The Development of DTP3: A GADD45β/MKK7 Inhibitor with Improved Bioavailability
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).
We further evaluated the modality and specificity of the binding of DTP3 to the GADD45β/MKK7 complex. Circular dichroism (CD) studies demonstrated that DTP3, but not a control scrambled (SCRB) D-tripeptide, can physically interact with and cause a significant conformational change of the structure of this complex, as well as of the isolated MKK7 protein, in a dose-dependent manner (Figure S5I). By contrast, DTP3 had no effect on the CD signal of GADD45β (Figure S5I). MALDI-TOF mass spectrometry confirmed the ability of DTP3 to bind to MKK7, but not to GADD45β (Figure S5J). Spectrofluorimetric analyses yielded similar results and, furthermore, established the 1:1 stoichiometry and low equilibrium dissociation constant (KD) value of the DTP3 interaction with MKK7 (Figure 5B; Figure S5K), thus underscoring the strength and specificity of this interaction. Conversely, the SCRB D-tripeptide had no impact on the fluorescence emission spectrum of MKK7, thus reaffirming the specificity of the effects of DTP3. Computational analyses predicted the presence of a pocket of MKK7, also found on the GADD45β/MKK7 complex, that can bind to DTP3 (Figures S5L–S5T). Collectively, these results demonstrate that DTP3 has the capacity to physically interact with MKK7, both in isolation and within the complex with GADD45β, and support a model whereby, upon binding to MKK7, DTP3 dissociates the GADD45β/MKK7 complex via an allosteric mechanism, potentially involving a conformational rearrangement of the kinase.