Samuele Di Cristofano

G protein–coupled receptors are major pharmacological targets, yet achieving subtype selectivity remains challenging when closely related receptors share highly conserved orthosteric binding sites. Here, we investigate the molecular determinants governing ligand recognition and unbinding at the adenosine A2A and A2B receptors, two closely related class A GPCRs with markedly different pharmacological profiles. We combine Funnel Metadynamics and adaptive infrequent metadynamics to characterize the thermodynamics and kinetics of three representative ligands: the non-selective antagonist theophylline (TEP), the A2A–selective inverse agonist ZM–241385 (ZMA), and the non–selective full agonist NECA. Across six ligand–receptor complexes, our simulations reproduce experimentally resolved binding modes, predict the unresolved binding poses of TEP and ZMA at A2B, and provide binding free energies consistent with experimental trends. Kinetic simulations further resolve ligand–specific unbinding pathways, metastable intermediates, residence times, rate–determining transitions, and their associated transition–state configurations. Comparison of A2A and A2B reveals how subtle differences within and around their highly conserved orthosteric sites are amplified into distinct thermodynamic and kinetic behaviors. In particular, we identify three major receptor–specific features: differences in hydration and polarity near TM1/TM2/TM7, differences in steric packing and pocket volume at the TM3/TM5/TM6 floor, and a more dynamic network of charged extracellular residues and lipids in A2B that modulates ligand egress. These features rationalize ligand–dependent differences in affinity, residence time, and subtype selectivity, including the preferential stabilization of ZMA–like antagonists at A2A. Overall, our results provide a dynamic atomistic map of the A2A and A2B orthosteric regions and demonstrate how thermodynamic and kinetic information can reveal pharmacologically relevant differences that are not apparent from static structures alone. This framework may support the rational design and repurposing of subtype–selective adenosine receptor ligands.

In this study, we explored the design of linear D‐tripeptides tailored to bind specific cavities of Gadd45β, chosen as a model protein target. To identify peptides that selectively interact with predicted binding sites, we combined computational modeling with biophysical experiments. Gadd45β was selected since it has emerged as a promising therapeutic target involved in multiple disease pathways, including cancer and inflammation. Computational analysis was first employed to characterize the structural features and potential binding sites of Gadd45β. Guided by these insights, linear D‐tripeptides were designed and optimized for specific interactions with the target surface. The resulting candidates were subsequently assessed through a series of biophysical assays to evaluate their binding affinity, selectivity, and potential therapeutic activity. Complementary computational simulations were employed to gain atomistic insight into the dynamics of peptide–protein recognition. This integrated computational–experimental strategy led to the identification of two D‐tripeptides, RYR and VWR, that bind Gadd45β at a biologically relevant site, illustrating a general framework for early‐stage peptide ligand discovery.

Inteins are proteins involved in the protein splicing mechanism, an autoprocessing event, where sequences (exteins) separated by inteins become ligated each other after recombination. Two kinds of inteins have been described, contiguous inteins and split inteins. The former ones are transcribed and translated as a single peptide along with their exteins, while the latter are fragmented between two different genes and are transcribed and translated separately. The aim of this study is to establish a method to obtain a fluorescent eukaryotic protein to analyze its cellular localization, using the natural split gp41‐1 inteins. We chose natural split inteins due to their distribution in all three domains of life. Two constructs were prepared, one containing the N‐terminal split intein along with the N‐moiety of the Red Fluorescent Protein (RFP) and a second construct containing the C‐terminal of split intein, the C‐moiety of RFP and the gene coding for Maspin, a tumor suppressor protein. The trans‐splicing was verified by transfecting both N‐terminal and C‐terminal constructs into mammalian cells. The success of the recombination event was highlighted through the fluorescence produced by reconstituted RFP after recombination, along with the overlap of the red fluorescence produced by recombined RFP and the green fluorescence produced by the hybridization of the recombinant Maspin with a specific antibody. In conclusion, we opted to use this mechanism of recombination to obtain a fluorescent Maspin instead to express a large fusion protein, considering that it could interfere with Maspin's structure and function.