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Interactions between proteins and identification of high affinity antagonists The relationship between the pathological processes of infection, inflammation and cancer is correlated to the role of the transcription factor NF-κB in cellular homeostasis. Therefore studying NF-κB target genes makes it is possible to understand the molecular mechanisms of inflammation, of tumor growth and progression. Gadd45β is one of the anti-apoptotic regulators controlled by NF-κB in response to pro-inflammatory stimuli and genotoxic stress exerting its functions by interacting with several partners such as MAP kinases and DNA clamp protein. The interaction between Gadd45β and MKK7 has been described as a molecular link between the NF-kB cytoprotective effects and the suppression of the signaling of JNK, one of the main MKK7 substrates that strongly promotes apoptosis. Understanding this complex network of interactions and finding effective antagonists is of preeminent importance for elucidating the balance between cell death and cell survival and to modulate cell homeostasis for therapeutic applications. For this reason, this PhD project has been focused on the structural and functional characterization of the Gadd45β-MKK7 complex and on the identification of compounds able to disrupt this interaction. In fact, developing molecules able to block the pro-survival action of NF-κB without significantly compromising the innate activation of the immune system is an effective way to suppress the proliferative effects induced by Gadd45β expression. Biochemical analyses confirmed that Gadd45β exists in solution prevalently as a non covalent dimer in a 1:1 stoichiometry. The self-association dissociation constant was estimated to be about 100 nM and by limited proteolysis we identified the regions of Gadd45β involved in the self-association as corresponding to the predicted helix 1 (H1) and helix 5 (H5) of the protein. The biochemical analysis of Gadd45β-MKK7 complex has been investigated and provided a new context for dissecting the structure-activity relationship of this interaction which exhibited a KD of about 13 nM. To select antagonists of the Gadd45β-MKK7 interaction, a combinatorial chemistry approach has been followed. By this process, Lead Compounds 1 and 2 have been selected from deconvolution of two generations of tetrapeptide libraries. These novel antagonists can disrupt very efficiently the interaction between Gadd45β and MKK7 at concentrations in the low nanomolar range. Further investigations by co-immunoprecipitation and kinase assays have confirmed that the measured antagonistic activity matches perfectly with the IC50 values from ELISA data. Additionally, the results suggest that the selected peptides disrupt the interaction between Gadd45β and MKK7 without directly interfering with the kinase activity. Indeed several kinase assays have confirmed that both Lead Compounds do not block JNK phosphorylation, therefore they do not enter the kinase ATP binding site. These results suggest that Lead Peptides 1 and 2 have the potential of becoming very useful new drug candidates, since the selective inhibition of NF-kB downstream targets, such as Gadd45β, could be a way to convert inflammation-driven tumor growth into inflammation–induced tumor suppression.

GADD45&beta, /MKK7 complex is a non-redundant, cancer cell-restricted survival module downstream of the NF-kB survival pathway, and it has a pathogenically critical role in multiple myeloma, an incurable malignancy of plasma cells. The first-in-class GADD45&beta, /MKK7 inhibitor DTP3 effectively kills MM cells expressing its molecular target, both in vitro and in vivo, by inducing MKK7/JNK-dependent apoptosis with no apparent toxicity to normal cells. DTP3 combines favorable drug-like properties, with on-target-specific pharmacology, resulting in a safe and cancer-selective therapeutic effect, however, its mode of action is only partially understood. In this work, we have investigated the molecular determinants underlying the MKK7 interaction with DTP3 by combining computational, NMR, and spectroscopic methods. Data gathered by fluorescence quenching and computational approaches consistently indicate that the N-terminal region of MKK7 is the optimal binding site explored by DTP3. These findings further the understanding of the selective mode of action of GADD45&beta, /MKK7 inhibitors and inform potential mechanisms of drug resistance. Notably, upon validation of the safety and efficacy of DTP3 in human trials, our results could also facilitate the development of novel DTP3-like therapeutics with improved bioavailability or the capacity to bypass drug resistance.

GADD45β is selectively and constitutively expressed in Multiple Myeloma cells, and this expression correlates with an unfavourable clinical outcome. GADD45β physically interacts with the JNK kinase, MKK7, inhibiting its activity to enable the survival of cancer cells. DTP3 is a small peptide inhibitor of the GADD45β/MKK7 complex and is able to restore MKK7/JNK activation, thereby promoting selective cell death of GADD45β-overexpressing cancer cells. Enzymatic MS foot-printing and diazirine-based chemical cross-linking MS (CX-MS) strategies were applied to study the interactions between GADD45β and MKK7 kinase domain (MKK7_KD) and between DTP3 and MKK7_KD. Our data show that the binding between GADD45β and MKK7 largely occurs between GADD45β loop 2 (region 103-117) and the kinase enzymatic pocket. We also show that DTP3 interferes with this GADD45β/MKK7 interaction by contacting the MKK7 peptides, 113-136 and 259-274. Accordingly, an MKK7_KD Δ(101-136) variant lacking Trp135 did not produce a fluorescence quenching effect upon the binding of DTP3. The assessment of the interaction between GADD45β and MKK7 and the elucidation of the recognition surfaces between DTP3 and MKK7 significantly advance the understanding of the mechanism underlying the inhibition of the GADD45β/MKK7 interaction by DTP3 and pave the way to the design of small-molecule DTP3 analogues. GADD45β is selectively and constitutively expressed in Multiple Myeloma cells, and this expression correlates with an unfavourable clinical outcome. GADD45β physically interacts with the JNK kinase, MKK7, inhibiting its activity to enable the survival of cancer cells. DTP3 is a small peptide inhibitor of the GADD45β/MKK7 complex and is able to restore MKK7/JNK activation, thereby promoting selective cell death of GADD45β-overexpressing cancer cells. Enzymatic MS foot-printing and diazirine-based chemical cross-linking MS (CX-MS) strategies were applied to study the interactions between GADD45β and MKK7 kinase domain (MKK7_KD) and between DTP3 and MKK7_KD. Our data show that the binding between GADD45β and MKK7 largely occurs between GADD45β loop 2 (region 103–117) and the kinase enzymatic pocket. We also show that DTP3 interferes with this GADD45β/MKK7 interaction by contacting the MKK7 peptides, 113–136 and 259–274. Accordingly, an MKK7_KD Δ(101–136) variant lacking Trp135 did not produce a fluorescence quenching effect upon the binding of DTP3. The assessment of the interaction between GADD45β and MKK7 and the elucidation of the recognition surfaces between DTP3 and MKK7 significantly advance the understanding of the mechanism underlying the inhibition of the GADD45β/MKK7 interaction by DTP3 and pave the way to the design of small-molecule DTP3 analogues.