The target of LYN-1604 is UNC-51-like kinase 1 (ULK1), with an EC₅₀ value for activating ULK1 kinase activity determined by kinase assay; key amino acid residues involved in binding are LYS50, LEU53, and TYR89 of ULK1 [1]

LYN-1604 has the potential to be an agonist for ULK1 (enzymatic activity = 195.7% at 100 nM and IC50 = 1.66 μM against MDA-MB-231 cells) [1]. With an affinity for binding in the nanomole range (KD=291.4 nM), LYN-1604 binds to wild-type ULK1 [1]. On MDA-MB-231 cells, LYN-1604 (0.5, 1.0, and 2.0 μM) causes cell death through the ULK complex[1].. hours) dramatically increases Beclin-1 expression, degrades p62, and causes LC3-I to become LC3-II in MDA-MB-231 cells[1]. Via the ULK complex, LYN-1604 triggers autophagy that is ATG5-dependent[1]. Additionally, LYN-1604 has the ability to trigger apoptosis and boost caspase3 cleavage[1].

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1. Activation of ULK1 kinase activity: LYN-1604 binds to the kinase domain of ULK1 through hydrophobic interactions, significantly enhancing ULK1 kinase activity in a dose-dependent manner. The EC₅₀ value was calculated from the fitted curve of ULK1 kinase activity. Mutations at LYS50, LEU53, or TYR89 residues of ULK1 abolished the activating effect of LYN-1604, as demonstrated by ADP-Glo kinase assay and in vitro phosphorylation assay (impaired phosphorylation of mATG13 at Ser318). Surface plasmon resonance (SPR) confirmed the binding affinity between LYN-1604 and wild-type ULK1, which was reduced in mutant ULK1 [1]
2. Induction of autophagy in MDA-MB-231 cells: LYN-1604 (0.5μM, 1.0μM, 2.0μM) dose-dependently increased the number of MDC-positive autophagic vesicles (detected by fluorescence microscopy and flow cytometry). Western blot analysis showed that LYN-1604 upregulated Beclin-1 and LC3 (LC3-I to LC3-II conversion) and downregulated p62 in a concentration-dependent manner. Transfection with GFP/mRFP-LC3 plasmid revealed enhanced GFP-LC3 puncta formation after LYN-1604 (2.0μM) treatment, which was further increased by co-incubation with Bafilomycin A₁ (10nM), indicating increased autophagic flux [1]
3. Inhibition of cell viability and induction of apoptosis: LYN-1604 reduced the viability of MDA-MB-231 cells (IC₅₀ values determined by MTT assay). Pretreatment with 3-methyladenine (3-MA, 1mM, an autophagy inhibitor) significantly reversed the cell viability reduction induced by LYN-1604 (2.0μM), confirming autophagy-dependent cell death. Flow cytometry with Annexin-V/PI double staining showed that LYN-1604 (2.0μM) induced apoptosis in a time-dependent manner. Western blot analysis revealed cleavage of caspase3 and PARP, markers of apoptosis [1]
4. Regulation of ULK complex and downstream molecules: LYN-1604 (2.0μM) upregulated the phosphorylation of ULK1 at Ser317 (detected by immunocytochemistry and western blot) and enhanced the expression of ULK complex components (ULK1, mATG13, FIP200, ATG101). Silencing ULK1 or ATG5 by siRNA abolished LYN-1604-induced LC3 conversion and p62 degradation, confirming ATG5-dependent autophagy via the ULK complex. Microarray analysis identified ATF3, RAD21, and caspase3 as ULK1 interactors; LYN-1604 upregulated ATF3 and cleaved-caspase3, and downregulated RAD21, which was reversed by ULK1 silencing [1]

Targeting ULK1-modulated cell death, LYN-1604 (low dose: 25 mg/kg; medium dose: 50 mg/kg; high dose: 100 mg/kg) is an intragastric drug administered once daily for 14 days that reduces the growth of xenograft TNBC [1].

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1. Antitumor activity in TNBC xenograft model: Nude mice bearing MDA-MB-231 xenografts (n=6 per group) were treated with LYN-1604 or vehicle. LYN-1604 significantly reduced tumor volume and tumor weight compared to the control group (p<0.001, p<0.05). Immunohistochemistry of tumor tissues showed a significant increase in the positive ratio of p-ULK1 (Ser317) in LYN-1604-treated groups. Western blot analysis of tumor tissues revealed upregulation of ULK1, p-ULK1 (Ser317), LC3-II, and Beclin-1, and cleavage of PARP, consistent with in vitro autophagy and apoptosis induction [1]
2. In vivo safety profile: LYN-1604 treatment did not cause significant changes in relative body weight, liver index, spleen index, or kidney index compared to the vehicle group, indicating good in vivo tolerability [1]

1. ULK1 kinase activity assay (ADP-Glo assay): Purified ULK1 kinase was incubated with LYN-1604 at various concentrations (including 100nM for initial screening) in reaction buffer. After incubation, ADP-Glo reagent was added to terminate the reaction and convert ADP to ATP. Luminescence (RLU) was measured to quantify ULK1 kinase activity (blank group: no ULK1, 0% activity; control group: no LYN-1604, 100% activity). The EC₅₀ value was calculated from the dose-response curve of normalized kinase activity [1]
2. In vitro phosphorylation assay: Wild-type or mutant (K50A, L53A, Y89A) ULK1 was purified from HEK-293T cells. Purified mATG13 was used as the substrate, and the reaction was performed in the presence or absence of LYN-1604. Phosphorylation of mATG13 at Ser318 was detected by specific antibody via western blot, with equal loading of enzymes and substrate confirmed [1]
3. Surface plasmon resonance (SPR) assay: Wild-type or mutant ULK1 was immobilized on a Biacore sensor chip. LYN-1604 at various concentrations was injected over the chip, and binding affinities were evaluated by measuring the SPR signal. Data were analyzed to assess the interaction between LYN-1604 and ULK1 [1]
4. Molecular dynamics simulation: Computational simulation of LYN-1604 binding to wild-type or mutant ULK1 was performed for 10ns. The binding conformation was stabilized for wild-type and K50A/L53A mutants, but not for the Y89A mutant, confirming the key role of Y89 in binding [1]

Cell Viability Assay[1]
Cell Types: MDA-MB-231 cells
Tested Concentrations: 0.5, 1.0 and 2.0 μM
Incubation Duration:
Experimental Results: Induced cell death. Autophagy ratio was increased in a dose-dependent manner.

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Western Blot Analysis[1]
Cell Types: MDA-MB-231 cells
Tested Concentrations: 0, 0.5, 1, and 2 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Induced remarkable up -regulation of Beclin-1 and degradation of p62, as well as transformation of LC3-I to LC3-II.

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1. Cell viability assay (MTT assay): MDA-MB-231 cells were seeded in 96-well plates and treated with LYN-1604 at various concentrations for 24h. For autophagy inhibition experiments, 3-MA (1mM) was added 1h before LYN-1604 treatment. MTT reagent was added, and absorbance was measured to calculate cell viability. IC₅₀ values were determined using Prism 6.0 [1]
2. Autophagic vesicle detection (MDC staining): MDA-MB-231 cells were treated with LYN-1604 (0.5μM, 1.0μM, 2.0μM) and stained with MDC. Fluorescence microscopy was used to observe autophagic vesicles, and flow cytometry was used to quantify the MDC-positive cell ratio [1]
3. Autophagic flux assay: MDA-MB-231 cells were transfected with GFP/mRFP-LC3 plasmid and treated with LYN-1604 (2.0μM) in the presence or absence of Bafilomycin A₁ (10nM). Fluorescence microscopy was used to observe GFP-LC3 puncta. Western blot analysis was performed to detect p62 and LC3 expression to assess autophagic flux [1]
4. Western blot analysis: MDA-MB-231 cells were treated with LYN-1604 at specified concentrations and times. Cell lysates were prepared, and proteins were separated by electrophoresis, transferred to membranes, and probed with antibodies against Beclin-1, p62, LC3, ULK1, p-ULK1 (Ser317/Ser757), ATF3, RAD21, caspase3, PARP, and β-actin (loading control). Immunoreactive bands were detected and quantified [1]
5. Immunocytochemistry: MDA-MB-231 cells were treated with LYN-1604 (2.0μM) and fixed, permeabilized. Cells were incubated with primary antibodies against p-ULK1 (Ser317) or LC3B, followed by fluorescently labeled secondary antibodies. DAPI was used for nuclear staining. Fluorescence microscopy was used to observe and quantify protein expression [1]
6. Apoptosis assay (Annexin-V/PI double staining): MDA-MB-231 cells were treated with LYN-1604 (2.0μM) for specified times. Cells were harvested, stained with Annexin-V and PI, and analyzed by flow cytometry to determine the apoptosis ratio [1]
7. siRNA transfection assay: MDA-MB-231 cells were transfected with control siRNA, ULK1 siRNA, or ATF5 siRNA using transfection reagents. After transfection, cells were treated with LYN-1604 (2.0μM) for 24h. Western blot analysis or immunocytochemistry was used to detect target protein expression and assess the effect of gene silencing on LYN-1604-induced autophagy and apoptosis [1]
8. Microarray analysis: MDA-MB-231 cells were transfected with ULK1 siRNA or pcDNA3.1-ULK1. Total RNA was extracted, and microarray analysis was performed to identify differentially expressed genes with opposite expression changes. Core ULK1-regulated genes (including ATF3, RAD21, caspase3) were verified by western blot [1]

Animal/Disease Models: 24 female nude mice (BALB/c, 6-8 weeks, 20-22 g)[1]
Doses: Low dose, 25 mg/kg; median dose, 50 mg/kg; high dose, 100 mg/kg
Route of Administration: intragastric (po) administration; one time/day for 14 days
Experimental Results: Dramatically inhibited the growth of xenograft MDA-MB-231 cells. The body weights of mice were stable. By the end of the experiment, the liver and spleen weight indexes of mice were slightly increased in parts of the groups, while the kidney weight index was not affected in all dose groups.

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1. TNBC xenograft model establishment: MDA-MB-231 cells in log-phase growth were harvested and resuspended in appropriate medium. Nude mice were inoculated subcutaneously with MDA-MB-231 cells to establish xenograft tumors. Mice were randomly divided into control (vehicle) and LYN-1604-treated groups (n=6 per group) after tumor formation [1]
2. Drug administration: LYN-1604 was dissolved in a suitable vehicle (not specified in detail). The drug was administered to mice via an unspecified route at a median dose and other doses (not explicitly stated) for a specified treatment duration until the study endpoint [1]
3. Data collection and analysis: Tumor volume was measured regularly using calipers (tumor volume = length × width² / 2). At the end of treatment, mice were euthanized, and tumor weight was measured. Relative body weight was recorded throughout the study. Liver, spleen, and kidney were collected to calculate organ indices. Tumor tissues were fixed for immunohistochemistry (detection of p-ULK1) or frozen for western blot analysis (detection of ULK1, p-ULK1, LC3, Beclin-1, PARP) [1]

[1]. Discovery of a small molecule targeting ULK1-modulated cell death of triple negative breast cancer in vitro and in vivo. Chem Sci. 2017 Apr 1;8(4):2687-2701.

1. Background: ULK1 is a key initiator of autophagy, and its expression is significantly downregulated in breast cancer tissues, especially in triple-negative breast cancer (TNBC). Activation of ULK1-regulated autophagy is a potential therapeutic strategy for TNBC [1]. 2. Chemical structure: Figure 5A in reference [1] provides the chemical structure of LYN-1604. 3. Mechanism of action: LYN-1604 acts as a ULK1 agonist, binding to ULK1 through the residues LYS50, LEU53, and TYR89, thereby activating its kinase activity. It induces ATG5-dependent autophagy through the ULK complex (ULK1-mATG13-FIP200-ATG101) and triggers apoptosis by regulating ATF3, RAD21 and caspase3, ultimately leading to TNBC cell death [1]. 4. Therapeutic potential: LYN-1604 exhibits strong antitumor activity both in vitro and in vivo, and is well tolerated, making it a promising small molecule drug candidate for TNBC [1].

C33H43CL2N3O2

584.63

583.273

2088939-99-3

LYN-1604 dihydrochloride;2310109-38-5;LYN-1604 hydrochloride;2216753-86-3

131801113

Colorless to light yellow oil

7.6

0

4

12

40

750

0

DVNVYWLKGWAELS-UHFFFAOYSA-N

InChI=1S/C33H43Cl2N3O2/c1-24(2)19-37(20-25(3)4)22-33(39)38-15-13-36(14-16-38)21-32(30-12-11-29(34)18-31(30)35)40-23-26-9-10-27-7-5-6-8-28(27)17-26/h5-12,17-18,24-25,32H,13-16,19-23H2,1-4H3

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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