C. Rumancev, S. K. Vellas, A. Gräfenstein, D. Obitz, J. Garrevoet, G. Falkenberg, N. Metzler-Nolte, A. Rosenhahn
The intracellular localization of metal-based anticancer agents can provide important indications for their biological mode of action and intracellular targets. Here, we investigated the uptake, the intracellular distribution and cellular responses to three cytotoxic rhenium 3 complexes with different ligand environments in HeLa cells using cryogenic X-ray fluorescence (XRF) nanoanalysis combined with fluorescence microscopy. Despite their distinct ligand functionalities and expected targeting properties, all three Re complexes showed a similar intracellular distribution. The close agreement between fluorescence microscopy and XRF imaging of Re and Br supports the preservation of the spatial association of the complexes and their ligands following cellular uptake. Quantitative pixel-by-pixel XRF analysis at the single-cell level revealed elevated intracellular potassium levels after treatment, consistent with a global cellular stress response, but no local correlation between Re and K. In contrast, Zn showed a spatial correlation with Re, pointing to Zn-rich cellular structures as potential sites of interaction. The presented work characterizes the uptake of medicinally relevant metal-based drug candidates containing unnatural metals with different ligand spheres and correlates the data with the resulting endogeneous biometal levels and their localization. The observed Re–Zn association provides a basis for investigating potential Zn-associated targets of emerging metallodrugs.
G-protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins involved in cellular signaling. Although X-ray crystallography and cryo-Electron Microscopy (cryo-EM) have provided valuable structural insights into GPCRs, these experimental techniques primarily capture static conformations and cannot fully resolve the dynamic transitions that underlie activation, allostery, ligand recognition, and signaling of GPCRs. In this perspective, we highlight Molecular Dynamics (MD) simulations of GPCRs using enhanced sampling methods. These computational approaches have provided mechanistic insights into receptor activation and deactivation mechanisms, allosteric communication networks, ligand and peptide binding and dissociation pathways, and GPCR–G protein interaction kinetics while revealing transient intermediate states that are often inaccessible to experiments. Together, these advances provide a powerful framework for understanding GPCR signaling mechanisms and accelerating the rational design of selective therapeutics with improved efficacy and reduced side effects.
María Alejandra Dagrosa, Marina Carpano, Antonella Pastini, Estanislao Pavlik, Andrés Bechis, María Silvina Olivera, Pablo Vallecorsa, Belén Dávila, Luciano Parodi, Esther Yardena Salem, María Sol Espain, Agustina Mariana Portu, Laura Todaro, Alejandro Urtreger, Pablo Menendez, Hugo Cerecetto, Marcos Couto
Boron neutron capture therapy (BNCT) is a binary therapeutic modality based on the nuclear reaction between boron-10 (10 B) and low-energy neutrons, generating high-linear-energy-transfer (LET) α-particles (4 He) and recoiling lithium-7 (7 Li) nuclei with a very short path length, thereby inducing highly localized tumor cell damage. However, the clinically available boron delivery agents, 10 B-boronophenylalanine (BPA) and sodium 10 B-borocaptate (BSH), remain limited by suboptimal tumor selectivity, cellular uptake, and boron retention. This study aimed to (i) evaluate the intrinsic antitumor activity of Cmpd. 1 in cutaneous melanoma (CM) and non-small cell lung cancer (NSCLC) models; (ii) assess its ability to deliver boron and mediate BNCT effects in vitro; and (iii) evaluate its antitumor activity and boron biodistribution in vivo. Cmpd. 1 was investigated in CM and NSCLC models using complementary in vitro and in vivo approaches, including analysis of EGFR and LAT1 expression, cellular boron uptake, neutron autoradiography, BNCT-mediated cytotoxicity, cell death, tumor growth inhibition, and biodistribution. NCI-H125 cells exhibited higher EGFR and LAT1 transcript levels, which may contribute to their greater sensitivity to Cmpd. 1 and to the higher BPA uptake observed in this model. Despite being supplied at an approximately ninefold lower total boron concentration than BPA, Cmpd. 1 produced greater cellular boron accumulation after 24 h and substantially higher uptake when normalized to the amount of boron added to the culture medium. In vitro BNCT significantly reduced cell viability in both tumor models, with apoptosis contributing substantially to the response in NCI-H125 cells mainly for Cmpd. 1. Repeated in vivo administration of Cmpd. 1 delayed tumor growth in CM and NSCLC xenografts without significant body-weight loss. In the NSCLC model, co-administration of Cmpd. 1 and BPA resulted in a tumor boron concentration of approximately 23 μg B/g tissue and a tumor-to-blood boron ratio of 6.3. These findings identify Cmpd. 1 as a promising EGFR-targeted boron delivery agent that combines intrinsic antitumor activity with efficient boron delivery, supporting its further development as a bifunctional platform for BNCT.
Michele Larocca, Giuseppe Floresta, Daniele Verderese, Agostino Cilibrizzi
The stability and flexibility of peptide structures are strongly influenced by environmental conditions, particularly pH. In this study, we investigate how changes in protonation states of pH-responsive residues modulate non-covalent interaction networks and drive conformational transitions. Using a β-hairpin peptide (PDB ID: 1K43) as a model system, we developed a tailored extension of the Main Mechanical Forces (MMFs) approach that explicitly accounts for side-chain protonation and its effects on non-covalent interaction patterns. Molecular dynamics (MD) simulations were performed to validate the MMFs-derived pH-related structural predictions and to provide complementary insights into pH-dependent conformational stability and flexibility of a peptide structure. The overall agreement between MMFs and MD results is an indication of the reliability of the proposed methodology. Overall, we present a refined MMFs-based methodological framework capable of predicting pH-dependent conformational transitions by systematically modelling changes in side-chain interaction patterns and non-covalent networks. This approach provides an effective calculation strategy for studying pH-responsive systems, which has potential to inform future simulation platforms focusing on peptide folding mechanisms.
T cell receptor engineering holds great promise for cancer immunotherapy, yet the identification and optimization of high-affinity, functionally effective TCRs remain challenging. While advances in artificial intelligence have enabled large-scale exploration of TCR sequence space, these approaches often lack mechanistic interpretability and generate extensive candidate pools that are difficult to prioritize. Conversely, physics-based methods such as molecular dynamics simulations provide detailed insight into binding energetics and conformational dynamics but are not suited for high-throughput screening. Here, we propose an integrated, mechanism-aware framework that bridges AI-driven design, MD-based refinement, and experimental validation. In this strategy, biologically informed selection of initial TCRs is followed by AI-driven generation of variants, while molecular dynamics simulations act as a low-throughput, high-information filter to uncover the molecular determinants of TCR-pMHC interactions, including allosteric communication. Importantly, MD is positioned not only as a selection tool but also as a generator of mutation hypotheses. Experimental validation using surface plasmon resonance and functional cellular assays such as RAPTR enables the identification of TCRs that are both high-affinity and functionally active. This integrated approach establishes a closed-loop, multi-scale design paradigm that combines exploration, mechanistic understanding, and functional validation, providing a promising strategy for the rational development of therapeutic TCRs.
