From a therapeutic perspective, it is interesting that ISA27 in combination with the conventional chemotherapy drug TMZ inhibited U87MG cell growth. This combination worked in a synergistic manner as confirmed by isobolographic GDC-0199 Bcl-2 inhibitor analysis. This result suggests the possibility of lowering the dose of TMZ used in the treatment of GBM. In conclusion, our data show that ISA27 disrupts the MDM2p53 interaction and releases the powerful antitumor capacities of p53 in GBM cells. The use of this MDM2 inhibitor could offer a novel therapy for the treatment of GBM patients by inhibiting tumor growth. Proteases catalyze the hydrolysis of peptide bonds in proteins and are involved in digestive as well as regulatory processes. In the human genome, approximately 2% of the genes code for proteases. While most proteases are soluble, a small fraction is membrane-embedded. These intramembrane proteases differ from soluble proteases in a variety of aspects: They are composed of a number of transmembrane domains which harbor the catalytic residues with their active sites buried several A ? into the membrane. Their substrates are transmembrane proteins that reside inside the membrane in a dormant form. Upon cleavage, most substrates release a soluble part into the cytosol or extracellular space. It is therefore not surprising that intramembrane proteases are involved in various signaling pathways. There are three families of intramembrane proteases, classified according to their catalytic mechanism: intramembrane metalloproteases, intramembrane aspartic proteases, and intramembrane serine proteases. The latter belong to the family of rhomboid proteins, containing active intramembrane proteases and inactive homologs. Rhomboids are found in all kingdoms of life, but are functionally diverse. They take part in various distinct cellular processes such as the EGFR-signaling pathway in the fruit fly Drosophila melanogaster, quorum sensing in the Gram negative bacterium Providencia stuartii and host cell infection by apicomplexan parasites. Structurally, rhomboids are the best characterized intramembrane proteases. Several different crystal forms of the E. coli rhomboid GlpG have provided insight into the mechanism of intramembrane proteolysis. However, a detailed picture of the rhomboid-substrate interaction is not available. As an alternative, crystal structures of covalent inhibitors bound to GlpG have revealed which areas and residues may play a role in primed and non-primed site interaction, and oxyanion stabilization. The availability of inhibitors is also important for future functional studies. Moreover, potent and BAY 73-4506 selective inhibitors may serve as lead structures for future drug design. Up to date, rhomboid inhibitors have been reported based on three distinct scaffolds: 4-chloro-isocoumarins, fluorophosphonates, and N-sulfonylated beta-lactams. However, these are not selective enough to inhibit only rhomboids within the entire proteome. In addition, these inhibitors are also not promiscuous enough to inhibit rhomboids from different organisms equally well. Therefore, it is still of great interest to find new types of inhibitors. In order to facilitate this search, various screening methods have been employed so far. All of these have relied on monitoring the cleavage of a substrate through gel-based, FRET or MALDI mass spectrometry techniques. However, a limitation of these methods is the availability of a matching protein or polypeptide substrate. Rhomboids from one species may cleave substrates from another species, but this is not a general rule. We therefore reasoned that it would be beneficial to develop an inhibitor assay for rhomboid proteases that does not rely on a substrate at all. A few years ago Cravatt and co-workers developed a highthroughput inhibitor screening method that uses fluorescent activity-based probes. ABPs are small molecules that covalently bind to the active form of an enzyme, but not to an inactivated or zymogen form. ABPs generally consist of a tag, a spacer and an electrophilic group that traps an active site nucleophile. The binding event can be detected by a variety of techniques, such as gel-scanning, biotin blot or fluorescent microscopy, depending on the tagging moiety. When appended to a fluorescent dye, the binding of an ABP can be detected by fluorescence polarization. This so-called fluorescence polarization activity-based protein profiling has been used in inhibitor high-throughput screens for a variety of poorly characterized enzymes.