YL939 binds to prohibitin 2 (PHB2) with a Kd value of 3.43 μM as determined by surface plasmon resonance (SPR) assay. The binding induces a thermal shift (ΔTm) of 2.65°C in differential scanning fluorimetry (DSF) assay. Molecular docking predicted that YL939 forms two hydrogen bonds: one between the nitrogen atom of the pyrazole ring and residue D82, and another between the nitrogen atom of the piperidine ring and residue D127. The distal phenyl group forms hydrophobic interactions with residues V43 and I80. Mutation of D82A and D127A almost abolished the binding of YL939 to PHB2. [1]
YL-939 targets the Prohibitin 2 (PHB2) protein. It binds directly to PHB2 with a Kd of 3.43 microM as determined by surface plasmon resonance (SPR) assays. PHB2 is a multifunctional scaffold protein involved in various cellular processes, including mitochondrial function, cell cycle regulation, and apoptosis. YL-939 specifically protects cells from ferroptosis by modulating the PHB2/ferritin/iron axis, distinguishing it from inhibitors of other cell death pathways.

YL939 protected cells from erastin-induced ferroptosis with EC50 values: ES-2 cells 0.09 μM, HT1080 cells 0.14 μM, Miapaca-2 cells 0.25 μM, Calu-1 cells 0.16 μM, HCT116 cells 0.16 μM, and SHSYSY cells 0.24 μM. [1]
YL939 also protected cells from other ferroptosis inducers including RSL3 and ML210. [1]
YL939 did not rescue cell death in bortezomib-induced apoptosis, TNFα/Smac-mimetic/zVAD-FMK (TSZ)-induced necroptosis, nigericin-induced pyroptosis, or elesclomol/CuCl2-induced cuproptosis, indicating specificity for ferroptosis. [1]
Transmission electron microscopy showed that YL939 (5 μM, 10 h) protected erastin-induced mitochondrial cristae disappearance and outer membrane rupture. [1]
YL939 treatment did not impact GSH levels but substantially reduced malondialdehyde (MDA) levels. YL939 treatment substantially diminished cytosolic ROS (H2DCFDA), lipid ROS (C11-BODIPY), and mitochondrial ROS (MitoSox) induced by erastin. [1]
YL939 treatment dose-dependently up-regulated ferritin protein (FTH1 and FTL) and their mRNA expression, and dose-dependently reduced intracellular iron levels elevated by erastin treatment. YL939 treatment increased NCOA4 and ferritin levels in erastin-treated cells in a concentration-dependent manner, indicating inhibition of ferritinophagy. [1]
YL939 did not show obvious cytotoxicity against six normal cell lines (L02, LX-2, Beas-2b, HUVEC, Arpe, and hTERT-HPNE) at concentrations less than 3 μM, but displayed evident cytotoxicity at concentrations greater than 3 μM. [1]

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YL-939 (0.01-10 μM) and HT-1080, Miapaca-2, Calu-1, HCT116, and SHSY5Y cells significantly protect cells against ferroptosis (IC50 values of 0.14 μM, 0.25 μM, 0.16 μM, 0.16 μM). 0.24 micrograms per milligram[1]. Reduces ROS levels in cytosol and membrane lipids with YL-939 (5 μM; 10 hours; ES-2 cells)[1]. PHB2 is a biological target of YL-939 (5 μM; 1-10 h; ES-2 cells) [1]. The concentration-dependent inhibition of ferritin autophagy is achieved by YL-939 (3 μM; 10 h; ES-2 cells) through its improvement of ferritin expression and its ability to block autophagosomes/lysosomes.

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In vitro, YL-939 specifically protects cells from erastin-induced ferroptosis in various cell lines. It shows potent protective effects with EC50 values of 0.14 microM in HT1080 cells and 0.09 microM in ES-2 cells. YL-939 does not inhibit apoptosis, necroptosis, pyroptosis, or cuprotosis, highlighting its selectivity for ferroptosis. It dose-dependently reduces erastin-elevated intracellular iron levels, up-regulates ferritin protein and mRNA expression of FTH1 and FTL, and blocks autophagosomes/lysosomes, thereby inhibiting ferritinophagy. YL-939 does not impact currently known signal pathways regulating ferroptosis.

YL939 ameliorated liver damage in an acetaminophen (APAP)-induced acute liver injury model. Male C57BL/6 mice were pretreated with YL939 (3 mg/kg, intraperitoneal) 2 hours before APAP injection (500 mg/kg, intraperitoneal). Six hours after APAP injection, YL939 treatment significantly reduced serum AST and ALT levels compared to APAP alone. YL939 also reduced serum and hepatic MDA levels. Hepatic ferritin protein (FTH1 and FTL) and mRNA expression were increased by YL939 treatment. Histological analysis (H&E staining) showed that YL939 ameliorated APAP-induced liver damage. [1]

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In an acute liver injury model induced by acetaminophen (APAP), YL-939 (3 mg/kg; i.p.; single injection) ameliorates liver injury [1].

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In vivo, YL-939 (3 mg/kg) ameliorates liver damage in an acetaminophen (APAP)-induced acute liver injury model, a condition where ferroptosis is known to be involved. It protects PHB2 from degradation by pronase, demonstrating a direct interaction. YL-939 has no effect on the PHB2 isoform PHB1, nor on VDAC1 and VDAC2, indicating specificity for PHB2. Its in vivo efficacy in reducing acute liver injury highlights its potential as a therapeutic agent for ferroptosis-related diseases.

YL939 Surface plasmon resonance (SPR) assay: PHB2¹⁻¹⁹⁴ protein was immobilized on a CMS sensor chip. Various concentrations of YL939 were flowed over the chip. The flow rate was 10 μL/min, with contact time of 120 seconds and dissociation time of 100 seconds. The Kd value for YL939 binding to PHB2 was 3.43 μM. YL447 (an inactive analogue) was used as a negative control and showed no binding. [1]
Differential scanning fluorimetry (DSF) assay: 9.8 μL of protein and SYPRO Orange mixture was added per well, mixed with 0.2 μL of compound (final concentration 200 μM compound, 10 μM protein in 20 mM Hepes pH 7.5, 150 mM NaCl). Thermal denaturation was achieved by a temperature ramp from 25°C to 95°C (1°C per minute). YL939 induced a thermal shift (ΔTm) of 2.65°C. [1]
DPPH free radical scavenging assay: YL939 (50 μM) showed no antioxidant activity. [1]
Ferrozine-based colorimetric iron chelation assay: YL939 (50 μM) showed no iron-chelating ability. [1]

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The direct binding of YL-939 to PHB2 is assessed using Surface Plasmon Resonance (SPR) assays. Recombinant PHB2 protein is immobilized on a sensor chip. Increasing concentrations of YL-939 (e.g., 0.1-100 microM) are injected over the chip surface. The resulting sensorgrams are used to calculate the equilibrium dissociation constant (Kd). The assay confirmed a Kd of 3.43 microM for the YL-939-PHB2 interaction, quantifying the direct binding affinity of the compound for its target.

YL939 Cell viability assay (MTT/CCK-8): Cells were seeded in 96-well plates, treated with compounds for 48 h, then MTT or CCK-8 reagent was added and incubated for 2-4 h. Absorbance was measured at 490 nm (MTT) or 450 nm (CCK-8). EC50 values were calculated using GraphPad Prism. [1]
Dual staining with CytoCalcein Violet 450 (living cells) and 7-AAD (dead cells): ES-2 and HT1080 cells were treated with or without erastin (10 μM) and YL939 for 10 h, then stained and imaged. [1]
Transmission electron microscopy: ES-2 cells were treated with DMSO, erastin (10 μM), or erastin plus YL939 (5 μM) for 10 h, fixed with 3% glutaraldehyde, then 1% osmium tetroxide, embedded in epoxy resin, sectioned (50 nm), stained with uranyl acetate and lead citrate, and visualized. [1]
Intracellular ROS detection: Cells were harvested after 10 h treatment, resuspended in HBSS, stained with H2DCFDA (5 μM) for cytosolic ROS, C11-BODIPY (2 μM) for lipid ROS, or MitoSOX (5 μM) for mitochondrial ROS, incubated for 10 min at 37°C, and analyzed by flow cytometry (≥10,000 cells). [1]
Intracellular MDA detection: Cells were lysed, mixed with MDA working solution (0.37% TBA plus antioxidants), heated at 100°C for 15 min, cooled, centrifuged, and absorbance measured at 532 nm. [1]
Intracellular GSH detection: Cells were lysed, and GSH levels were measured using a GSH detection kit according to manufacturer's instructions. [1]
Western blot analysis: Cells were lysed in NP-40 lysis buffer with protease inhibitor cocktail and PMSF. Proteins were separated by 12% SDS-PAGE, transferred to PVDF membranes, blocked with 5% milk, incubated with primary antibodies (1:1000) at 4°C overnight, then with HRP-conjugated secondary antibodies (1:5000) at 37°C for 1 h, and visualized with ECL. Antibodies used: PHB2, VDAC1, VDAC2, PHB1, α-Tublin, β-actin, SLC7A11, GPX4, TFR1, FPN, DMT1, Ferritin, NCOA4, FTH1, FTL. [1]
RT-qPCR: Total RNA was extracted, reverse transcribed to cDNA, and qPCR performed using SYBR Green. Primers used: PHB2, FTH1, FTL, DMT1, TFR1, FSP1, DHODH, GCH1, GAPDH. Expression was normalized to GAPDH. [1]
siRNA transfection: ES-2 cells were transfected with 50 nM siPHB2 using lipofectamine 2000 for 24 h. PHB2 siRNA#1 sequence: 5'-CUACAGAUGGUAGAAUUCUTT-3'; siRNA#2: 5'-CUUGAGAAGCCAAAACCAGUTT-3'; siRNA#3: 5'-CACAGAAGCUGAUCUAUUCUTT-3'. [1]
Lentiviral transfection: shPHB2#1 sequence: 5'-CCAGAATATCTCCAAGACGAT-3'; shPHB2#2 sequence: 5'-AAGAACCCTGGCTACATAAA-3'. For PHB2 overexpression, PHB2 sequence was inserted into pLVX-IRES-Zsgreen1 vector. [1]
Fe²⁺ detection: Intracellular iron was detected using a Fe²⁺ detection kit or fluorescent imaging probe (BioTracker 575 Red Fe2+ Dye) according to manufacturer's instructions. [1]

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Western Blot Analysis[1]
Cell Types: ES-2 cells
Tested Concentrations: 5 μM
Incubation Duration: 1, 5, 7.5, and 10 hrs (hours)
Experimental Results: Pulled down PHB2 protein by the probe.

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Western Blot Analysis[1]
Cell Types: ES- 2 cells
Tested Concentrations: 3 μM
Incubation Duration: 10 hrs (hours)
Experimental Results: Increased the expression of nuclear receptor coactivator 4(NCOA4) in a dose-dependent manner.

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For ferroptosis protection assays, cells such as HT1080 (fibrosarcoma) or ES-2 (ovarian clear cell carcinoma) are seeded in 96-well plates. The cells are pre-treated with varying concentrations of YL-939 (e.g., 0.001-10 microM) for 1 hour, followed by the induction of ferroptosis with a lethal concentration of erastin (e.g., 5-10 microM). After 24-48 hours of incubation, cell viability is measured using a standard MTT or CellTiter-Glo assay. The EC50 for protection is calculated by fitting a dose-response curve. Key readouts include lipid ROS levels (e.g., with C11-BODIPY 581/591 dye) and intracellular iron levels.

YL939 Acetaminophen (APAP)-induced acute liver injury model: Male C57BL/6 mice (4-5 weeks old) were used. YL939 was dissolved in vehicle (5% DMSO + 3% HS-15 Solutol + 92% saline) and administered via intraperitoneal injection at 3 mg/kg. Two hours later, APAP (500 mg/kg dissolved in saline) was injected intraperitoneally. Six hours after APAP injection, mice were sacrificed. Serum was isolated for AST and ALT detection using an automatic biochemical analyzer. Livers were harvested: one portion was rapidly frozen in liquid nitrogen for protein and mRNA expression analysis, and another portion was fixed in 4% paraformaldehyde for H&E staining and immunohistochemistry. Eight mice were used per group (n=4 for serum AST/ALT detection, n=6 for MDA detection, n=2-3 for protein and mRNA expression). [1]

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Animal/Disease Models: Acetaminophen (APAP)-induced male C57BL /6J mouse[1]
Doses: 3 mg/kg
Route of Administration: intraperitoneal (ip)injection; single injection
Experimental Results: Inhibited the cell death and inflammatory infiltration in the liver tissues of male C57BL/J6 mice that received APAP.

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For the acetaminophen (APAP)-induced acute liver injury model, male C57BL/6 mice are fasted overnight. YL-939 is administered via intraperitoneal injection at a dose of 3 mg/kg. One hour later, hepatotoxicity is induced by an intraperitoneal injection of a hepatotoxic dose of APAP (e.g., 300 mg/kg). Mice are euthanized 6-24 hours post-APAP injection. Serum is collected to measure liver damage markers like alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Liver tissues are harvested for histopathological analysis (H&E staining) and to measure markers of ferroptosis such as lipid peroxidation (malondialdehyde levels) and iron content.

Detailed pharmacokinetic (PK) data for YL-939 is limited in public literature. As a small molecule with a molecular weight of 426.52 g/mol, it is expected to have properties amenable to in vivo administration. The compound is typically formulated in a vehicle such as 10% DMSO in saline for in vivo studies, as per its solubility profile. Researchers should perform pilot PK studies to determine parameters like plasma half-life, Cmax, and bioavailability for their specific experimental models.

YL939 did not show obvious cytotoxicity against six normal cell lines (L02, LX-2, Beas-2b, HUVEC, Arpe, and hTERT-HPNE) at concentrations less than 3 μM, but displayed evident cytotoxicity at concentrations greater than 3 μM. [1]
In the APAP-induced acute liver injury model, no mortality or significant adverse effects were reported at the dose of 3 mg/kg intraperitoneal. Body weight changes were not specifically reported. [1]

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Published toxicology data for YL-939 is limited to in vitro cell viability studies. In cell-based assays, YL-939 is non-toxic at concentrations typically used for ferroptosis protection (e.g., 0.09-0.14 microM). At higher concentrations, cell viability may be assessed to establish a therapeutic window. In the acute liver injury animal study at 3 mg/kg, no overt signs of systemic toxicity were reported. Comprehensive toxicological profiling has not been publicly released.

[1]. Non-classical ferroptosis inhibition by a small molecule targeting PHB2. Nat Commun. 2022 Dec 3;13(1):7473.

YL939 was discovered through a stepwise optimization process starting from hit compound Cpd-015. The optimization focused on three subgroups: 4-methoxybenzene (R1), 3,6-dihydro-2H-pyran (R2), and 1-methyl-4-phenylpiperazine (R3). [1]
Mechanistically, YL939 binding to PHB2 promotes the expression of the iron storage protein ferritin, hence reduces the iron content, thereby decreasing susceptibility to ferroptosis. PHB2 knockdown or YL939 treatment blocked autophagosomes/lysosomes and inhibited ferritinophagy. [1]
YL939 did not impact the expression of key components of the GPX4 signal pathway (SLC7A11, GPX4) nor the mRNA expression of FSP1, GCH1, and DHODH (core components of the other three ferroptosis defense pathways). [1]
Sichuan University has applied for Chinese patents covering YL939 and related compounds. [1]

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YL-939 is a research-grade chemical probe and is not approved for clinical use. It has no ongoing clinical trials. Its mechanism of action is distinct from classical ferroptosis inhibitors, as it targets the PHB2/ferritin/iron axis rather than GPX4. This unique mechanism makes it a valuable tool for dissecting the roles of PHB2 and ferritinophagy in ferroptosis. The compound is typically stored at -20degC, protected from light and moisture. For research use only.

C25H26N6O

426.513544559479

426.216809

C, 70.40; H, 6.14; N, 19.70; O, 3.75

3023925-68-7

Off-white to light yellow solid powder

O1CC=C(CC1)C1C2=NC(C3C=CC=CC=3)=CN2C=C(C2C=NN(C=2)C2CCNCC2)N=1

YL-939; YL939; orb1744106; SCHEMBL31339950;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :~100 mg/mL (~234.46 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.86 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)