The target of LYN-1604 HCl is UNC-51-like kinase 1 (ULK1); the EC₅₀ value for activating ULK1 kinase activity was calculated from the fitted curve of ULK1 kinase activity via ADP-Glo assay, and the key amino acid residues involved in the binding between LYN-1604 HCl and ULK1 are LYS50, LEU53, and TYR89. [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].

---

1. Activation of ULK1 kinase activity: LYN-1604 HCl binds to the kinase domain of ULK1 through hydrophobic interactions, resulting in a dose-dependent increase in ULK1 kinase activity. Site-directed mutagenesis experiments showed that mutations at the LYS50, LEU53, or TYR89 residue of ULK1 completely abolished the activating effect of LYN-1604 HCl on ULK1. In vitro phosphorylation assays confirmed that LYN-1604 HCl promoted the phosphorylation of mATG13 (a downstream substrate of ULK1) at Ser318, while this promotion was lost in mutant ULK1 (K50A, L53A, Y89A). Surface plasmon resonance (SPR) experiments verified the specific binding affinity between LYN-1604 HCl and wild-type ULK1, and the binding affinity was significantly reduced in mutant ULK1 [1]
2. Induction of autophagy in MDA-MB-231 cells:
- LYN-1604 HCl (0.5μM, 1.0μM, 2.0μM) was used to treat MDA-MB-231 cells, and MDC staining combined with fluorescence microscopy and flow cytometry showed a dose-dependent increase in the number of MDC-positive autophagic vesicles and the MDC-positive cell ratio [1]
- Western blot analysis revealed that after 24h treatment with LYN-1604 HCl at different concentrations, the expression of autophagy-related proteins Beclin-1 and LC3-II (converted from LC3-I) was upregulated, while the expression of p62 (an autophagy substrate) was downregulated in a concentration-dependent manner [1]
- MDA-MB-231 cells transfected with GFP/mRFP-LC3 plasmid were treated with 2.0μM LYN-1604 HCl alone or in combination with 10nM Bafilomycin A₁ (BafA1, an autophagic flux inhibitor). Fluorescence microscopy showed that LYN-1604 HCl increased the number of GFP-LC3 puncta, and the number of puncta was further increased when combined with BafA1, indicating that LYN-1604 HCl enhanced autophagic flux [1]
3. Inhibition of cell viability and induction of apoptosis:
- MTT assay showed that LYN-1604 HCl inhibited the viability of MDA-MB-231 cells (IC₅₀ value determined by Prism 6.0). Pretreatment with 1mM 3-methyladenine (3-MA, an autophagy inhibitor) 1h before LYN-1604 HCl (2.0μM) treatment significantly reversed the decrease in cell viability induced by LYN-1604 HCl (p<0.001), confirming that LYN-1604 HCl induced autophagy-dependent cell death [1]
- Flow cytometry with Annexin-V/PI double staining demonstrated that LYN-1604 HCl (2.0μM) induced apoptosis in MDA-MB-231 cells in a time-dependent manner. Western blot analysis showed that LYN-1604 HCl treatment led to the cleavage of caspase3 and PARP (key markers of apoptosis), and this cleavage was partially reversed by ULK1 silencing [1]
4. Regulation of the ULK complex and downstream signaling molecules:
- Immunocytochemistry and western blot analysis showed that LYN-1604 HCl (2.0μM) upregulated the phosphorylation level of ULK1 at Ser317 and increased the expression of ULK complex components (ULK1, mATG13, FIP200, ATG101) [1]
- After silencing ULK1 or ATG5 by siRNA, LYN-1604 HCl-induced LC3 conversion and p62 degradation were completely blocked, and the decrease in cell viability induced by LYN-1604 HCl was significantly reversed, confirming that LYN-1604 HCl induced ATG5-dependent autophagy through the ULK complex [1]
- Comparative microarray analysis identified ATF3, RAD21, and caspase3 as potential ULK1 interactors. Western blot verification showed that LYN-1604 HCl upregulated the expression of ATF3 and cleaved-caspase3, and downregulated the expression of RAD21; these regulatory effects were abolished 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].

---

1. Antitumor activity in triple-negative breast cancer (TNBC) xenograft models: Nude mice bearing MDA-MB-231 xenografts (n=6 per group) were treated with LYN-1604 HCl or vehicle. Compared with the vehicle group, LYN-1604 HCl significantly reduced tumor volume and tumor weight (p<0.001, p<0.05) [1]
2. Regulation of autophagy and apoptosis-related proteins in tumor tissues:
- Immunohistochemistry of tumor tissues showed that the positive ratio of p-ULK1 (Ser317) in the LYN-1604 HCl treatment group was significantly higher than that in the vehicle group (p<0.001) [1]
- Western blot analysis of tumor tissues revealed that LYN-1604 HCl upregulated the expression of ULK1, p-ULK1 (Ser317), LC3-II, and Beclin-1, and promoted the cleavage of PARP, which was consistent with the in vitro regulatory effects on autophagy and apoptosis [1]
3. In vivo safety performance: During the treatment period, there was no significant difference in the relative body weight, liver index, spleen index, or kidney index between the LYN-1604 HCl treatment group and the vehicle group, indicating good in vivo tolerability of LYN-1604 HCl [1]

1. ULK1 kinase activity assay (ADP-Glo assay):
- Purified wild-type or mutant ULK1 kinase was incubated with LYN-1604 HCl at different concentrations in a reaction buffer. After a specific incubation time, ADP-Glo reagent was added to terminate the kinase reaction and convert the generated ADP into ATP. Luminescent reagent was then added, and the luminescence intensity (RLU) was measured to quantify the kinase activity. The blank group (without ULK1 kinase) was set as 0% kinase activity, and the control group (without LYN-1604 HCl) was set as 100% kinase activity. The EC₅₀ value of LYN-1604 HCl for activating ULK1 was calculated by fitting the dose-response curve of normalized kinase activity [1]
2. In vitro ULK1 substrate phosphorylation assay:
- Wild-type or mutant ULK1 (K50A, L53A, Y89A) was expressed and purified from HEK-293T cells, and purified mATG13 protein was used as the substrate. The phosphorylation reaction was carried out in the presence or absence of LYN-1604 HCl. After the reaction, western blot analysis was performed using a specific antibody against phosphorylated mATG13 (Ser318) to detect the phosphorylation level of mATG13. The loading amounts of ULK1 (enzyme) and mATG13 (substrate) were verified by western blot with corresponding antibodies to ensure equal input [1]
3. Surface plasmon resonance (SPR) binding assay:
- Wild-type or mutant ULK1 protein was immobilized on a Biacore sensor chip. LYN-1604 HCl solutions of different concentrations were injected into the chip channel at a constant flow rate. The SPR signal (response unit, RU) was recorded in real time to obtain the binding curve. The binding affinity (KD value) between LYN-1604 HCl and ULK1 was calculated by fitting the binding curve using Biacore evaluation software [1]
4. Molecular dynamics simulation of ULK1-LYN-1604 HCl binding:
- The initial binding model of LYN-1604 HCl and wild-type/mutant ULK1 was constructed based on the crystal structure of ULK1. A 10ns molecular dynamics simulation was performed using simulation software under physiological conditions. The stability of the binding conformation was evaluated by analyzing the root mean square deviation (RMSD) of the protein-ligand complex. The results showed that the binding conformation of LYN-1604 HCl with wild-type ULK1 and K50A/L53A mutant ULK1 was stable after 10ns simulation, while the binding conformation with Y89A mutant ULK1 was unstable [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.

---

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.

---

1. Cell viability assay (MTT assay):
- MDA-MB-231 cells were seeded in 96-well plates at a certain density and cultured overnight. LYN-1604 HCl solutions of different concentrations were added to the wells, and each concentration was set with 3 replicate wells. For the autophagy inhibition group, 1mM 3-MA was added 1h before LYN-1604 HCl treatment. After 24h of incubation, MTT reagent was added to each well and incubated for 4h. The absorbance at 490nm was measured using a microplate reader, and the cell viability was calculated as the percentage of the absorbance of the treatment group to the control group (without LYN-1604 HCl). The IC₅₀ value was calculated using Prism 6.0 software based on the dose-response curve of cell viability [1]
2. Autophagic vesicle detection (MDC staining):
- MDA-MB-231 cells were seeded on coverslips or in 6-well plates and treated with LYN-1604 HCl (0.5μM, 1.0μM, 2.0μM) for 24h. After washing with PBS, MDC staining solution was added and incubated at 37°C for 30min. For fluorescence microscopy observation, cells on coverslips were mounted with anti-fluorescence quenching mounting medium, and images were captured under a fluorescence microscope to count the number of MDC-positive autophagic vesicles. For flow cytometry analysis, cells in 6-well plates were digested, resuspended in PBS, and the MDC-positive cell ratio was detected using a flow cytometer [1]
3. Autophagic flux assay:
- MDA-MB-231 cells were transfected with GFP/mRFP-LC3 plasmid using a transfection reagent. After 24h of transfection, the cells were treated with 2.0μM LYN-1604 HCl alone or in combination with 10nM BafA1. After 24h of treatment, the cells were fixed, and the distribution of GFP-LC3 puncta was observed under a confocal fluorescence microscope to analyze the accumulation of autophagic vesicles. At the same time, the expression levels of p62 and LC3 in the cells were detected by western blot to evaluate the effect of LYN-1604 HCl on autophagic flux [1]
4. Western blot analysis for protein expression:
- MDA-MB-231 cells were treated with LYN-1604 HCl at specified concentrations and times, or transfected with siRNA (control, ULK1, or ATG5) followed by LYN-1604 HCl treatment. The cells were lysed with RIPA lysis buffer to extract total protein. The protein concentration was determined using a BCA protein assay kit. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk, then incubated with primary antibodies against ULK1, p-ULK1 (Ser317/Ser757), mATG13, FIP200, ATG101, ATG5, Beclin-1, p62, LC3, caspase3, PARP, ATF3, RAD21, and β-actin (internal reference) overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. The protein bands were visualized using an ECL chemiluminescence kit, and the band intensity was quantified using ImageJ software [1]
5. Immunocytochemistry:
- MDA-MB-231 cells were seeded on coverslips and treated with 2.0μM LYN-1604 HCl for 24h, or transfected with control/siULK1 followed by LYN-1604 HCl treatment. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA. Primary antibodies against p-ULK1 (Ser317) or LC3B were added and incubated overnight at 4°C. After washing, TRITC-conjugated (for p-ULK1) or FITC-conjugated (for LC3B) secondary antibodies were added and incubated for 1h at room temperature. The cell nuclei were stained with DAPI. The coverslips were mounted, and the protein expression and distribution were observed under a fluorescence microscope. The number of LC3B puncta per cell was counted to evaluate autophagy levels [1]
6. Apoptosis assay (Annexin-V/PI double staining):
- MDA-MB-231 cells were treated with 2.0μM LYN-1604 HCl for different times (0h, 12h, 24h, 48h). For the autophagy inhibition group, 1mM 3-MA was added 1h before LYN-1604 HCl treatment. The cells were digested with trypsin (without EDTA), washed with cold PBS, and resuspended in binding buffer. Annexin-V-FITC and PI staining solutions were added sequentially and incubated in the dark for 15min. The apoptosis ratio (early apoptosis: Annexin-V-positive/PI-negative; late apoptosis: Annexin-V-positive/PI-positive) was detected using a flow cytometer [1]
7. siRNA transfection assay:
- MDA-MB-231 cells were seeded in 6-well plates and cultured until the confluency reached 50%-60%. Control siRNA, ULK1 siRNA, or ATG5 siRNA was transfected into the cells using a transfection reagent according to the manufacturer's instructions. After 48h of transfection, the cells were treated with 2.0μM LYN-1604 HCl for 24h. The silencing efficiency of ULK1/ATG5 and the expression of autophagy/apoptosis-related proteins were detected by western blot. The effect of siRNA transfection on LYN-1604 HCl-induced autophagy was observed by immunocytochemistry (LC3B puncta), and the effect on cell viability was detected by MTT assay [1]
8. Microarray analysis for ULK1-regulated genes:
- MDA-MB-231 cells were divided into three groups: control group, ULK1 siRNA transfection group, and pcDNA3.1-ULK1 overexpression group. After 48h of transfection, total RNA was extracted from the cells using Trizol reagent. The RNA quality and integrity were verified using a NanoDrop spectrophotometer and agarose gel electrophoresis. The RNA was labeled and hybridized to a gene expression microarray. The differentially expressed genes (DEGs) were screened with the criteria of |log2 fold change| >1 and p<0.05. The DEGs with opposite expression trends between the ULK1 silencing group and the overexpression group were identified as core ULK1-regulated genes. Among these genes, ATF3, RAD21, and caspase3 were selected for verification by western blot to confirm their regulation by ULK1 and LYN-1604 HCl [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.

---

1. Establishment of TNBC xenograft model:
- MDA-MB-231 cells in the logarithmic growth phase were harvested and resuspended in a mixture of serum-free medium and Matrigel (volume ratio 1:1) to a final concentration of 5×10⁶ cells/mL. Female nude mice (4-6 weeks old) were used, and 0.2mL of the cell suspension was injected subcutaneously into the right flank of each mouse. The mice were raised under specific pathogen-free (SPF) conditions, and the tumor volume was measured every 3 days using calipers. When the tumor volume reached approximately 100mm³, the mice were randomly divided into the vehicle group and the LYN-1604 HCl treatment group (n=6 per group) [1]
2. Drug administration and sample collection:
- LYN-1604 HCl was dissolved in a suitable vehicle (the specific solvent was not explicitly stated in the literature) to prepare drug solutions of different doses. The drug was administered to the mice via an unspecified route (the literature did not clearly state the administration route) at a certain frequency (the administration frequency was not explicitly mentioned) for a total treatment duration of 21 days. During the treatment period, the body weight of the mice was measured every 3 days to monitor the general condition. After the treatment, the mice were euthanized by cervical dislocation. The tumors were excised and weighed, and the tumor volume was calculated using the formula: tumor volume = length × width² / 2. The liver, spleen, and kidneys were also excised, weighed, and the organ indices (organ weight/body weight × 100%) were calculated. Part of the tumor tissue was fixed in 4% paraformaldehyde for immunohistochemistry, and the remaining part was stored at -80°C for western blot analysis [1]
3. Immunohistochemistry of tumor tissues:
- The paraformaldehyde-fixed tumor tissues were dehydrated, embedded in paraffin, and sectioned into 4μm-thick slices. The slices were dewaxed in xylene and rehydrated in gradient ethanol. Antigen retrieval was performed by boiling the slices in citrate buffer. The slices were blocked with 3% H₂O₂ to eliminate endogenous peroxidase activity, then blocked with 5% BSA. A primary antibody against p-ULK1 (Ser317) was added and incubated overnight at 4°C. After washing, a biotin-conjugated secondary antibody was added and incubated for 1h at room temperature. The slices were then incubated with streptavidin-peroxidase complex, and the immunoreactive signals were visualized using a DAB chromogenic kit. The slices were counterstained with hematoxylin, dehydrated, cleared, and mounted. The positive ratio of p-ULK1 was quantified by counting the number of positive cells in 10 random high-power fields (×100 magnification) using Image-Pro Plus software [1]

1. In vitro toxicity: MTS assay showed that at the tested concentration, LYN-1604 HCl only inhibited the viability of MDA-MB-231 cells (tumor cells) without affecting the viability of normal cells (the specific normal cell type was not mentioned in the literature) [1] 2. In vivo toxicity: After treatment with LYN-1604 HCl, there were no significant differences in relative body weight, liver index, spleen index, and kidney index between the treatment group and the control group. No obvious pathological changes were observed in the liver, spleen, and kidney tissues (the literature did not provide detailed pathological section results), indicating that at the tested dose, LYN-1604 HCl had no obvious acute toxicity to the major organs of mice. [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 Information: ULK1 is a key regulatory protein that initiates autophagy. TCGA database analysis and tissue microarray (TMA) analysis showed that ULK1 expression was significantly downregulated in breast cancer tissues, especially in triple-negative breast cancer (TNBC) tissues. This downregulation leads to impaired autophagy function in tumor cells, thereby promoting tumor progression. Therefore, activating ULK1-regulated autophagy is considered a potential therapeutic strategy for TNBC [1]. 2. Mechanism of Action: LYN-1604 HCl is a ULK1-specific agonist. It binds to ULK1 through LYS50, LEU53 and TYR89 residues, activating ULK1 kinase activity and promoting the formation of the ULK complex (ULK1-mATG13-FIP200-ATG101). The activated ULK complex induces ATG5-dependent autophagy, leading to autophagic death of tumor cells. Meanwhile, LYN-1604 HCl regulates the expression of ATF3, RAD21 and caspase3 through ULK1 and promotes tumor cell apoptosis by activating the caspase3-PARP signaling pathway. Therefore, LYN-1604 HCl exerts its anti-tumor effect by inducing autophagy and apoptosis in TNBC cells [1]. 3. Therapeutic potential: LYN-1604 HCl showed significant anti-tumor activity in both in vitro MDA-MB-231 cell models and in vivo TNBC xenograft models, and had good in vivo tolerance. It is a potential small molecule candidate drug for the treatment of triple-negative breast cancer [1]. 4. Chemical structure: The chemical structure of LYN-1604 HCl in the literature is shown in Figure 5A. It belongs to small molecule compounds that target ULK1 [1].

C₃₃H₄₄CL₃N₃O₂

621.08

619.249

C, 63.82; H, 7.14; Cl, 17.12; N, 6.77; O, 5.15

2216753-86-3

LYN-1604 dihydrochloride;2310109-38-5;LYN-1604;2088939-99-3

131801112

White to off-white solid powder

1

4

12

41

750

0

ClC1C=C(C=CC=1C(CN1CCN(C(CN(CC(C)C)CC(C)C)=O)CC1)OCC1C=CC2C=CC=CC=2C=1)Cl.Cl

KWFDUNOLEJSFBQ-UHFFFAOYSA-N

InChI=1S/C33H43Cl2N3O2.ClH/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;1H

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.03 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (4.03 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (4.03 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 900 μL of corn oil and mix evenly.

---

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