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Cancer-selective targeting of the NF-κB survival pathway with GADD45β/MKK7 inhibitors.

Tornatore L, Sandomenico A, Raimondo D, Low C, Rocci A, Tralau-Stewart C, Capece D, D'Andrea D, Bua M, Boyle E, van Duin M, Zoppoli P, Jaxa-Chamiec A, Thotakura AK, Dyson J, Walker BA, Leonardi A, Chambery A, Driessen C, Sonneveld P, Morgan G, Palumbo A, Tramontano A, Rahemtulla A, Ruvo M, Franzoso G - Cancer Cell (2014)

Bottom Line: Using a drug-discovery strategy, we developed DTP3, a D-tripeptide, which disrupts the GADD45β/MKK7 complex, kills MM cells effectively, and, importantly, lacks toxicity to normal cells.Notably, DTP3 ablates myeloma xenografts in mice with no apparent side effects at the effective doses.Hence, cancer-selective targeting of the NF-κB pathway is possible and, at least for myeloma patients, promises a profound benefit.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Centre for Cell Signalling and Inflammation, Imperial College London, London W12 0NN, UK.

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The Potent Activity and Stability of D-Peptide Inhibitors of GADD45β/MKK7 In Vitro(A and B) ELISA GADD45β/MKK7 competition assays showing the IC50 values of the active L-tetrapeptides (Ac-LTP1, Ac-LTP2) and D-tetrapeptides (Ac-DTP1, Ac-DTP2), before (A) and after (B) a 48 hr preincubation with human serum. Values express the percentage of inhibition of GADD45β binding to MKK7 relative to the binding in the absence of peptide and denote means ± SD (n = 3). Ac-LNC, acetylated (Ac) negative control L-tetrapeptide (LNC); Ac-DNC, Ac negative control D-tetrapeptide (DNC).(C) Coimmunoprecipitations (IP) performed with cell lysates prepared from human embryonic kidney 293T cells expressing ectopic HA-tagged GADD45β (HA-hGADD45β) and FLAG-tagged MKK7 (FLAG-hMKK7) and incubated with anti-FLAG antibody in the presence or absence of bioactive (Ac-DTP1 and Ac-DTP2) or inactive (Ac-DNC, Ac-DNC2, Ac-DNC3 and Ac-DNC4) D-tetrapeptides, as indicated. Western blots were developed using anti-HA or anti-MKK7 antibodies. -, incubation without D-tetrapeptides.(D) Kinase assays (K.A.) showing MKK7 activity before (-) and after incubation with Ac-DTP1, Ac-DTP2 or control D-tetrapeptides as in (C), in the presence (+) or absence of recombinant human (h)GADD45β. T/I, TPA/ionomycin; UT, untreated.See also Figure S3 and Tables S1 and S2.
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fig3: The Potent Activity and Stability of D-Peptide Inhibitors of GADD45β/MKK7 In Vitro(A and B) ELISA GADD45β/MKK7 competition assays showing the IC50 values of the active L-tetrapeptides (Ac-LTP1, Ac-LTP2) and D-tetrapeptides (Ac-DTP1, Ac-DTP2), before (A) and after (B) a 48 hr preincubation with human serum. Values express the percentage of inhibition of GADD45β binding to MKK7 relative to the binding in the absence of peptide and denote means ± SD (n = 3). Ac-LNC, acetylated (Ac) negative control L-tetrapeptide (LNC); Ac-DNC, Ac negative control D-tetrapeptide (DNC).(C) Coimmunoprecipitations (IP) performed with cell lysates prepared from human embryonic kidney 293T cells expressing ectopic HA-tagged GADD45β (HA-hGADD45β) and FLAG-tagged MKK7 (FLAG-hMKK7) and incubated with anti-FLAG antibody in the presence or absence of bioactive (Ac-DTP1 and Ac-DTP2) or inactive (Ac-DNC, Ac-DNC2, Ac-DNC3 and Ac-DNC4) D-tetrapeptides, as indicated. Western blots were developed using anti-HA or anti-MKK7 antibodies. -, incubation without D-tetrapeptides.(D) Kinase assays (K.A.) showing MKK7 activity before (-) and after incubation with Ac-DTP1, Ac-DTP2 or control D-tetrapeptides as in (C), in the presence (+) or absence of recombinant human (h)GADD45β. T/I, TPA/ionomycin; UT, untreated.See also Figure S3 and Tables S1 and S2.

Mentions: Given 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).


Cancer-selective targeting of the NF-κB survival pathway with GADD45β/MKK7 inhibitors.

Tornatore L, Sandomenico A, Raimondo D, Low C, Rocci A, Tralau-Stewart C, Capece D, D'Andrea D, Bua M, Boyle E, van Duin M, Zoppoli P, Jaxa-Chamiec A, Thotakura AK, Dyson J, Walker BA, Leonardi A, Chambery A, Driessen C, Sonneveld P, Morgan G, Palumbo A, Tramontano A, Rahemtulla A, Ruvo M, Franzoso G - Cancer Cell (2014)

The Potent Activity and Stability of D-Peptide Inhibitors of GADD45β/MKK7 In Vitro(A and B) ELISA GADD45β/MKK7 competition assays showing the IC50 values of the active L-tetrapeptides (Ac-LTP1, Ac-LTP2) and D-tetrapeptides (Ac-DTP1, Ac-DTP2), before (A) and after (B) a 48 hr preincubation with human serum. Values express the percentage of inhibition of GADD45β binding to MKK7 relative to the binding in the absence of peptide and denote means ± SD (n = 3). Ac-LNC, acetylated (Ac) negative control L-tetrapeptide (LNC); Ac-DNC, Ac negative control D-tetrapeptide (DNC).(C) Coimmunoprecipitations (IP) performed with cell lysates prepared from human embryonic kidney 293T cells expressing ectopic HA-tagged GADD45β (HA-hGADD45β) and FLAG-tagged MKK7 (FLAG-hMKK7) and incubated with anti-FLAG antibody in the presence or absence of bioactive (Ac-DTP1 and Ac-DTP2) or inactive (Ac-DNC, Ac-DNC2, Ac-DNC3 and Ac-DNC4) D-tetrapeptides, as indicated. Western blots were developed using anti-HA or anti-MKK7 antibodies. -, incubation without D-tetrapeptides.(D) Kinase assays (K.A.) showing MKK7 activity before (-) and after incubation with Ac-DTP1, Ac-DTP2 or control D-tetrapeptides as in (C), in the presence (+) or absence of recombinant human (h)GADD45β. T/I, TPA/ionomycin; UT, untreated.See also Figure S3 and Tables S1 and S2.
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Related In: Results  -  Collection

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fig3: The Potent Activity and Stability of D-Peptide Inhibitors of GADD45β/MKK7 In Vitro(A and B) ELISA GADD45β/MKK7 competition assays showing the IC50 values of the active L-tetrapeptides (Ac-LTP1, Ac-LTP2) and D-tetrapeptides (Ac-DTP1, Ac-DTP2), before (A) and after (B) a 48 hr preincubation with human serum. Values express the percentage of inhibition of GADD45β binding to MKK7 relative to the binding in the absence of peptide and denote means ± SD (n = 3). Ac-LNC, acetylated (Ac) negative control L-tetrapeptide (LNC); Ac-DNC, Ac negative control D-tetrapeptide (DNC).(C) Coimmunoprecipitations (IP) performed with cell lysates prepared from human embryonic kidney 293T cells expressing ectopic HA-tagged GADD45β (HA-hGADD45β) and FLAG-tagged MKK7 (FLAG-hMKK7) and incubated with anti-FLAG antibody in the presence or absence of bioactive (Ac-DTP1 and Ac-DTP2) or inactive (Ac-DNC, Ac-DNC2, Ac-DNC3 and Ac-DNC4) D-tetrapeptides, as indicated. Western blots were developed using anti-HA or anti-MKK7 antibodies. -, incubation without D-tetrapeptides.(D) Kinase assays (K.A.) showing MKK7 activity before (-) and after incubation with Ac-DTP1, Ac-DTP2 or control D-tetrapeptides as in (C), in the presence (+) or absence of recombinant human (h)GADD45β. T/I, TPA/ionomycin; UT, untreated.See also Figure S3 and Tables S1 and S2.
Mentions: Given 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).

Bottom Line: Using a drug-discovery strategy, we developed DTP3, a D-tripeptide, which disrupts the GADD45β/MKK7 complex, kills MM cells effectively, and, importantly, lacks toxicity to normal cells.Notably, DTP3 ablates myeloma xenografts in mice with no apparent side effects at the effective doses.Hence, cancer-selective targeting of the NF-κB pathway is possible and, at least for myeloma patients, promises a profound benefit.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Centre for Cell Signalling and Inflammation, Imperial College London, London W12 0NN, UK.

Show MeSH
Related in: MedlinePlus