PMC:4197335 / 13006-17267 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/4197335","sourcedb":"PMC","sourceid":"4197335","source_url":"http://www.ncbi.nlm.nih.gov/pmc/4197335","text":"Development of D-Tetrapeptide Inhibitors of the GADD45β/MKK7 Complex\nGiven the essential antiapoptotic role of GADD45β in MM and our previous results showing that GADD45β suppresses JNK signaling and apoptosis by blocking MKK7 via direct physical interaction (De Smaele et al., 2001; Papa et al., 2004, 2007), we aimed to develop selective inhibitors of this protein-protein interaction in order to induce cytotoxic JNK signaling in MM cells. We screened a simplified combinatorial library of 20,736 L-tetrapeptides to select compounds capable of disrupting the GADD45β/MKK7 complex (Figure S3A and Table S1). Iterative deconvolution of this library in ELISA competition assays, followed by secondary screening and optimization of the resulting hits, yielded two acetylated L-tetrapeptides of similar structure, namely, Ac-LTP1 and Ac-LTP2, which disrupted the GADD45β/MKK7 complex, in vitro, with remarkable half-maximal inhibitory concentration (IC50) values in the subnanomolar range (Figure 3A; Figure S3B and Table S2), in line with the top-end potencies of other hits isolated from similar peptide library screens (Houghten et al., 1999).\nNext, we examined whether these two L-tetrapeptides retained in vitro potency upon synthesis in the D configuration, a strategy used successfully in some cases to render small peptides resistant to proteolysis (Zhou et al., 2002; Nickl et al., 2010). Strikingly, as shown in Figures 3A and 3B, the D-enantiomers of Ac-LTP1 and Ac-LTP2 (termed Ac-DTP1 and Ac-DTP2, respectively) displayed no loss of activity in vitro and, unlike their L counterparts, were highly stable in human serum, even after prolonged incubation. Coimmunoprecipitation assays confirmed the potent and specific inhibitory effect of Ac-DTP1 and Ac-DTP2 on GADD45β/MKK7 complex stability in vitro (Figure 3C). By contrast, control D-tetrapeptides had no such effect on stability, thus demonstrating the specificity of the inhibitory activities of Ac-DTP1 and Ac-DTP2. Crucially, the disruption of the complex by these two active D-peptides completely reversed the GADD45β-mediated inhibition of MKK7, fully restoring the kinase catalytic activity (Figure 3D, top). Importantly, none of Ac-DTP1, Ac-DTP2, or any of the control D-tetrapeptides affected MKK7 activity in the absence of GADD45β (Figure 3D, bottom). Collectively, these findings identify a downstream, drug-targetable module in the NF-κB pathway and confirm the potential of our pharmacological approach for inducing cytotoxic MKK7/JNK signaling in cells that rely on GADD45β for restraining MKK7 activation.\nTo verify the therapeutic potential of GADD45β/MKK7 inhibitors, we aimed to improve their cell penetration. We replaced the N-terminal acetyl group of Ac-DTP1 and Ac-DTP2 with a benzyloxycarbonyl [Z] group, thereby generating z-DTP1 and z-DTP2, respectively (Table S3), which retained high activity and stability in vitro (Figures S4A and S4B) and, importantly, also exhibited potent cytotoxic activity across a panel of genetically heterogeneous MM cell lines (Figures 4A and 4B; Figures S4C–S4F and Table S3). Significantly, z-DTP1 and z-DTP2, but not a control D-tetrapeptide, induced potent and dose-dependent toxicity in all of the MM cell lines tested, except the two expressing nearly undetectable levels of GADD45B and low levels of MKK7 (further discussed below), exhibiting IC50 values in the sensitive MM cell lines in the low nanomolar to low micromolar range (Figures 4A and 4B; Figures S2G, S4E, and S4F). Consistent with the protective mechanism mediated by GADD45β (Figure S2G), the z-DTP1- and z-DTP2-afforded killing of sensitive MM cells was due to the induction of apoptosis (Figure 4C; Figures S4G and S4H). Importantly, both active D-peptides retained potent and cancer-selective activity in primary PCs from MM patients (Figure 4D, left). Crucially, these D-peptides also exhibited an apparently complete lack of toxicity to normal cells, even when used at very high concentrations (i.e., 100 μM; Figure 4D, right; Figure S4I). Hence, D-tetrapeptide antagonists of the GADD45β/MKK7 complex show exceptionally high activity and cancer cell specificity in terms of apoptosis induction in MM cells, without displaying any apparent toxicity to normal cells.","divisions":[{"label":"Title","span":{"begin":0,"end":68}}],"tracks":[{"project":"2_test","denotations":[{"id":"25314077-11713530-17318411","span":{"begin":303,"end":307},"obj":"11713530"},{"id":"25314077-14743220-17318411","span":{"begin":303,"end":307},"obj":"14743220"},{"id":"25314077-17485467-17318411","span":{"begin":303,"end":307},"obj":"17485467"},{"id":"25314077-10508425-17318412","span":{"begin":1138,"end":1142},"obj":"10508425"},{"id":"25314077-11880384-17318413","span":{"begin":1389,"end":1393},"obj":"11880384"},{"id":"25314077-20018259-17318413","span":{"begin":1389,"end":1393},"obj":"20018259"}],"attributes":[{"subj":"25314077-11713530-17318411","pred":"source","obj":"2_test"},{"subj":"25314077-14743220-17318411","pred":"source","obj":"2_test"},{"subj":"25314077-17485467-17318411","pred":"source","obj":"2_test"},{"subj":"25314077-10508425-17318412","pred":"source","obj":"2_test"},{"subj":"25314077-11880384-17318413","pred":"source","obj":"2_test"},{"subj":"25314077-20018259-17318413","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ec939d","default":true}]}]}}