RESUMEN
Aberrant FGF2/FGFR signaling is implicated in lung squamous cell carcinoma (LSCC), posing treatment challenges due to the lack of targeted therapeutic options. Designing drugs that block FGF2 signaling presents a promising strategy different from traditional kinase inhibitors. We previously reported a ColVα1-derived fragment, HEPV (127AA), that inhibits FGF2-induced angiogenesis. However, its large size may limit therapeutic application. This study combines rational peptide design, molecular dynamics simulations, knowledge-based prediction, and GUV and FRET assays to identify smaller peptides with FGF2-blocking properties. We synthesized two novel peptides, HBS-P1 (45AA) and HBS-P2 (66AA), that retained the heparin-binding site. Both peptides demonstrated anti-LSCC and antiangiogenesis properties in cell viability and microvessel network induction assays. In two LSCC subcutaneous models, HBS-P1, with its affinity for FGF2 and enhanced penetration ability, demonstrated substantial therapeutic potential without apparent toxicities. Our study provides the first evidence supporting the development of collagen V-derived natural peptides as FGF2-blocking agents for LSCC treatment.
Asunto(s)
Carcinoma de Células Escamosas , Diseño de Fármacos , Factor 2 de Crecimiento de Fibroblastos , Neoplasias Pulmonares , Péptidos , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/química , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Humanos , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/metabolismo , Animales , Péptidos/farmacología , Péptidos/química , Péptidos/síntesis química , Ratones , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Línea Celular Tumoral , Simulación de Dinámica Molecular , Ratones DesnudosRESUMEN
Exosomal biomarker detection holds great importance in the field of in vitro diagnostics, offering a non-invasive and highly sensitive approach for early disease detection and personalized treatment. Here, we proposed an "APPROACH" strategy, combining aptamer-mediated proximity ligation assay (PLA) with rolling circle amplification (RCA) and time-resolved Förster resonance energy transfer (TR-FRET) for the sensitive and semi-homogenous detection of exosomal biomarkers. PLA probes consisted of a cholesterol-conjugated oligonucleotide, which anchored to the membrane of an exosome, and a specific aptamer oligonucleotide that recognized a target protein of the exosome; the proximal binding of pairs of PLA probes to the same exosome positioned the oligonucleotides in the vicinity of each other, guiding the hybridization and ligation of two subsequently added backbone and connector oligonucleotides to form a circular DNA molecule. Circular DNA formed from PLA underwent rolling circle amplification (RCA) for signal amplification, and the resulting RCA products were subsequently quantified by TR-FRET. The limits of detection provided by APPROACH for the exosomal biomarkers CD63, PD-L1, and HER2 were 0.46 ngâµL-1, 0.77 ngâµL-1, and 1.1 ngâµL-1, respectively, demonstrating excellent analytical performance with high sensitivity and quantification accuracy. Furthermore, the strategy afforded sensitive detection of exosomal CD63 with a LOD of 1.56 ngâµL-1 in complex biological matrices, which underscored its anti-interference capability and potential for in vitro detection. The proposed strategy demonstrates wide-ranging applicability in quantifying diverse exosomal biomarkers while exhibiting robust analytical characteristics, including high sensitivity and accuracy.