In vitro
This study presents a novel chemical platform for installing carbon-14 (¹⁴C) or tritium (³H) radiolabeled lysine residues directly onto solid-supported peptides, circumventing the high cost and complexity of traditional radiolabeling methods. The researchers developed a two-step workflow: first, a mild hydroformylation reaction converts allylglycine residues — already incorporated into the peptide on a solid support — into a labeled allysine intermediate using either ¹⁴CO (generated from solid precursors) or ³H₂ gas. Second, reductive amination converts allysine into a radiolabeled lysine residue, with the final labeled peptide released upon cleavage from the solid support. The study reports that the optimized conditions are compatible with diverse peptide sequences and were successfully applied to analogs of semaglutide, a complex pharmaceutical peptide. The platform's key advantages highlighted by the authors include late-stage isotope introduction, flexibility in choosing the radiolabel, and avoidance of lengthy multi-step synthesis. Limitations include that this is a synthetic chemistry methods paper with no biological or clinical testing; all work was conducted in vitro at the bench-chemistry level. No pharmacological, pharmacokinetic, or efficacy data in animals or humans are reported.
Nature communications · Jun 2026DOI ↗ In vitro
This study presents a novel chemical strategy for delivering protein-degrading molecules (PROTACs) selectively to pancreatic β-cells by exploiting the glucagon-like peptide-1 receptor (GLP-1R). The researchers engineered a modified GLP-1 peptide using a tryptophan-mediated multicomponent Petasis reaction (TMPR), a modular "stapling" technique that locks the peptide into a stable α-helical conformation. This stapled analogue was reported to show enhanced structural stability and improved GLP-1R potency compared with the wild-type peptide. The stapling strategy also incorporated a chemical handle allowing conjugation to a PROTAC — a bifunctional molecule designed to degrade bromodomain-containing protein 4 (BRD4), a transcriptional regulator implicated in various diseases. The resulting GLP-1–PROTAC conjugate reportedly retained GLP-1R agonist activity and selectively induced BRD4 degradation in GLP-1R-expressing cells, consistent with receptor-mediated uptake and intracellular degrader activation. The study was conducted entirely in cellular (in vitro) systems; no animal or human data were reported. Key limitations include the absence of in vivo validation, and the translational relevance to human β-cell biology remains to be established.
Angewandte Chemie (International ed. in English) · Jun 2026DOI ↗ In vitro
This study describes the development of a scalable, chemically defined protocol for differentiating human pluripotent stem cells (hPSCs) into hypothalamic neurons enriched for pro-opiomelanocortin (POMC)-expressing cells, which are key regulators of appetite, energy, and glucose balance. The researchers validated neuronal identity using multiple high-resolution techniques — including MERFISH single-cell transcriptomics, RNA-Seq, and ATAC-Seq — and benchmarked results against human hypothalamic tissue. The protocol was tested across multiple hPSC lines and demonstrated consistent induction of ventral diencephalon and hypothalamic markers, and was designed to be compatible with robotic, high-throughput cell culture platforms. Functional assays showed that derived neurons responded to insulin and the GLP-1 receptor agonist Exendin-4, and displayed transcriptional changes under altered glucose conditions. ATAC-Seq analysis identified candidate regulatory genomic regions associated with hypothalamic development and metabolic traits, and BMI-associated gene enrichment was observed in the derived neurons. Limitations include that this is an in vitro cell model and may not fully recapitulate the complexity of the intact human hypothalamus. No human clinical outcomes were assessed. The platform is positioned as a tool for studying the mechanisms underlying metabolic disease and for therapeutic screening.
Stem cell reports · Jun 2026DOI ↗ In vitro
This study presents a novel chemical synthesis methodology called Aryl Selenoester Aminolysis Ligation (ASAL), designed to overcome key limitations of existing peptide manufacturing approaches. Traditional solid-phase peptide synthesis (SPPS) is costly and resource-intensive, while tag-assisted peptide synthesis (TAPS) is generally restricted to peptides of 20 residues or fewer. Fragment condensation methods for longer peptides often produce problematic epimerization (structural errors). The researchers integrated ASAL into a TAPS workflow to enable convergent, fragment-based assembly of longer therapeutic peptides in solution. They demonstrated the platform by successfully synthesizing three clinically relevant peptides of increasing complexity: teriparatide (34 residues, used in osteoporosis treatment), a sulfated thrombin-inhibiting anticoagulant TTI (32 residues), and tirzepatide (39 residues, used for type 2 diabetes and weight management). The method reportedly minimized epimerization, reduced reagent and solvent consumption, and showed promise for scalability. Limitations include the study being purely a chemistry/methods paper with no biological testing in cells, animals, or humans — all findings are based on synthetic yield, purity, and chemical characterization metrics alone.
Journal of the American Chemical Society · Jun 2026DOI ↗ In vitro
This study investigated whether the gastric pentadecapeptide BPC-157 and two newly designed hybrid analogs (CIARA-1 and CIARA-2) could inhibit acetylcholinesterase (AChE), an enzyme targeted in the treatment of neurodegenerative conditions such as Alzheimer's disease. The hybrid peptides were rationally designed by combining a BPC-157-derived fragment with an arginine-rich C-terminal sequence intended to enhance binding at both the catalytic and peripheral sites of AChE. Inhibitory activity was measured in vitro using a modified Ellman colorimetric assay, with kinetic parameters derived from Lineweaver-Burk plots. All three compounds showed competitive inhibition—meaning they raised the Michaelis-Menten constant (Km) without affecting maximum velocity (Vmax)—indicating direct competition with the substrate at the enzyme's active site. CIARA-1 showed the strongest inhibition (Ki = 0.24 mM; IC50 = 2.52 mM), followed by CIARA-2 and then BPC-157 itself. Results were supported by molecular docking predictions. A key limitation is that inhibitory potencies were substantially lower than those of approved AChE inhibitors. No animal or human data were included. The authors suggest these peptides represent a structural scaffold for further optimization rather than immediate therapeutic candidates.
International journal of molecular sciences · May 2026DOI ↗ In vitro
This study describes the computational design and preliminary laboratory characterization of SR18, a rationally engineered 18-amino acid peptide candidate intended to act as a GLP-1 receptor (GLP-1R) agonist for potential use in Type 2 diabetes (T2DM). The researchers used in silico methods—including molecular docking and 1-microsecond molecular dynamics (MD) simulations—to design SR18 to be resistant to DPP-4 cleavage and to retain key amino acids that interact with GLP-1R, similar to endogenous GLP-1 and approved drugs like Semaglutide and Liraglutide. In laboratory experiments, circular dichroism confirmed that synthesized SR18 adopts a stable α-helical conformation across various solvent conditions. Dynamic light scattering, cytotoxicity, and hemolytic assays suggested acceptable basic pharmaceutical and safety properties for a lead peptide candidate. Computational analyses indicated that SR18 may bind GLP-1R with comparable or favorable affinity relative to GLP-1 and Semaglutide. Limitations include the absence of any cell-based receptor activation assays, animal studies, or human data; the evidence remains entirely in silico and in vitro. No conclusions about clinical efficacy can be drawn at this stage.
Frontiers in pharmacology · May 2026DOI ↗ In vitro
This study investigated whether five FDA-approved glucagon-like peptide-1 receptor agonists (GLP-1RAs) — semaglutide, tirzepatide, liraglutide, and two others — could directly inhibit the aggregation of the 42-residue amyloid-β peptide (Aβ42), a key process implicated in Alzheimer's disease (AD). Using in vitro aggregation kinetics and microscopic analysis, researchers found that semaglutide, tirzepatide, and liraglutide inhibited Aβ42 aggregation, primarily by targeting the primary nucleation step — the initial formation of amyloid seeds. Semaglutide and tirzepatide delayed aggregation with submicromolar potency, while liraglutide showed the strongest suppression of primary nucleation and additionally modestly inhibited secondary nucleation. Liraglutide also altered fibril structure — producing less mature, more tortuous, and longer fibrils — and reduced the ability of fibrils to self-replicate (template). The study was conducted entirely in vitro (no cell, animal, or human data), which is a significant limitation for clinical translation. The authors conclude that certain GLP-1RAs can directly interfere with molecular steps of Aβ42 aggregation, and call for further studies to determine whether these mechanisms contribute to potential AD-protective effects observed in preclinical and clinical research.
Journal of the American Chemical Society · May 2026DOI ↗ In vitro
This cell-culture study investigated whether the endogenous tripeptide Lysine-Proline-Valine (KPV), derived from α-melanocyte-stimulating hormone, could protect liver cells from fat accumulation. Using the human hepatocellular carcinoma cell line HepG2, researchers induced a hepatic steatosis model by exposing cells to oleic acid (OA), which mimics lipid overload seen in non-alcoholic fatty liver disease (NAFLD). The study found that OA treatment increased intracellular lipid deposits and upregulated fatty acid synthase (FAS), a key enzyme in fat production. KPV treatment was reported to attenuate these effects without causing cytotoxicity. Mechanistically, the authors propose that KPV reduced reactive oxygen species (ROS), which in turn blunted activation of ERK, reduced AKT/mTORC1 phosphorylation, and normalized phosphorylation of PPARγ — a transcription factor central to de novo lipogenesis — ultimately suppressing FAS expression. The study is limited to a single in vitro cell line model, with no animal or human data, meaning findings cannot yet be extrapolated to living organisms. Concentrations used in cell culture do not translate directly to physiological dosing. Results are hypothesis-generating and require further validation in vivo.
Cytotechnology · Apr 2026DOI ↗ In vitro
This computational study introduces the concept of "carbonless" biomolecular design, in which all carbon atoms in amino acids and peptides are systematically replaced by boron and nitrogen atoms under an isoelectronicity constraint. Using glycine, histidine, lysine, and the copper-binding tripeptide Gly-His-Lys (GHK) as model systems, the researchers applied density functional theory (DFT) calculations with aqueous solvation modeling and conformer sampling to identify the most stable carbonless analogues (cGly, cHis, cLys, and cGHK) among all possible boron-nitrogen constitutional isomers. The study predicted that cGHK displays a broader conformational landscape than native GHK under physiological aqueous conditions, suggesting enhanced structural flexibility. Copper(II) binding was modeled using an experimentally informed coordination motif, and thermodynamic calculations indicated that cGHK binds Cu(II) more favorably than GHK by approximately 6.24 kcal/mol. The work is entirely theoretical; no synthesis, cell-based, animal, or human experiments were conducted. Limitations include the absence of experimental validation of the proposed carbonless structures, reliance on computational approximations for solvation and conformational sampling, and uncertainty about whether these novel BN-substituted molecules could be synthesized or would exhibit biological stability.
Physical chemistry chemical physics : PCCP · Apr 2026DOI ↗ In vitro
This study investigated the catalytic properties of GHK-Cu (a copper-bound tripeptide composed of glycine, histidine, and lysine) as a mimic of the enzyme laccase, which is naturally used in the detection and degradation of phenolic compounds. Researchers characterized GHK-Cu's enzyme-like kinetics, finding a maximum reaction velocity (Vmax) of 1.735 × 10⁻⁴ mM·s⁻¹ and a Michaelis constant (Km) of 0.061 mM, suggesting strong substrate affinity and catalytic efficiency compared to natural laccase. Building on this, the team developed colorimetric assays to detect two phenolic compounds—epinephrine (EP) and 2-aminophenol (2-AP)—across defined concentration ranges in ultrapure water and seawater. Additionally, a portable cotton-based sensor paired with a smartphone platform was constructed to enable field-ready detection of 2-AP in seawater. The study is conducted entirely in vitro and focuses on analytical chemistry applications rather than biological or therapeutic effects of GHK-Cu. Key limitations include the absence of any cell-based, animal, or human testing; findings are restricted to a controlled laboratory sensing context. The work proposes GHK-Cu's copper-coordination structure as a template for designing novel synthetic laccase mimetics.
Biosensors · Apr 2026DOI ↗ In vitro
This in vitro study examined the effects of three incretin-based therapies — semaglutide (GLP-1 receptor agonist), tirzepatide (dual GLP-1/GIP agonist), and cagrilintide (amylin analogue) — on mitochondrial function in C2C12 skeletal muscle myotubes under both normal and lipotoxic conditions. Lipotoxicity was induced using palmitic acid (PA), which significantly reduced basal oxygen consumption rate and ATP production in treated cells. The study used Seahorse XFp metabolic flux analysis, mitochondrial DNA copy number quantification (qPCR), and oxidative phosphorylation complex protein expression (western blotting), with key findings replicated in primary human skeletal muscle cells. The researchers found that semaglutide and cagrilintide transiently reduced basal respiration in healthy myotubes, while tirzepatide demonstrated more sustained improvements in mitochondrial respiration under both healthy and lipotoxic conditions. The study's primary limitations include its reliance on cell culture models, meaning findings may not directly translate to whole-organism physiology, and the use of a single lipotoxic stimulus. The partial replication in human primary cells adds some translational relevance, but in vivo validation remains absent.
Journal of cachexia, sarcopenia and muscle · Apr 2026DOI ↗ In vitro
This study characterized two novel pan-estrogen-related receptor (ERR) agonists, SLU-PP-332 and SLU-PP-915, which are being investigated as potential "exercise mimetics" — compounds that trigger physiological responses similar to physical exercise. Because of this property, the researchers assessed their relevance for sports anti-doping programs. Using liquid chromatography–high resolution tandem mass spectrometry (LC-HRMS/MS), the team analyzed the chemical profiles of both compounds and mapped their in vitro metabolic transformation products generated via human liver S9 fractions and human liver microsomes. For SLU-PP-332, nine metabolites were identified: six Phase-I and three Phase-II conjugates. For SLU-PP-915, seven Phase-I metabolites were identified. Three SLU-PP-915 metabolites were independently confirmed through chemical synthesis and nuclear magnetic resonance (NMR) spectroscopy. The study is limited to in vitro methodology and does not include in vivo human or animal pharmacokinetic data. The authors suggest these findings could serve as a foundation for developing urine or blood detection methods to identify illicit use of these compounds in competitive sports, but no conclusions about human efficacy or safety are drawn.
Rapid communications in mass spectrometry : RCM · Apr 2026DOI ↗ In vitro
This in vitro study investigated how Thymosin α1 (Tα1) may help reduce the HIV-1 viral reservoir by acting on immune cells. Researchers differentiated THP-1 cells into monocyte-derived dendritic cells (MoDCs) and co-cultured them with peripheral blood mononuclear cells (PBMCs) obtained from people living with HIV-1 (PLWH). The study found that Tα1 stimulation of MoDCs triggered secretion of the IL-15/IL-15 receptor alpha (IL-15/RA) complex, which was associated with enhanced CD8+ T cell and NK cell functionality — including increased secretion of IFN-γ, TNF-α, and granzyme B (GZMB) — along with reductions in intracellular HIV-1 p24 levels and integrated HIV-1 DNA. Notably, these effects were only observed in PBMCs from immunological responders (CD4+ T cell count >350 cells/µL) and not in non-responders. Key limitations include reliance on an in vitro cell line model (THP-1) rather than primary human dendritic cells, lack of an in vivo component, and the correlational nature of many associations. The authors suggest that Tα1's IL-15 pathway activation in dendritic cells could be a candidate mechanism for functional HIV cure strategies, warranting future clinical investigation.
Virulence · Mar 2026DOI ↗ In vitro
This study addressed the growing global burden of obesity and type 2 diabetes by designing novel peptide-based triagonists that simultaneously activate three receptors: GLP-1R (glucagon-like peptide-1 receptor), GCGR (glucagon receptor), and GIPR (glucose-dependent insulinotropic polypeptide receptor). The researchers used computer-aided drug design, bioinformatics analyses, and molecular dynamics simulations to rationally identify key sequence determinants, optimal modification sites, and to understand how peptide "stapling" (a structural stabilization technique) affects alpha-helical stability and conformational rigidity. Based on these computational insights, 22 novel triagonist peptide structures were designed and synthesized. Their activity was evaluated in vitro using fluorescence membrane potential assays, with pharmacological balance assessed via a "balanced triagonism score" measuring consistency of activity across all three receptors. Most compounds showed highly balanced activity profiles. The lead compound, P2-L4, demonstrated low-nanomolar potency across all three receptor targets, with efficacy reported as comparable to existing incretin-based reference therapeutics. The study is limited to in vitro findings only, with no animal or human data reported. Further preclinical and clinical validation is required before any therapeutic conclusions can be drawn.
RSC medicinal chemistry · Mar 2026DOI ↗ In vitro
This study presents the first comprehensive structure-activity relationship (SAR) analysis of SLU-PP-332, a synthetic agonist of estrogen-related receptors (ERRα and ERRγ) — nuclear receptors that regulate mitochondrial metabolism and exercise-responsive gene transcription. The researchers synthesized a library of SLU-PP-332 analogues and systematically varied core pharmacophoric elements, evaluating their effects using cell-based functional assays, downstream gene-expression profiling, and computational modeling (docking and molecular dynamics simulations). The study found that specific structural features of the SLU-PP-332 scaffold govern ERR potency, transcriptional efficacy, selectivity, ligand efficiency, solubility, and metabolic stability. While SLU-PP-332 remained a strong benchmark, certain analogues showed comparable or context-dependent transcriptional activity alongside improvements in ligand efficiency, solubility, or metabolic stability. Computational analyses helped explain how subtle chemical modifications influence receptor engagement and downstream signaling. Limitations include reliance on cell-based and in silico methods with no animal or human data reported. The work establishes design principles for next-generation ERR agonists and positions these compounds as potential exercise-mimetic therapeutics, though extensive preclinical and clinical validation remains to be done.
International journal of biological macromolecules · Mar 2026DOI ↗ 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 the metabolic profiles of three amylin receptor agonists — pramlintide, cagrilintide, and KBP-066 — in the context of sports anti-doping research. Motivated by growing concerns about misuse of weight-loss peptide hormones in athletic disciplines where weight management is performance-relevant, researchers used comprehensive in vitro models (human skin and kidney S9 fractions, biological fluids) to characterize how these compounds are broken down. High-resolution tandem mass spectrometry (HRMS/MS) was used to identify metabolites, and authentic post-administration rat plasma samples were analyzed for cagrilintide to assess in vivo relevance. The study found that all three peptides underwent N-terminal and C-terminal degradation, producing multiple stable metabolites considered suitable as analytical detection targets. Metabolites predicted from in vitro experiments for cagrilintide were confirmed in rat plasma samples. The researchers developed and validated an LC-MS/MS-based detection method applicable to anti-doping screening. Limitations include the primary reliance on in vitro models; the only in vivo data came from rat samples, not humans. This represents the first systematic metabolic characterization of these three compounds in an anti-doping context and lays groundwork for future human monitoring programs.
Journal of pharmaceutical and biomedical analysis · Feb 2026DOI ↗ In vitro
This study investigated the in vitro metabolic profile of SLU-PP-332, a synthetic agonist of estrogen-related receptors alpha, beta, and gamma (ERRα/β/γ), developed as an "exercise mimetic" — a compound designed to replicate some physiological benefits of physical exercise, such as increased fatty acid oxidation, oxidative muscle fiber development, and improved exercise endurance. Because the World Anti-Doping Agency (WADA) prohibits metabolic modulators in competitive sports, identifying the metabolites of SLU-PP-332 is an important step for anti-doping surveillance. The researchers incubated SLU-PP-332 with pooled human liver S9 fractions to simulate Phase I and Phase II hepatic metabolism, then analyzed the resulting products using liquid chromatography-high-resolution mass spectrometry (LC-MS/HRMS). They identified 22 metabolites in total: five monohydroxylated, three dihydroxylated, four reduced dihydroxylated, and several glucuronidated and sulfated conjugates. Eight metabolites (M1, M7, M9, M10, M13, M14, M19, and M20) were identified as the most abundant and were flagged as priority targets for doping-control testing. The authors note that full structural elucidation of all metabolites requires further investigation. No human or animal subjects were involved; all findings are limited to an in vitro cell-free system.
Drug testing and analysis · 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 whether Thymosin α1 (Tα1), an endogenous thymic peptide known to modulate immune function, could enhance CD8+ T cell-mediated killing of breast cancer cells. Researchers isolated CD8+ T cells from peripheral blood of ten healthy donors and tested them under four conditions: unstimulated, CD3/CD28-stimulated, Tα1-treated, or exhaustion-rescue. Cytotoxic activity was assessed against MDA-MB-231 breast cancer cells and CD44+ cancer stem-like cells. The study reported that Tα1 treatment significantly increased cancer cell apoptosis, suppressed tumor cell proliferation, and boosted granzyme B secretion compared to CD3/CD28 stimulation alone. In artificially exhausted T cells, Tα1 partially restored effector function and reduced expression of exhaustion markers PD-1, TIM-3, and LAG-3. Complementary bioinformatic analysis of TCGA-BRCA data (n=1,112) using a four-gene Tα1 Response Index correlated with antigen presentation and cytotoxic gene programs. Key limitations include the small donor sample (n=10), use of healthy donor rather than patient-derived T cells, an in vitro experimental design, and the exploratory nature of the transcriptomic index. Results may not directly translate to in vivo or clinical settings.
Human immunology · Jan 2026DOI ↗