Biology subjects

Di Cristofano, S.

Publications and source records attributed to Di Cristofano, S..

3 recordsLinked to original sources

bioRxiv2026

Thermodynamic, Kinetic, and Structural Determinants of Ligand Selectivity in A2A and A2B Adenosine Receptors

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.

biophysics↗

bioRxiv2025

Exploring the mechanism and pattern of bone formation during RANKL inhibition in a mouse model of fibrous dysplasia

Fibrous dysplasia (FD) of bone is a genetic fibro-osseous disorder with increased bone remodeling activity. Inhibition of RANKL modifies FD lesions by inducing the replacement of the fibrous tissue with bone. This effect was observed in FD murine models receiving anti-mouse RANKL antibodies or small molecule RANKL inhibitors and in FD patients treated with denosumab. However, in neither case the mechanism and pattern of deposition of the newly formed bone were clarified. We performed radiographic, morphological and molecular analyses on EF1-GsR201C (FD) mice receiving an anti-mouse RANKL antibody. We observed that RANKL inhibition caused a decrease in the expression of genes involved in osteogenesis, osteoclastogenesis, matrix remodeling and osteoblast-osteoclast cross-talk in affected skeletal segments. Nonetheless, intra-lesional bone surfaces were covered by a continuous layer of osteoid, indicating that bone formation was actively ongoing in the pathological tissue in spite of the treatment. Accordingly, all bone surfaces within FD lesions showed calcein labeling which was never detected in the fibrous tissue far from bone. These results indicate that in the absence of RANKL activity, bone formation in FD tissue does not occur diffusely or stochastically. In contrast, it is restricted to bone surfaces where osteoprogenitor cells are orderly recruited from the adjacent fibrosis, progressively converting it into bone. Clinically, this suggests that the volume of pre-treatment bone in FD lesions may be a determinant of the skeletal improvement that individual patients may achieve during the same denosumab treatment course. As a consequence, it may also be a determinant of the severity of the rebound effect that they can experience upon treatment discontinuation.

pathology↗

bioRxiv2025

Pharmacological inhibition of CXCR4 increases the anti-tumor activity of conventional and targeted therapies in B-cell lymphoma models

BackgroundCXCR4 is a chemokine receptor frequently implicated in the pathogenesis and treatment resistance of B-cell lymphomas and other tumor types. Thus, CXCR4 targeting is explored using various approaches, including small molecules, antibodies, and short peptides. Here, we investigated the antitumor effects of the CXCR4 pharmacological inhibition with the synthetic peptides SPX5551 and balixafortide, as single agents and in combination, in various B-cell lymphoma models. MethodsBinding modalities were assessed via molecular docking and simulation. In vitro assays evaluated single-agent and combinatorial effects of CXCR4 antagonists with BTK, PI3K, and conventional therapies across 20 lymphoma cell lines, including models with acquired resistance. Transcriptomic analyses and mechanistic studies elucidated the pathways modulated by combined CXCR4 and BTK inhibition. ResultsStructural modeling confirmed similar CXCR4 binding modes for SPX5551 and balixafortide. SPX5551 displayed minimal single-agent activity but restored sensitivity to BTK and PI3K inhibitors in resistant marginal zone lymphoma (MZL) models. Across mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), and diffuse large B-cell lymphoma (DLBCL) models, SPX5551 enhanced the efficacy and/or potency of ibrutinib, copanlisib, rituximab, and R-CHOP. In MCL, co-treatment with SPX5551 and ibrutinib synergistically induced apoptosis, inhibited NF-{kappa}B and AKT signaling, and led to broader transcriptomic repression of tumor-promoting pathways compared to either agent alone. ConclusionsAlthough CXCR4 inhibitors alone show limited cytotoxicity, their combination with standard and targeted therapies significantly enhances anti-lymphoma effects, particularly in drug-resistant settings. These findings provide a strong rationale for clinical evaluation of CXCR4 blockade as a combinatorial strategy in B-cell lymphomas.

cancer biology↗