Prostacyclin analogues are effective treatments in pulmonary arterial hypertension (PAH), especially in advanced stages. Treprostinil, a stable prostacyclin analogue, can be administered as subcutaneous and intravenous infusions, oral extended-release tablets and inhalation. Inhalation offers several advantages over other routes of administration, including direct access to the lungs for localized therapy, reduced infection risk, and a painless, convenient mode of delivery. However, small lipophilic molecules like treprostinil are absorbed into the bloodstream within minutes after inhalation, resulting in a short duration of action in the lungs and systemic side effects. To address these limitations, we developed a novel strategy involving a double treprostinil prodrug tailored for pulmonary delivery. The prodrug consists of treprostinil di-esterified at its carboxylic acid with a polyethylene glycol (PEG) chain, and at its C11 hydroxyl group with butyric acid. The prodrug exhibited sustained treprostinil release in bronchoalveolar lavage fluid and serum, supporting its suitability for pulmonary delivery. It was cleaved by initial hydrolysis of the PEG chain, followed by subsequent cleavage of the short-chain fatty acid. Ex vivo studies in isolated pulmonary artery rings showed a delayed and prolonged vasorelaxation effect of the conjugate compared to the free drug. In vivo studies demonstrated significant lung retention, with detectable quantities of the compound remaining in the lungs 24 h after administration, and a markedly reduced peak serum concentration following inhalation. This double-prodrug approach represents a promising strategy for improving PAH treatment by optimizing local, sustained treprostinil delivery while minimizing systemic exposure.

The development of acid-responsive fluorescent probes with additional chemical functionality remains of considerable interest in chemical biology. Herein, we report an acid-responsive rhodamine-based fluorescent probe, 2PCA-RhB, which exhibits a pronounced fluorescence “turn-on” response under acidic aqueous conditions through spirolactam ring opening. The probe displays favorable photophysical properties, good aqueous compatibility, and enables fluorescence imaging in living cells. Beyond its fluorescence response, 2PCA-RhB incorporates a 2-pyridinecarboxaldehyde (2PCA) moiety that enables N-terminal-selective protein labeling, while its allyl carbonate functionality is compatible with palladium-catalyzed Tsuji–Trost allylic alkylation under aqueous conditions. These studies establish 2PCA-RhB as an acid-responsive fluorescent probe with both N-terminal labeling capability and compatibility with aqueous transition-metal-mediated transformation. The present work provides a versatile molecular scaffold for the future development of fluorescent chemical tools for protein modification and bioorthogonal applications in complex biological environments.

Lactate dehydrogenase (LDH) catalyzes the reversible conversion of pyruvate, NADH, and H+ into lactate and NAD+, maintaining the energy metabolism and the cellular redox balance. It also plays a key role in metabolic reprogramming processes observed in cancer and other proliferative conditions. Consequently, LDH has long been considered as an attractive therapeutic target, and numerous inhibitors have been developed, albeit with limited clinical success. Most of these efforts have focused on the highly conserved active site, a strategy often hampered by poor selectivity and off-target effects. Here, we explored an alternative approach based on targeting LDH oligomerization, which may help circumvent some limitations associated with active-site inhibition. Our research focuses on optimizing a perfluoroaryl cysteine-stapled octapeptide named macrocycle 7, which was previously identified to bind the LDH tetramerization site with micromolar affinity. Through systematic structure-activity relationship studies and the incorporation of unnatural amino acids, we report the design and synthesis of improved analogs with enhanced binding affinity, stability, and inhibitory efficacy. Biophysical and enzymatic assays confirmed that these optimized peptides effectively destabilize LDH and inhibit its catalytic activity. In cellular models, the most potent analogs significantly altered LDH thermal stability, confirming target engagement, and 72-h treatments in HCT116 and MDA-MB-231 human cancer cells further revealed significant cytotoxic anticancer effects. These optimized peptides demonstrate the potential of targeting protein oligomerization as a general strategy to regulate LDH activity through structural destabilization rather than active site competition.

The active form of lactate dehydrogenase B (LDHB) is a homotetramer, and disruption of this quaternary structure represents a promising strategy for therapeutic intervention, particularly in cancer metabolism. However, accurate characterization of LDHB oligomerization is challenging due to the weak, reversible, and environmentally sensitive nature of subunit interactions. Here, we establish a robust size exclusion chromatography coupled with multi angle light scattering (SEC–MALS) workflow for analysis of LDHB oligomerization under physiologically relevant conditions.Wild type LDHB and three single point interface mutants (I9A, L71A, and F72A), covering oligomeric states from tetramers to monomers, were analyzed. SEC–MALS enabled clear separation and absolute molar mass determination of coexisting oligomeric species at micromolar protein concentrations, capturing concentration dependent equilibria inaccessible to techniques requiring nanomolar levels. The wild type enzyme remained exclusively tetrameric and remarkably stable, whereas the mutants displayed heterogeneous, dynamic oligomeric distributions.Systematic evaluation of experimental parameters demonstrated that LDHB oligomerization is highly sensitive to buffer composition. While the wild type protein was largely unaffected, variations in salt nature, ionic strength, pH, organic solvent, and arginine selectively destabilized mutant assemblies. Sodium chloride emerged as the most appropriate salt for preserving native oligomeric states while minimizing ion specific perturbations.Together, these results establish SEC–MALS as a powerful and essential method for characterizing dynamic protein oligomerization equilibria under biologically relevant conditions and provide critical insights into the structural determinants governing LDHB assembly and stability.

IntroductionUrsolic acid (UA), the antimalarial triterpenic mixture 8TTE (containing C-27 feruloyl and coumaroyl esters of ursane and oleane skeletons), and the semi-synthetic antitrypanosomal derivative ursolic acid O-phenyl propionate (UAOPP) exhibit high lipophilicity, which may limit their oral bioavailability. This study aimed to develop lipid nanocapsules (LNCs) to improve the solubility, intestinal permeability, and antiparasitic activity of these triterpenic compounds for potential oral delivery.MethodsLNCs formulations containing UA, 8TTE, and UAOPP were prepared and evaluated. A sensitive UPLC\x{2013}MS method was developed and validated for selected triterpenes quantification during transport studies across Caco-2 cell monolayers [limit of detection (LOD): 2 nM; limit of quantification (LOQ): 25 nM]. Cytotoxicity and permeability studies were conducted on Caco-2 cells to assess formulation safety and intestinal transport. In vitro antiparasitic activity of free and formulated compounds was evaluated against Plasmodium falciparum and Trypanosoma brucei brucei (Tbb).Results and discussionThe formulations were non-toxic to Caco-2 cells at concentrations up to 2 mg/mL. Permeability studies demonstrated enhanced transport for the formulated triterpenic esters, with permeability increases of up to 2.68-fold, shifting their classification from poorly absorbed to moderately absorbed compounds in humans [apparent permeability coefficient (Papp) > 1 × 10–6 cm/s]. Free UA showed the highest Papp value (4.95 × 10–6 cm/s ± 1.29 × 10–7), but caused epithelial integrity disruption after 2 h of incubation and during the following 48 h, whereas formulated UA induced minimal integrity loss at the same concentration. In antiparasitic assays, blank LNCs exhibited maximum non-toxic concentrations of 165 μg/mL against P. falciparum and 65 μg/mL against Tbb. At these maximum concentrations, formulated UA and 8TTE showed enhanced antiplasmodial activity; however, blank LNCs produced comparable effects. In contrast, UAOPP-loaded LNCs showed significantly improved antitrypanosomal activity by approximately 20% at 2.15 μM (cell viability: 38.20% ± 5.41) compared with free UAOPP (20.38% ± 8.80). These findings suggest that LNCs represent promising oral delivery systems for lipophilic triterpenes. Further in vivo pharmacokinetic and efficacy studies are needed to confirm their therapeutic potential.

Acyl-coenzyme A synthetase long-chain family member 4 (ACSL4), a pivotal enzyme in lipid metabolism, has emerged as a therapeutic target for ferroptosis-related conditions and cancer. However, its reference inhibitor, rosiglitazone, has off-target activity on peroxisome proliferator-activated receptor gamma (PPARγ), a key regulator of lipid homeostasis. Here, the discovery of LIBX-A401, a potent ACSL4 inhibitor derived from rosiglitazone devoid of PPARγ activity, is reported. Its binding to ACSL4 is ATP-dependent, stabilizing the C-terminal domain and altering the fatty acid gate region, as shown by Hydrogen-Deuterium Exchange Mass Spectrometry. Photoaffinity labeling identified A329 within the fatty acid binding site, while molecular dynamics and mutagenesis highlighted Q302 as critical for LIBX-A401 binding. LIBX-A401 exhibits anti-ferroptotic properties in cells, supported by target engagement. These findings establish LIBX-A401 as a valuable tool to study ACSL4 in ferroptosis and cancer, while its elucidated binding mode paves the way for the rational design of improved inhibitors.

The Alternative Lengthening of Telomeres (ALT) mechanism enables telomere maintenance, contributing to the immortality of certain cancer cells. Disrupting the interaction between testis-specific Y-encoded-like protein 5 (TSPYL5) and ubiquitin-specific protease 7 (USP7) has emerged as a promising strategy to target ALT-dependent cancers. While the N-terminal MATH domain of USP7 mediates the protein interaction, the regions of TSPYL5 involved in binding remain unclear. Here, we present a structural analysis of the TSPYL5-USP7 interaction to guide targeted therapeutic strategies. We showed that TSPYL5 is intrinsically disordered, with an unfolded N-terminal region and partial structure in the C-terminal half. In vitro, recombinantly expressed TSPYL5 binds USP7 with nanomolar affinity and is prone to C-terminal truncation. However, the truncated form retained a similar binding affinity for USP7, suggesting the primary interaction site resides in the N-terminal region of TSPYL5. We identified three key binding hotspots within TSPYL5: residues 65-97, residues 210-262, and residues 368-388. Moreover, TSPYL5 forms trimers that further assemble into hexamers. This study provides the first structural and quantitative analysis of the TSPYL5-USP7 interaction, highlighting these three binding sites. These findings lay the groundwork for the development of novel inhibitors targeting ALT-dependent cancers.

Objectives To evaluate the efficacy of Bacopa monnieri (BM) containing Bacopaside II, a specific Aquaporin 1 (AQP1)-blocker, on systemic oxidative stress. Background AQP1, is a peroxiporin which facilitates hydrogen peroxide transmembrane passage. It is predominantly expressed in endothelial cells and erythrocytes. Methods BM extract was administered orally for 6 weeks to 20 healthy volunteers (Group A/B: 400/800 mg/day). Assessments occurred at baseline (V0), after 6 weeks of treatment (V4), and 4 weeks post-treatment (V6). Primary endpoint: ROS levels in erythrocytes post-H2O2 exposure (DCFDA fluorescence). Secondary endpoints: Oxidative stress and safety biomarkers, blood pressure monitoring. Bacopaside II metabolites in plasma were identified using liquid chromatography-mass spectrometry (LC-MS). Results BM intake reduced ROS levels in RBCs in Group B (T40 min: Mean Fluorescence Intensity of DCF V0=381 +/- 43 a.u vs V4= 187 +/- 69 a.u, p<0.01). Methemoglobin and oxidized Methionine 148 of Apolipoprotein A-1 levels decreased (Methemoglobin group B: V0= 0.900 +/- 0.105 a.u vs V4= 0.233 +/- 0.047 a.u; p<0.001, M148-ox/M148 ratio group B: V0= 0.06 +/- 0.01 a.u. vs V4= 0.02 +/- 0.00 a.u.; p<0.05). A reduction in blood pressure was observed in Group B (Systolic Blood Pressure V0=131 +/- 15 mmHg vs SBP V4=116 +/- 7 mmHg; p < 0.05). Two potential Bacopaside II metabolites with putative binding pockets on AQP1 were identified during the treatment. Conclusion A six-week oral intake of BM reduced systemic oxidative stress in healthy volunteers in a dose-dependent manner. Pharmacological blocking of AQP1 may help restore redox balance in the vasculature.

Arginase-1 (ARG-1) is a promising target for cancer immunotherapy, but the small size and the highly polar nature of its catalytic site present significant challenges for inhibitor development. An alternative strategy to induce enzyme inhibition by targeting protein oligomerization has been developed recently, offering several advantages such as increased selectivity, promotion of protein degradation, and potential substoichiometric inhibition. In this study, we demonstrated that only trimeric ARG-1 is active, which was confirmed by producing monomeric arginase-1. Through in silico-driven site-directed mutagenesis, we identified an allosteric site involving five key amino acids responsible for ARG-1 trimerization. We further demonstrated the covalent modification of a key arginine residue within this pocket using phenylglyoxal disrupted ARG-1 oligomerization. Although phenylglyoxal has limited potency, it effectively supports the concept of ARG-1 inhibition via homomeric disruption, validating this allosteric targeting approach.

Lactate dehydrogenase (LDH) is a key enzyme in cancer metabolism, with isoforms LDH5 and LDH1 supporting glycolysis and oxidative lactate metabolism, respectively. While the development of competitive LDH inhibitors has faced diverse challenges, allosteric strategies targeting LDH tetramerization have recently attracted increasing attention. To further explore this alternative, we investigated the factors influencing LDH tetramerization and enzymatic activity using a truncated form of human LDH-B (LDHBtr), which was reported to exist predominantly as a dimer. Unexpectedly, LDHBtr exhibited measurable activity at high concentrations, correlating with increased protein stability and a structural transition to the tetrameric form. Preincubation with NADH further enhanced LDHBtr activity, stability, and self-association, consistent with cofactor-promoted tetramer assembly. Crystallographic studies confirmed the tetrameric structure of LDHBtr bound to NADH. Furthermore, reported LDH allosteric inhibitors, including cGmC9 and fluoxetine, preferentially inhibited LDHBtr compared to the native LDHB, by preventing tetramer formation. Overall, this work highlights the central role of tetramerization in regulating LDH activity, and the therapeutic potential of targeting this process. It also establishes LDHBtr as a valuable tool for screening tetramerization disruptors, paving the way for next-generation LDH inhibitors to target cancer metabolism.

Treprostinil (TRE) is a prostacyclin analogue approved for the treatment of pulmonary arterial hypertension (PAH). Despite its effectiveness, TRE has a short half-life, necessitating frequent or continuous administration to maintain therapeutic levels while minimizing adverse effects. To improve pharmacokinetics and pulmonary targeting of TRE, we designed a series of polyethylene glycol (PEG) ester conjugates for inhalation. The increase in molecular size achieved through polymer conjugation prevents the passive diffusion of TRE across the alveolar-capillary barrier, a common drawback of small, lipophilic drugs delivered to the lungs. The ester bond between TRE and PEG enables a gradual release of the active compound within the alveolar space. TRE was chemically modified with seven different alkyne-bearing linkers and subsequently conjugated to PEG-azide 6 kDa via click chemistry. These linkers were strategically designed to modulate the chemical environment around the cleavable ester bond, allowing for the systematic evaluation of the steric and electronic effects on the stability of the PEG-TRE conjugates. Drug release studies in bronchoalveolar lavage from healthy rats demonstrated that sterically hindered and electronically stabilized linkers significantly slowed the release of TRE. Moreover, conjugate stability was dependent on the enzymes availability, with a higher conjugate-enzyme ratio leading to slower release, suggesting enzyme saturation as a potential mechanism for controlled drug release. Overall, these findings demonstrate a tunable strategy for releasing drugs from polymer-drug conjugates in biological media.

Ferroptosis, an iron-dependent regulated cell death, is implicated in several diseases, including cancer and neurodegeneration. While most ferroptosis inhibitors act as radical-trapping antioxidants, direct modulation of pro-ferroptotic enzymes remains underexplored. Acyl-coenzyme A synthetase long-chain family member 4 (ACSL4), a key regulator of ferroptosis, has emerged as a promising therapeutic target. Here, we report a fragment-based screening that identified a benzofuran hit (compound 8, IC50 = 33 μM), leading to the discovery of two selective ACSL4 inhibitors: compound 15b (LIBX-A402, IC50 = 0.33 μM) and compound 21 (LIBX-A403, IC50 = 0.049 μM). Compound 21 is the most potent ACSL4 inhibitor reported to date and shows no activity against ACSL3. Molecular modeling and mutagenesis support its binding in the ACSL4 fatty acid pocket. The strong antiferroptotic activity of both compounds in cells, together with confirmed target engagement for 21, underscores the relevance of ACSL4 as a target for ferroptosis modulation.

The grafting of a diisopropylaminobenzyl substituent onto an N-Alloc protecting group significantly accelerates Tsuji–Trost deallylation, enabling intramolecular capture of the π-allylpalladium intermediate.

Lipid metabolism affects many cellular processes essential for homeostasis, and its disruption is linked to various diseases. A key enzyme in these processes, acyl-coenzyme A synthetase long-chain family member 4 (ACSL4), is a promising target for treating conditions involving ferroptosis and certain cancers. Rosiglitazone (ROSI) is a known ACSL4 inhibitor but its potent activity on peroxisome proliferator-activated receptor gamma (PPARγ), a nuclear receptor strongly involved in lipid metabolism constitutes an important limitation. This study focuses on developing novel ACSL4 inhibitors derived from ROSI, which lack PPARγ activity. Binding of the most potent compound of this series (9) relies on the prior binding of ATP. Hydrogen-Deuterium Exchange Mass Spectrometry (HDx-MS) demonstrated that ATP binding stabilizes the ACSL4 C-terminus, an effect enhanced by compound 9, which also alters important peptide sequences, including the fatty acid gate-domain. Photoaffinity Labeling (PAL) with a diazirine-based probe identified residue A329 in the fatty acid pocket. Molecular dynamics simulations and site-directed mutagenesis highlighted Q302 as critical for compound 9 binding. Thus, compound 9 (LIBX-A401) is a promising tool for studying ACSL4 in ferroptosis-related diseases and cancer, and the elucidation of its binding mode paves the way to the rational design of optimized inhibitors

Human tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) are two important targets in cancer immunotherapy. Extensive research has led to a large number of potent IDO inhibitors; in addition, 52 structures of IDO in complex with inhibitors with a wide array of chemical scaffolds have been documented. In contrast, progress in the development of TDO inhibitors has been limited. Only four structures of TDO in complex with competitive inhibitors that compete with the substrate L-tryptophan for binding to the active site have been reported to date. Here we systematically evaluated the structures of TDO in complex with competitive inhibitors with three types of pharmacophores, imidazo-isoindole, indole-tetrazole, and indole-benzotriazole. The comparative assessment of the protein-inhibitor interactions sheds new light into the structure-based design of enzyme-selective inhibitors.

Targeting enzymes involved in lipid metabolism is increasingly recognized as a promising anticancer strategy. Efficient inhibition of diacylglycerol O-transferase 1 (DGAT1) can block fatty acid (FA) storage. This, in turn, triggers an increase in free polyunsaturated FA concentration, leading to peroxidation and ferroptosis. In this study, we report the development of a pH-sensitive peptide (pHLIP)-drug conjugate designed to selectively deliver DGAT1 inhibitors to cancer cells nested within the acidic microenvironment of tumors. We utilized two previously established pHLIP sequences for coupling with drugs. The study of DGAT1 conjugates in large unilamellar vesicles (LUVs) of different compositions did not reveal enhanced pH-dependent insertion compared to POPC LUVs. However, using in vitro 3D tumor spheroids, significant antiproliferative effects were observed upon exposure to pHLIP-T863 (DGAT1 inhibitor) conjugates, surpassing the inhibitory activity of T863 alone. In conclusion, our study provides the first evidence that pHLIP-based conjugates with DGAT1 inhibitors have the potential to specifically target the acidic compartment of tumors. Moreover, it sheds light on the limitations of LUV models in capturing the pH-dependency of such conjugates.

The palladium-catalyzed uncaging of alloc-protected amines in living cells has been extensively stud-ied mostly in the form of nanostructures or mesostructures. However, the scarcity of kinetic and mechanistic studies of discrete palladium complexes for deallylation reactions has hindered progress. Herein, we report the development of a series of discrete palladium complexes bearing acetanilide lig-ands, which exhibit a good balance between reactivity and stability in living cells. We investigated the catalytic activity and cytotoxicity of these complexes in SiHa cells and found that acetanilide[tri(2-furyl)phosphine]palladium(II) triflate showed promising results. Under physiological conditions, this pal-ladium complex exhibited a second-order reaction rate of 30 M -1 s -1, and liquid chromatography-mass spectrometry (LC-MS) studies suggested the possibility of a tandem Heck/Tsuji-Trost mechanism. Our re-sults demonstrate the potential of using these discrete palladium complexes for bioorthogonal chemistry studies in living cells. (c) 2023 Elsevier B.V. All rights reserved.

Background Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan-dioxygenase (TDO) are enzymes catabolizing the essential amino acid tryptophan into kynurenine. Expression of these enzymes is frequently observed in advanced-stage cancers and is associated with poor disease prognosis and immune suppression. Mechanistically, the respective roles of tryptophan shortage and kynurenine production in suppressing immunity remain unclear. Kynurenine was proposed as an endogenous ligand for the aryl hydrocarbon receptor (AHR), which can regulate inflammation and immunity. However, controversy remains regarding the role of AHR in IDO1/TDO-mediated immune suppression, as well as the involvement of kynurenine. In this study, we aimed to clarify the link between IDO1/TDO expression, AHR pathway activation and immune suppression. Methods AHR expression and activation was analyzed by RT-qPCR and western blot analysis in cells engineered to express IDO1/TDO, or cultured in medium mimicking tryptophan catabolism by IDO1/TDO. In vitro differentiation of naïve CD4 + T cells into regulatory T cells (Tregs) was compared in T cells isolated from mice bearing different Ahr alleles or a knockout of Ahr , and cultured in medium with or without tryptophan and kynurenine. Results We confirmed that IDO1/TDO expression activated AHR in HEK-293-E cells, as measured by the induction of AHR target genes. Unexpectedly, AHR was also overexpressed on IDO1/TDO expression. AHR overexpression did not depend on kynurenine but was triggered by tryptophan deprivation. Multiple human tumor cell lines overexpressed AHR on tryptophan deprivation. AHR overexpression was not dependent on general control non-derepressible 2 (GCN2), and strongly sensitized the AHR pathway. As a result, kynurenine and other tryptophan catabolites, which are weak AHR agonists in normal conditions, strongly induced AHR target genes in tryptophan-depleted conditions. Tryptophan depletion also increased kynurenine uptake by increasing SLC7A5 (LAT1) expression in a GCN2-dependent manner. Tryptophan deprivation potentiated Treg differentiation from naïve CD4 + T cells isolated from mice bearing an AHR allele of weak affinity similar to the human AHR. Conclusions Tryptophan deprivation sensitizes the AHR pathway by inducing AHR overexpression and increasing cellular kynurenine uptake. As a result, tryptophan catabolites such as kynurenine more potently activate AHR, and Treg differentiation is promoted. Our results propose a molecular explanation for the combined roles of tryptophan deprivation and kynurenine production in mediating IDO1/TDO-induced immune suppression.

Targeting enzymes involved in tumor metabolism is a promising way to tackle cancer progression. The inhibition of carnitine palmitoyltransferase 1 (CPT1) by etomoxir (Eto) efficiently slows down the growth of various cancers. Unfortunately, the clinical use of this drug was abandoned because of hepatotoxic effects. We report the development of pH-sensitive peptide (pHLIP)-drug conjugate to deliver Eto selectively to cancer cells exposed to acidic microenvironmental conditions. A newly designed sequence for the pHLIP peptide, named pHLIPd, was compared with a previously published reference pHLIP peptide, named pHLIPr. We showed that the conjugate between pHLIPd and Eto has a better pH-dependent insertion and structuration than the pHLIPr-based conjugate inside POPC vesicles. We observed antiproliferative effects when applied on acid-adapted cancer cells, reaching a larger inhibitory activity than Eto alone. In conclusion, this study brings the first evidence that pHLIP-based conjugates with a CPT1 inhibitor has the potential to specifically target the tumor acidic compartment and exert anticancer effects while sparing healthy tissues.

Ferroptosis, first coined in 2012, is an iron-dependent regulated cell death (RCD) characterized by the accu-mulation of lipid peroxides to toxic levels. This mechanism is currently being evaluated as a target for a variety of diseases offering new opportunities for drug design and development. Recent reports uncovered acyl-CoA syn-thetase long-chain 4 (ACSL4) as a critical contributor to ferroptosis execution. Therefore, ACSL4 inhibitors are emerging as attractive anti-ferroptotic agents. Herein, we developed a robust screening cascade with orthogonal biophysical and biochemical techniques to identify original human ACSL4 inhibitors. By screening an FDA -approved drug library, we were able to identify and validate new inhibitors with micromolar-range activities against ACSL4. With an IC50 of 280 nM against hACSL4, antifungal agent sertaconazole is to our knowledge, the most potent ACSL4 inhibitor identified so far. In addition, sertaconazole significantly reduced lipid peroxidation and ferroptosis in human differentiated dopaminergic neurons (Lund human mesencephalic LUHMES cells), demonstrating that it is a valuable chemical tool for further investigating the role of ACSL4 in ferroptosis. This study highlights the phenethyl-imidazole scaffold as a novel and promising starting point for the development of anti-ferroptotic agents targeting ACSL4.