Phd in GENETICS AND MOLECULAR BIOLOGY

Thesis title: "Probing intermolecular interactions using computational methods"

In Chapter 4, “Multiple Myeloma”, CD38 and Gadd45β, two proteins involved in the onset and progression of Multiple Myeloma (MM), are studied. In Chapter 4.1, the interaction between between CD38 enzyme and cGAMP cyclic di- nucleotide is studied. CD38 is an extracellular ectoenzyme characterized by a remarkable promiscuity toward different substrates, whose functionis crucial in innate immunity [1] . On the other hand, cGAMP is an extracellular “immunotransmitter” with powerful immunostimulatory activity which demands intensive investigation about its modes of regulation [2]. In vitro experiments demonstrated that doxorubicin increases the release of extracellular cGAMP by myeloma cells and CD38 inhibition causes the accumulation of extracellular cGAMP [2]. Thus, we performed extensive in silico molecular modeling to preliminarly investigate a direct interaction netween CD38 and cGAMP. Computational results are validated through cell-free biochemical assays and further experiments using different cell lines and clinical samples demonstrated a link between CD38 and extracellular cGAMP activity. Indeed, we demonstrated that CD38 inhibits extracellular cGAMP activity through its direct binding. In the context of MM microenvironment, is plausible that the restraint of cGAMP activity exherted by CD38 may represent a relevant immunoevasive mechanism. CD38 is also abundantly expressed by most immune cells upon activation [3]; therefore, the CD38-cGAMP interaction may play a regulatory role in immune cell-highly infiltrated tumor microenvironments, where cGAMP can be released by cancer cells spontaneously or upon treatment [4-6]. Further work will be required to unveil the physiological consequences of such interaction in different contexts. In Chapter 4.2, we designed short peptides binders that target Gadd45β protein. Gadd45β play critical roles in stress signaling, influencing processes such as apoptosis, cell cycle arrest, DNA repair, cell survival, and senescence [7-10]. The biological functions of Gadd45β are mediated through its protein interactions, which are regulated by factors such as expression levels, cellular localization, and post-translational modifications of both Gadd45β and its interacting partners [11]. Understanding the modulation of these interactions through Gadd45β binders is a priority in ongoing research. Notably, selectively targeting Gadd45β / MKK7 interaction in NF-κB signaling induced apoptosis in multiple myeloma cells with minimal impact on normal tissue, as confirmed by in vivo and ex vivo studies [12]. In this study, we aimed to explore Gadd45β as a target for developing D-tripeptide binders, which could serve as selective probes for bioassays and potentially guide small-molecule drug development. Peptides have emerged as a significant frontier in drug discovery, offering a unique chemical space between small molecules and biologics, with the potential to harness the advantages of both [13-15]. Advances in peptide synthesis, structural modification, and machine learning-based predictions have accelerated peptide drug development [16]. However, traditional combinatorial chemistry approaches for ligand identification face limitations, including the cost of synthesizing large numbers of single molecules and the risk of producing side products in mixture-based screening. In this chapter, we present results from the rational design of ranked sets of short peptide binders for Gadd45β. These binders address some of the limitations of traditional screening methods by offering a platform for generating focused libraries of small peptides that can be synthesized and screened rapidly [17-19]. In order to provide a structural characterization of Gadd45β engagement by the designed peptides, we devised a computational workflow, using NMR data to reduce the number of the putative binding modes, that allowed us to obtain an accurate binding pose for two selected tightly binders. Biochemical experiments and in vitro assays confirmed our prediction and demonstrated the ability of a designed peptide to reduces cell survival in neoplastic cells over-expressing Gadd45β. In Chapter 5, we studied two protein:protein interactions that play a fundamental role in breast (BRCA2:MEILB2) cancer and ovarian (Pin1:Notch3) cancer. In Chapter 5.1, we demonstrated that the Pin1:Notch3 axis drives platinum-based chemotherapy resistance by High-grade Serous Ovarian Cancer (HSOG). Among the four Notch paralogs encoded by the mammal genome, Notch3 (N3) is frequently altered in a wide panel of OC [27]. Accumulating evidence has disclosed its pivotal role in OC stem cells [28] and Platinum resistance [29,30], hence evaluating the efficacy of Notch3 specific inactivation to restore chemo-sensitivity in HGSOC [31,32]. Notably, given that Notch signaling plays a key role in tumoral, stromal, and immune compartments, as well as in healthy tissues, pan-Notch inhibition led to off-target effects in several clinical trials [33]. To overcome these problems, research is moving towards Notch- specific targeted therapies [34] even if a lot of shortcomings have been come across in clinical trials too [35], thereby underpinning the need of finding novel strategies. In this scenario, one appealing candidate as a fine-tuner of N3 might be the peptidyl-prolyl cis/trans isomerase Pin1 which is overexpressed in several cancers, including OC [36]. By binding and catalyzing the cis/trans conversion of specific motifs, Pin1 regulates the activity of a plethora of cancer-driving pathways [37], including Notch receptor [38]. Here, we utilized computational simulations to provide a broader context for our experimental findings. This approach revealed the specific phospho-site that maintained a stable association with Pin1. This stability is likely due to its sequence and favourable conformational dynamics. Indeed, we were able to explain how the sequence differences in the phospho-sites are structurally and dynamically reflected in binding, identifying specific residues responsible for the molecular recognition between the two partners, as demonstrate by the rational design of mutant phospho-site. Overall, these findings not only illuminate the critical role of Pin1 in regulating Notch3 but also underscore the complex interplay of structural and dynamic factors that govern protein interactions. This research enhances our understanding of the molecular mechanisms underlying Notch signaling, potentially offering new avenues for therapeutic interventions in related diseases. In this scenario, we suggested a novel role of Pin1/N3 axis in predicting Platinum-response in HGSOC patients, that could be exploited to develop new diagnostic and therapeutic tools, thus opening innovative perspectives in supporting clinicians in the selection of the most effective front-line treatment. In Chapter 5.2, we identified a founder variant through a germline BRCA screening among BC and/or OC affected women in a specific ethnical group. The mutation lies in the BRCA2 Ovarian Cancer Cluster Region (OCCR) region [20-22] and linked to Fanconi anemia in newborns with genetic disease [23, 24]. BRCA2 is essential for genome integrity through various mechanisms [25] and interacts with the MEILB-2complex, formerly known as HSF2BP, in a highly evolutionarily conserved manner. This interaction occurs within residues 2270-2337, between the BRC and the DNA Binding Domain at the Carboxy (C) terminus of BRCA2, where the R2336P variant is located [26,27]. Pendlebury et al. [26] revealed a novel architecture for BRCA2 recruitment architecture to meiotic DSBs, involving salt bridges blocking BRCA2 between MEILB2 dimers and stacking MEILB2-BRCA2 interactions reinforcing MEILB2 homodimerization. We investigated the R2336P mutation's impact on the structure and dynamics of the MEILB2:BRCA2MBD complex using enhanced sampling MD. The mutant complex exhibited distinct dynamics, suggesting potential dysfunctionality in BRCA2-MEILB2 binding. Simulation analysis showed significant differences between wild-type and mutant complexes. Long-range allosteric effects induced by the mutation affected MEILB2 dynamics beyond the binding region. Principal component analysis revealed altered dynamics in the mutant complex, indicating impaired binding of BRCA2 to MEILB2, ultimately contributing to impaired meiotic BRCA2 recruitment and increased cancer susceptibility. These findings align with previous research showing complex disassembly in vitro and hindered recruitment to meiotic DSBs. In Chapter 6, the study of a more generic mechanism is presented: the production of reactive oxygen species (ROS), shared by many types of cancer. Here, we provide our contribution to begin bridging the gap in understanding the structure– dynamics relationship of the hNOX2 protein, a key producer of ROS. This project originates from a collaboration with the research groups guided by Antonello Mai at Sapienza University of Rome and Andrea Mattevi from the University of Pavia, aiming to establish the structural foundations for the rational optimization of previously identified human NOXs inhibitors [39]. Despite the physiological importance of hNOX2, its structure remains poorly understood. During the initial stages of this project we noted that recent structural information on hNOX2 were incomplete, and a dynamic view of the complex was missing. Thus, in this section we present an in-depth study of hNOX2 structure and dynamics. These public informations can be also used by other research groups as a platform to dock potential inhibitors (i.e.: ensemble docking on the different conformational states of the hNOX2 catalytic pocket generated in this project). In particular, in Chapter 6.1 we present: 1) the construction of a model to study the functional dynamics of hNOX2 in a context as close as possible to the physiological conditions; 2) a comparative analysis between the DH domain of csNOX5 and hNOX2; 3) an investigation of the hNOX2 complex dynamics in different molecular assemblies and 4) a comprehensive dynamic characterization of hNOX2 in its active state. In the last paragraph 5) we will briefly discuss the preliminary docking of hNOX2 inhibitors in the cryoEM structure [40], mentioning ongoing experiments and further perspectives.