GI254023X is a MMP9 and ADAM10 inhibitor with IC50 values ​​of 2.5 and 5.3 nM, respectively. GI254023X can also significantly inhibit the proliferation of Jurkat cells and induce apoptosis.

ADAM10:5.3 nM (cell free), MMP9:2.5 nM (cell free)

METHODS: Mouse mesothelioma cells were treated with GI254023X (5 μM, 4, 24, 48, 72 hours), and the proliferation of AB12 and PM27 cells was measured by CYQUANT analysis; the percentage of AB12 or PM27 cells in different phases of the cell cycle (G1, S or G2) was measured by FACS analysis and wound healing assay was performed to study its effects on mesothelioma cell proliferation, migration and invasion.
RESULTS GI254023X had no effect on the proliferation of either cell type; mouse mesothelioma cells treated with GI254023X showed weaker migration properties in transwell chambers; GI254023X showed a significant reduction in migration of mouse mesothelioma cells in the scratch assay (4, 6 and 8 hours); and the invasion of AB12 and PM27 cells measured in the spheroid assay was also significantly reduced. [3]

METHODS: C57BL/6N mice were subjected to a controlled cortical impact (CCI) model of TBI or sham surgery and received GI254023X or vehicle (40, 100 mg/kg, intraperitoneal injection, 30 minutes and 24 hours after TBI) in the acute phase of injury. The expression of brain mRNA and some inflammatory factors was measured by quantitative PCR to study its effects on neurological and histopathological outcomes in mice after experimental traumatic brain injury (TBI).
RESULTS GI254023X treatment did not improve neurological deficits from 1 to 7 days post-injury (DPI), but animals treated with GI254023X showed less brain damage compared with vehicle treatment. Brain mRNA expression measured by quantitative PCR showed that TBI-induced upregulation of Adam10 and Adam17 was not affected by GI254023X, but upregulation of matrix metalloproteinase genes Mmp2 and Mmp9 was attenuated; GI254023X also attenuated upregulation of proinflammatory markers Il6, Trfa, and Lcn2, but not pan-microglial marker Aif1, M2 microglial marker Arg1, and reactive astrocyte marker Gfap.[1]

Cell death is quantified based on plasma membrane permeabilization. When applying the ADAM10 (a-secretase) inhibitor GI254023X (5 mM), slices are cultured in the serum-/glucose-free medium for 48 h containing the inhibitor or its respective carrier (DMSO) as control. Round circles of identical size (? 500mm) are positioned in equivalent locations within the CA1 region of each hippocampus image and all PI-stained cells are counted using the software. Cell viability assays are performed with a commercial kit according to the manufacturer's instructions. The assay quantitates ATP levels, an indicator of metabolically active cells, photometrically with a fluorescence plate reader. Additionally, the live-dead cell staining kit are applied according to the manual. Cells are simultaneously stained with green fluorescent calcein-AM (4mM; ex/em: 495/515 nm) to detect intracellular esterase activity (viable cells) and red fluorescent ethidium homodimer-3 (2mM; ex/em: 530/635 nm) to indicate loss of plasma membrane integrity (dead cells) [3].