Biology subjects

Limongelli, V.

Publications and source records attributed to Limongelli, V..

10 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↗

bioRxiv2026

A CYFIP1-Inspired Peptidomimetic Modulates eIF4E-Dependent Translational Control in Cancer and Neurodevelopmental Disorders

The eukaryotic translation initiation factor 4E (eIF4E) is a central regulator of cap-dependent translation and a compelling pharmacological target in disorders marked by protein synthesis dysregulation, including cancer and Fragile X Syndrome (FXS). Among endogenous eIF4E regulators, the CYFIP1-eIF4E interaction is uniquely selective, offering a framework for designing targeted translation modulators. Here, we report Cy-9B, a rationally engineered, stapled peptidomimetic derived from CYFIP1 that binds eIF4E, disrupts eIF4E-eIF4G complex, and suppresses cap-dependent translation. Enhanced-sampling free-energy simulations reveal that Cy-9B engages eIF4E through a non-canonical binding mode. Cy-9B exhibits drug-like properties, including high proteolytic stability and nanomolar affinity. Functionally, Cy-9B inhibits lung cancer cell proliferation, migration, and invasion. In neurodevelopmental disease models, Cy-9B partially normalizes excessive translation in FXS hippocampal neurons and rescues social behavior deficits in a Cyfip1 haploinsufficient Drosophila melanogaster model, restoring wild-type-like performance. Cy-9B emerges as a first-in-class therapeutic candidate for disorders sharing translational dysregulation, highlighting targeted modulation of eIF4E as a broadly applicable and physiologically compatible therapeutic strategy.

biochemistry↗

bioRxiv2026

From Structure to Dynamics: Activation Mechanism of the G Protein-Coupled Bile Acid Receptor 1-Gs Complex

The G protein-coupled bile acid receptor 1 (GPBAR1, also known as TGR5) is a key mediator of bile acid signaling, exerting its physiological effects through coupling with the stimulatory G protein (Gs). This interaction is essential for stabilizing the receptors active conformation and triggering downstream signaling. Among endogenous ligands, lithocholic acid (LCA) is the most potent natural agonist. However, the dynamic features underlying its binding and activation mechanisms remain poorly defined. In this study, we investigated the molecular basis of the interaction between LCA and GPBAR1, as well as the functional consequences of this interaction on receptor activation by integrating homology modelling, molecular docking, and molecular dynamics (MD) simulations. Our calculations reveal that LCA binding stabilizes the active state of GPBAR1, biasing the conformational ensemble of TM5 and TM6, as well as the main microswitches. These ligand-induced rearrangements enhance the coupling interface with the 5 helix of Gs and facilitate allosteric communication between the orthosteric and intracellular sites. Overall, our findings provide dynamic insight into how LCA modulates GPBAR1 activation and G protein engagement, highlighting its role as a molecular effector in bile acid signaling, and furnishing molecular detail relevant to ongoing efforts in GPBAR1-targeted compound development.

bioinformatics↗

bioRxiv2025

Dual targeting of BTK and BCL2 enhances apoptosis in marginal zone lymphoma models: preclinical activity of BGB-16673 and sonrotoclax

BackgroundBTK and BCL2 represent key therapeutic targets in B-cell lymphomas, including marginal zone lymphoma (MZL). Here, we evaluated the novel BTK degrader BGB-16673, and the second-generation BCL2 inhibitor sonrotoclax in a panel of MZL cell lines, including models with acquired resistance to BTK, BCL2 and PI3K inhibitors, as single agents and in combination. MethodsCytotoxicity, transcriptomic changes, apoptosis, and protein expression were assessed for BGB-16673 alone and in combination with venetoclax, sonrotoclax, bendamustine, selinexor, lenalidomide, and tazemetostat. ResultsBGB-16673 demonstrated single-agent activity in selected MZL cell lines, inducing BTK degradation and transcriptional repression of BCR signaling and MYC targets. Its transcriptional and phenotypic effects overlapped with those of zanubrutinib but also included specific modulation of oxidative phosphorylation genes. Combination studies revealed that BGB-16673 synergized with other targeted agents, achieving the best results with sonrotoclax, venetoclax, bendamustine, lenalidomide, and rituximab. The mechanism of action of the combination with the BCL2 inhibitors was further evaluated. The combination with sonrotoclax consistently enhanced apoptosis across MZL models, outperforming venetoclax in potency in most cell lines. Sonrotoclax showed over 10-fold higher activity than venetoclax in three MZL lines and demonstrated synergistic interaction with BGB-16673. Mechanistically, the combination leads to BTK degradation and modulation of anti-apoptotic proteins such as BCL2, MCL1, and BCL-XL. ConclusionOur findings underscore the potential of BGB-16673 as a therapeutic agent, both as a monotherapy and in combination regimens, for treating MZL patients. Additionally, the results also identify the second-generation BCL2 inhibitor sonrotoclax as another drug to be explored in the same patient populations.

cancer biology↗

bioRxiv2025

Beyond the 1:1 Ligand-Protein Paradigm: An In Silico Assay for Competitive Ligand Binding

Competitive binding assays (CBAs) are widely used in drug discovery to quantify and compare ligand/receptor affinities. However, their molecular interpretation is often limited by the inherent complexity of ligand-receptor interactions and the transient nature of binding events. All-atom molecular dynamics (MD) simulations offer valuable mechanistic insights in ligand binding studies, but remain computationally prohibitive for capturing the long-timescale, multiligand behavior characteristic of CBAs. Therefore, characterizing the molecular aspects of CBAs remains a fundamental challenge in molecular biophysics. Here, we introduce a coarse-grained MD (CGMD) approach capable of recapitulating CBA-like dynamics between ligands of opposing efficacy-- the full agonist NECA and the inverse agonist ZM241385-- at the adenosine A2A receptor, a prototypical G protein-coupled receptor and a key pharmacological target. By simulating ligand mixtures at varying molar ratios, we capture hallmark features of experimental CBAs, including spontaneous binding, unbinding, and direct competition at the orthosteric site. Our simulations reveal an extracellular vestibular site that modulates ligand access to the binding pocket. Occupation of this site by NECA facilitates ZM241385 entry and prolongs its residence time, revealing a cooperative mechanism within an otherwise competitive process. These findings offer a molecular perspective on lig- and competition at GPCRs and demonstrate the potential of CGMD as a viable method for probing multiligand dynamics in drug discovery.

biochemistry↗

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↗

bioRxiv2023

Minute-timescale simulations of G Protein Coupled Receptor A2A activation mechanism reveals a receptor pseudo-active state

G protein coupled receptors (GPCRs) are membrane proteins of greatest pharmacological relevance, targeted by over one third of marketed drugs. These receptors are activated by orthosteric ligands and undergo large conformational changes that lead to coupling diverse effector proteins. To achieve a fine regulation of the drug pharmacological response, it is imperative to shed light on the yet poorly understood aspects of GPCRs activation. In this work, we elucidate the entire activation mechanism of the adenosine A2A receptor (A2AR), a class A GPCR, performing minute timescale molecular dynamics and free energy calculations. We have explored the entire conformational landscape of A2AR in its basal apo form and in differently ligated conditions, elucidating the ligand intrinsic activity and the receptors lowest energy functional states. Among these is a novel pseudo-active state (pAs) of the A2AR apo form stabilised by specific "microswitch" residues interactions, including the salt bridge between the class A conserved residues R5.66 and E6.30. In the pAs state, A2AR is able to couple {beta}-arrestin 1 over G proteins, providing unprecedented structural basis for receptor desensitization and G protein-alternative cellular pathways. Our simulation protocol is generalisable and can be applied to study the activation of any GPCR, resulting a precious tool for drug design and biased signaling studies.

biophysics↗

bioRxiv2023

Conformational plasticity and allosteric communication networks govern Shelterin protein TPP1 binding to human telomerase

The molecular binding interaction between the Shelterin complex protein TPP1 and human telomerase enzyme (TERT) triggers the telomerase maintenance mechanism that marks cell lifespan. The TPP1s structural element deputed to bind TERT is the OB-domain, which is able to interact with TERTs hTEN (TPP1 binding telomerase domain) through the TEL-patch, a group of amino acids whose mutations provoke harsh pathologies. Indeed, aberrations in the formation of TPP1-TERT het-erodimer can lead to severe diseases like Hoyeraal-Hreidarsson syndrome (HHS), whose patients are affected by short telomeres and extremely poor life expectancy. In the present study, we provide a thorough characterization of the structural properties of the TPP1s OB-domain by combining data coming from microsecond-long molecular dynamics calculations, time-series analyses, and graph-based networks. Our results show that the conformational plasticity of the TPP1s TEL-patch region is influenced by a network of long-range amino acid communications, needed for the proper TPP1-hTEN binding. Furthermore, we reveal that in the Glu169{Delta} and Lys170{Delta} TPP1 variants, responsible for HHS, the plasticity of the TEL-patch region is reduced, affecting the correct binding to hTEN and in turn the telomere processivity, which eventually leads to accelerated ageing of affected cells. Our study provides an unprecedented structural basis for the design of TPP1-targeting ligands with therapeutic potential against cancer and telomerase deficiency diseases.

biochemistry↗

bioRxiv2022

Molecular basis of uracil/H+ symport mechanism operated by the FurE/NCS1 transporter

Transporters mediate the uptake of solutes, metabolites and drugs across the cell membrane. The eukaryotic FurE nucleobase/H+ symporter of Aspergillus nidulans has been used as a model protein to address structure-function relationships in the APC transporter superfamily, members of which are characterized by the LeuT-fold and seem to operate by the so-called rocking-bundle mechanism. In this study, we reveal the binding mode, translocation and release pathway of uracil/H+ by FurE, using path collective variable, funnel metadynamics and rationally designed mutational analysis. Our study reveals a step-wise, induced-fit, mechanism of ordered sequential transport of proton and uracil, which in turn suggests that the FurE symporter, and probably structurally similar transporters, functions as a multi-step gated pore, rather than employing rocking of compact domains, as generally proposed for APC transporters. In addition, our work further supports the emerging concept that specific elements of cytosolic terminal regions of transporters might be functionally important.

molecular biology↗

bioRxiv2018

Accelerating the Calculation of Protein-Ligand Binding Free Energy and residence Times using Dynamically Optimized Collective Variables

Elucidation of the ligand/protein binding interaction is of paramount relevance in pharmacology to increase the success rate of drug design. To this end a number of computational methods have been proposed, however all of them suffer from limitations since the ligand binding/unbinding transitions to the molecular target involve many slow degrees of freedom that hamper a full characterization of the binding process. Being able to express this transition in simple and general slow degrees of freedom, would give a distinctive advantage, since it would require minimal knowledge of the system under study, while in turn it would elucidate its physics and accelerate the convergence speed of enhanced sampling methods relying on collective variables. In this study we pursuit this goal by combining for the first time Variation Approach to Conformational dynamics with Funnel-Metadynamics. In so doing, we predict for the benzamidine/trypsin system the ligand binding mode, and we accurately compute the absolute protein-ligand binding free energy and unbinding rate at unprecedented low computational cost. Finally, our simulation protocol reveals the energetics and structural details of the ligand binding mechanism and shows that water and binding pocket solvation/desolvation are the dominant slow degrees of freedom.

biophysics↗