC-γ isozymes through noncompetitive inhibition, raising the intriguing possibility of endogenous, nucleotide-dependent regulation of these phospholipases. 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| source | MEDLINE; ACS Publications |
| subjects | Animals Cell Membrane - enzymology Humans Isoenzymes - chemistry Isoenzymes - metabolism Phospholipase C gamma - chemistry Phospholipase C gamma - metabolism Signal Transduction - physiology |
| title | A High-Throughput Assay to Identify Allosteric Inhibitors of the PLC‑γ Isozymes Operating at Membranes |
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