In vitro
This study investigated how different photoaffinity crosslinkers influence the biological activity, labeling efficiency, and target protein preference of quinazolinone-based active molecules—specifically QDAU5, a compound previously shown to promote vascular normalization. The researchers constructed a library of photocrosslinkers and multifunctional photoaffinity probes using structures such as thiophene-substituted α-ketoamide, tetrazole, isoxazole, and 2-nitrobenzyl alcohol, then applied Photoaffinity Labeling–Affinity-Based Protein Profiling (PAL-AfBPP) combined with proteomic analysis to assess probe performance. Prior work had identified EphrinB2 as an intracellular target of QDAU5; this study further validated Thymosin β4 (Tβ4) as an additional potential target and began characterizing how QDAU5 interacts with Tβ4 at a molecular level. The findings suggest that the choice of photocrosslinker meaningfully affects both the retained bioactivity of the parent compound and which proteins are captured, offering practical guidance for PAL-AfBPP probe design. Limitations include that this is primarily a chemical biology and proteomics study conducted at the in vitro/biochemical level, with no human clinical data. The mechanistic link between QDAU5–Tβ4 interaction and angiogenesis regulation requires further validation in vivo.
Bioorganic chemistry · Feb 2026DOI ↗ In vitro
This study investigated, for the first time, how the small peptide Thymosin β4 (Tβ4) interacts with zinc ions (Zn²⁺) at physiological pH. Using a panel of biophysical techniques — including zeta potential analysis, dynamic light scattering (DLS), electrospray ionization mass spectrometry (ESI-MS), nuclear magnetic resonance (NMR) spectroscopy, and scanning electron microscopy with elemental mapping (SEM/EDS) — the researchers characterized the structural and chemical consequences of Zn²⁺ binding to this intrinsically disordered 43-amino-acid peptide. The study found that Zn²⁺ progressively neutralizes Tβ4's negative surface charge, triggering a sharp aggregation transition. ESI-MS identified peptide-to-zinc complexes at a 1:3 molar ratio, while DLS and SEM confirmed formation of compact, low-solubility supramolecular assemblies. NMR data indicated that Zn²⁺ binding does not induce folding of the peptide. Importantly, the study compared the experimentally determined critical aggregation concentration with known physiological extracellular Zn²⁺ levels, concluding that aggregation is unlikely under normal plasma or interstitial conditions but could occur in Zn-rich microenvironments such as the synaptic cleft. The authors propose that Zn²⁺-mediated Tβ4 assembly may be relevant in neurological or inflammatory contexts. This is a foundational biochemical characterization study with no direct in vivo or clinical component.
International journal of molecular sciences · Feb 2026DOI ↗ In vitro
This study investigated how Thymosin β4 (Tβ4), a 43-amino-acid secreted peptide, may protect against hypoxia-induced blood-brain barrier (BBB) disruption using human brain microvascular endothelial cells (hBMVECs) as an in vitro model. The researchers exposed hBMVECs to hypoxic conditions to simulate aspects of ischemic injury and traumatic brain injury (TBI), then examined whether Tβ4 pretreatment could reverse resulting damage. They measured gene expression of tight junction proteins, Sphingosine 1-phosphate receptor 1 (S1PR1), endothelial cell permeability, and tight junction dynamics. The study found that Tβ4 pretreatment appeared to reverse hypoxia-induced impairment of BBB components, and identified S1PR1 as a potential mechanistic target. Notably, when S1PR1 was pharmacologically inhibited, Tβ4 lost its protective effect, suggesting S1PR1 signaling is required for Tβ4's action. The authors conclude that S1PR1 pathway modulation is central to hypoxia-induced BBB pathophysiology and propose Tβ4 as a candidate therapeutic agent warranting further investigation. Key limitations include the exclusive use of cell culture models, absence of animal or human data, and lack of in vivo validation of the proposed mechanism.
Scientific reports · Dec 2025DOI ↗ In vitro
This study investigated whether Tβ4-17, a small bioactive peptide derived from the precursor protein thymosin β4 and identified via iTRAQ technology, could enhance the sensitivity of cisplatin (DDP)-resistant ovarian cancer cells to chemotherapy. Using in vitro cell line models of DDP-resistant ovarian cancer, the researchers tested the combination of Tβ4-17 with DDP on cell proliferation, migration, and apoptosis. Results indicated that the combination significantly inhibited proliferation and migration of resistant cells while promoting apoptosis compared to either treatment alone. Mechanistically, the study found that NF-κB (specifically the p65 subunit) was highly expressed in DDP-resistant ovarian cancer cells, and that Tβ4-17 appeared to downregulate NF-κB p65 protein expression. These findings were supported by qRT-PCR, Western blot, CCK-8 assays, EDU fluorescence proliferation assays, and scratch migration assays, as well as experiments using NF-κB inhibitors and activators. Key limitations include reliance solely on in vitro cell line models with no animal or human data, and the absence of pharmacokinetic or safety assessments. The study suggests a potential mechanism by which Tβ4-17 may overcome chemoresistance in ovarian cancer but requires substantial further validation.
Medical oncology (Northwood, London, England) · Nov 2025DOI ↗ In vitroPreprint
This study investigated whether Tβ4-17, a small bioactive peptide derived from thymosin β4 and identified via iTRAQ technology, could enhance the sensitivity of cisplatin (DDP)-resistant ovarian cancer cells to chemotherapy. Using in vitro models of DDP-resistant ovarian cancer cell lines, the researchers examined the effects of Tβ4-17 alone and in combination with DDP on cell proliferation, migration, and apoptosis. Multiple assays were employed, including CCK8 viability assays, EDU fluorescence proliferation assays, cell scratch (wound healing) assays, qRT-PCR, and Western blot. The study found that Tβ4-17 combined with DDP significantly inhibited proliferation and migration of resistant cells and promoted apoptosis compared to either agent alone. Mechanistically, the researchers reported that NF-κB p65 was highly expressed in DDP-resistant cells, and that Tβ4-17 down-regulated NF-κB p65 protein expression. Use of NF-κB inhibitors and activators further supported this proposed pathway. Key limitations include the exclusive use of cell-line models with no animal or human data, the preprint status of the work, and the absence of in vivo validation. Findings are preliminary and require further study.
Unknown journal · Jul 2025DOI ↗