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]

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.

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].

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]

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.

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.

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]

[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]

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.)