GI254023X is a potent MMP9 and ADAM10 inhibitor (IC50s: 2.5 and 5.3 nM) with 100-fold selectivity for the α-secretase ADAM10 over ADAM17 (TACE). GI254023X can remarkably inhibit the proliferation and induce the apoptosis of H929 cells, potentially through inhibition of Notch1 activation.

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

In the cellular assay, GI254023X (1-25 μM) strongly reduces constitutive RAGE shedding and also PACAP-inducing shedding of RAGE is significantly reduced. At a concentration of 100 nM, a slight inhibition of RAGE shedding is still observed. In in vitro assays with recombinant proteinases, GI254023X discriminates between ADAM17 (IC50: 541 nM) and ADAM10 (IC50: 5.3 nM)/MMP9 (IC50: 2.5 nM) [1]. CXCL16 shedding is inhibited by GI254023x. A2780 cells are incubated with the ADAM-10/ADAM-17 inhibitor TAPI-2, as well as the ADAM10-selective inhibitor GI254023x, as the level of expressed ADAM10, is on average 9.8-fold higher on mRNA level compare with ADAM17. In addition, GI254023x also prevents CXCL16 shedding from the cell membrane and is even more potent than TAPI-2[2]. When apply the specific ADAM10 (α-secretase) inhibitor GI254023X (5 mM) to serum/glucose-deprived slices, PI counts are significantly increased in comparison with DMSO (carrier)-treated controls [3].

Acute treatment with GI254023X in an AD mouse model substantially reduces brain LRP1 shedding and increases Aβ40 levels in the plasma, indicating enhanced Aβ transit from the brain to the periphery [4].

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].