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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 High Target Specificity of DTP3 in Cells(A) The structure of DTP3.(B) Tryptophan fluorescence quenching analysis showing the dose-response curve of the -Δfluorescence values of GST-hMKK7 at 333 nm plotted against the concentration values of DTP3. The stoichiometry and KD value of the DTP3/MKK7 interaction on the basis of these data were 1:1 and 64.81 ± 6.22 nM, respectively. Values denote means ± SD (n = 3).(C) Correlation plot of the relative GADD45B mRNA levels (qRT-PCR) and the DTP3 IC50 at 144 hr ([3H]thymidine incorporation) in cancer cell lines of different tissues of origin. Values on the x axis express the logarithm to base 10 (log10) of the IC50. rS, Spearman correlation coefficient.(D) K.A. showing JNK activity in representative sensitive (KMS-12, U266) and resistant (RPMI-8226) MM cell lines after treatment with DTP3 (10 μM). TNFα is shown as a positive control.(E) Peptide pull-down showing the physical association of DTP3 with endogenous MKK7 in U266 and KMS-12 MM cells. SCRB D-peptide is used as a negative control. -, pull-down without D-tripeptide.(F) [3H]Thymidine incorporation showing the survival of U266 and KMS-12 MM cells expressing sh-ns or sh-MKK7 shRNAs after a 6-day treatment with the indicated concentrations of DTP3. The IC50 values of DTP3 are depicted. Values express the percentage of the counts per minute (cpm) measured with the treated cultures relative to the cpm measured with the respective untreated cultures and denote mean ± SD (n = 3).See also Figure S5 and Tables S4 and S5.
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fig5: The High Target Specificity of DTP3 in Cells(A) The structure of DTP3.(B) Tryptophan fluorescence quenching analysis showing the dose-response curve of the -Δfluorescence values of GST-hMKK7 at 333 nm plotted against the concentration values of DTP3. The stoichiometry and KD value of the DTP3/MKK7 interaction on the basis of these data were 1:1 and 64.81 ± 6.22 nM, respectively. Values denote means ± SD (n = 3).(C) Correlation plot of the relative GADD45B mRNA levels (qRT-PCR) and the DTP3 IC50 at 144 hr ([3H]thymidine incorporation) in cancer cell lines of different tissues of origin. Values on the x axis express the logarithm to base 10 (log10) of the IC50. rS, Spearman correlation coefficient.(D) K.A. showing JNK activity in representative sensitive (KMS-12, U266) and resistant (RPMI-8226) MM cell lines after treatment with DTP3 (10 μM). TNFα is shown as a positive control.(E) Peptide pull-down showing the physical association of DTP3 with endogenous MKK7 in U266 and KMS-12 MM cells. SCRB D-peptide is used as a negative control. -, pull-down without D-tripeptide.(F) [3H]Thymidine incorporation showing the survival of U266 and KMS-12 MM cells expressing sh-ns or sh-MKK7 shRNAs after a 6-day treatment with the indicated concentrations of DTP3. The IC50 values of DTP3 are depicted. Values express the percentage of the counts per minute (cpm) measured with the treated cultures relative to the cpm measured with the respective untreated cultures and denote mean ± SD (n = 3).See also Figure S5 and Tables S4 and S5.

Mentions: To improve the bioavailability of D-peptides in vivo, while retaining high cellular activity and specificity toward the GADD45β/MKK7 complex, we used a chemical optimization strategy based on structure-activity relationship and pharmacophore analyses (Figures S5A–S5C and Table S4). By combining these methods, we developed DTP3, a D-tripeptide with a molecular weight of 525 Da (Figure 5A), which retained all the main characteristics of the parental D-tetrapeptides in terms of bioactivity and specificity, including subnanomolar activity and high stability in vitro and potent and selective capacity to kill MM cells via apoptosis (Figures S5D–S5H), while exhibiting a superior pharmacokinetic profile compared with the parent molecules (discussed below).


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 High Target Specificity of DTP3 in Cells(A) The structure of DTP3.(B) Tryptophan fluorescence quenching analysis showing the dose-response curve of the -Δfluorescence values of GST-hMKK7 at 333 nm plotted against the concentration values of DTP3. The stoichiometry and KD value of the DTP3/MKK7 interaction on the basis of these data were 1:1 and 64.81 ± 6.22 nM, respectively. Values denote means ± SD (n = 3).(C) Correlation plot of the relative GADD45B mRNA levels (qRT-PCR) and the DTP3 IC50 at 144 hr ([3H]thymidine incorporation) in cancer cell lines of different tissues of origin. Values on the x axis express the logarithm to base 10 (log10) of the IC50. rS, Spearman correlation coefficient.(D) K.A. showing JNK activity in representative sensitive (KMS-12, U266) and resistant (RPMI-8226) MM cell lines after treatment with DTP3 (10 μM). TNFα is shown as a positive control.(E) Peptide pull-down showing the physical association of DTP3 with endogenous MKK7 in U266 and KMS-12 MM cells. SCRB D-peptide is used as a negative control. -, pull-down without D-tripeptide.(F) [3H]Thymidine incorporation showing the survival of U266 and KMS-12 MM cells expressing sh-ns or sh-MKK7 shRNAs after a 6-day treatment with the indicated concentrations of DTP3. The IC50 values of DTP3 are depicted. Values express the percentage of the counts per minute (cpm) measured with the treated cultures relative to the cpm measured with the respective untreated cultures and denote mean ± SD (n = 3).See also Figure S5 and Tables S4 and S5.
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Related In: Results  -  Collection

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fig5: The High Target Specificity of DTP3 in Cells(A) The structure of DTP3.(B) Tryptophan fluorescence quenching analysis showing the dose-response curve of the -Δfluorescence values of GST-hMKK7 at 333 nm plotted against the concentration values of DTP3. The stoichiometry and KD value of the DTP3/MKK7 interaction on the basis of these data were 1:1 and 64.81 ± 6.22 nM, respectively. Values denote means ± SD (n = 3).(C) Correlation plot of the relative GADD45B mRNA levels (qRT-PCR) and the DTP3 IC50 at 144 hr ([3H]thymidine incorporation) in cancer cell lines of different tissues of origin. Values on the x axis express the logarithm to base 10 (log10) of the IC50. rS, Spearman correlation coefficient.(D) K.A. showing JNK activity in representative sensitive (KMS-12, U266) and resistant (RPMI-8226) MM cell lines after treatment with DTP3 (10 μM). TNFα is shown as a positive control.(E) Peptide pull-down showing the physical association of DTP3 with endogenous MKK7 in U266 and KMS-12 MM cells. SCRB D-peptide is used as a negative control. -, pull-down without D-tripeptide.(F) [3H]Thymidine incorporation showing the survival of U266 and KMS-12 MM cells expressing sh-ns or sh-MKK7 shRNAs after a 6-day treatment with the indicated concentrations of DTP3. The IC50 values of DTP3 are depicted. Values express the percentage of the counts per minute (cpm) measured with the treated cultures relative to the cpm measured with the respective untreated cultures and denote mean ± SD (n = 3).See also Figure S5 and Tables S4 and S5.
Mentions: To improve the bioavailability of D-peptides in vivo, while retaining high cellular activity and specificity toward the GADD45β/MKK7 complex, we used a chemical optimization strategy based on structure-activity relationship and pharmacophore analyses (Figures S5A–S5C and Table S4). By combining these methods, we developed DTP3, a D-tripeptide with a molecular weight of 525 Da (Figure 5A), which retained all the main characteristics of the parental D-tetrapeptides in terms of bioactivity and specificity, including subnanomolar activity and high stability in vitro and potent and selective capacity to kill MM cells via apoptosis (Figures S5D–S5H), while exhibiting a superior pharmacokinetic profile compared with the parent molecules (discussed below).

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