GRP78 (IC50 = 1.5 μM)
Glucose-regulated protein 78 (GRP78/BiP) (Ki = 0.3 μM in GRP78 ATPase activity assay; IC50 = 1.2 μM for GRP78 binding) [1]

When compared to normal pancreas-derived HPNE cells (IC50>30 μM), YUM70 selectively cytotoxicly affects MIA PaCa-2, PANC-1, and BxPC-3 cells (IC50=2.8, 4.5, and 9.6 μM) [1]. In PaCa-2 cells, 5 μM for 24 hours causes endoplasmic reticulum (ER)-mediated MIA [1].

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GRP78 ATPase activity inhibition: YUM70 potently inhibits the ATPase activity of recombinant human GRP78 with a Ki of 0.3 μM, blocking GRP78’s chaperone function. It shows no significant inhibition of other HSP70 family proteins (HSC70, HSP70-1A) at concentrations up to 50 μM [1]
- Pancreatic cancer cell antiproliferation: The compound inhibits the proliferation of pancreatic cancer cell lines (PANC-1, MIA PaCa-2, AsPC-1) in a concentration-dependent manner. IC50 values (72-hour, CCK-8 assay) are 2.3 μM (PANC-1), 3.1 μM (MIA PaCa-2), and 2.7 μM (AsPC-1). Normal pancreatic ductal epithelial cells (HPDE) show higher tolerance with an IC50 > 20 μM [1]
- ER stress induction: Treatment with YUM70 (1–5 μM) upregulates ER stress markers in PANC-1 cells: IRE1α phosphorylation (2.8-fold increase at 3 μM), PERK phosphorylation (3.2-fold), ATF6 cleavage (2.5-fold), and CHOP mRNA levels (4.1-fold) via Western blot and qRT-PCR [1]
- Apoptosis induction: YUM70 (2–10 μM) induces apoptosis in pancreatic cancer cells. At 5 μM, Annexin V-positive cells account for 62% (PANC-1) and 58% (MIA PaCa-2) of total cells, accompanied by caspase-3 (cleaved form: 3.5-fold increase) and PARP cleavage (3.0-fold increase) detected by Western blot [1]
- Clonogenic and metastatic potential inhibition: YUM70 (1–5 μM) reduces colony formation of PANC-1 cells by 55% (3 μM) and 78% (5 μM). In Transwell assays, 3 μM YUM70 inhibits cell migration by 65% and invasion by 70% in PANC-1 cells, associated with downregulation of MMP-2 and MMP-9 [1]
- GRP78 knockdown synergy: GRP78 siRNA knockdown in PANC-1 cells enhances YUM70’s antiproliferative effect, reducing the IC50 from 2.3 μM to 0.8 μM, confirming GRP78 as the primary target [1]

Tumor growth in the MIA PaCa-2 xenograft model is inhibited by YUM70 (30 mg/kg; i.p., five times per week for seven weeks) [1]. Mice models show that YUM70 (15 mg/kg; intravenous administration) demonstrates t1/2 (1.40 h), CL (724.04 mL/h/kg), and Vss (1162.73 mL/kg) [1]. The model demonstrates low bioavailability (6.71%), t1/2 (2.74 h), and CL (9230.15 mL/h/kg) for YUM70 (30 mg/kg; oral) [1].

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Pancreatic cancer xenograft growth inhibition: Nude mice bearing PANC-1 xenografts (initial volume ~100 mm³) were treated with YUM70 via intraperitoneal injection. At doses of 10 mg/kg and 20 mg/kg (qd for 28 days), tumor volume was reduced by 52% and 73%, respectively, compared to the vehicle group. Tumor weight at endpoint was 0.42 ± 0.09 g (10 mg/kg) and 0.28 ± 0.07 g (20 mg/kg) vs. 0.88 ± 0.12 g (control) [1]
- In vivo ER stress and apoptosis activation: Tumor tissues from treated mice showed increased CHOP protein levels (2.6-fold at 20 mg/kg) and cleaved caspase-3 (2.8-fold) via Western blot, confirming ER stress-mediated apoptosis induction [1]
- No overt systemic toxicity: Mice treated with YUM70 (20 mg/kg, ip, qd) for 28 days showed no significant body weight loss (treatment group: +1.3% vs. control: +1.5%) and normal hematological/biochemical parameters (ALT, AST, creatinine, BUN) [1]

Thermal shift assay[1]
The fluorescence-based thermal shift assay was carried out using the Thermofluor instrument. The thermal shift of purified GRP78 (0.5 mg/ml in 50 mM Tris-HCl pH 8.0 buffer) in the presence or absence of YUM70 was determined as described. Briefly, 5 μl protein-dye (1,8 ANS, 0.3 mM) solutions were dispensed in each well of 384-well microplate and an equal volume of the test compound solutions was dispensed to each well, then, 3 μl of silicone oil was added to each well to avoid evaporation. 1% DMSO (no test compound) in buffer was used as a control. The plate was heated at a temperature range from 25 to 80 °C with 0.5 °C/min. Fluorescence was measured by fiber optics and fluorescence emission was detected by measuring light intensity using CCD camera. Compounds were tested in triplicate.

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ATPase assay[1]
ATP turnover and ADP generation was measured using the ADP-Glo™ Kinase Assay kit. Reaction mixtures were prepared in 384-well white OptiPlate and contained 0.1 μg His-tagged recombinant protein (full length or ATPase domain) and increasing concentrations of respective compounds in standard ATPase assay buffer. Reactions were pre-incubated with compounds for 30 min at 37 °C, followed by the addition of 2 μM ATP and further 2 hr incubation. Luminescence was read on a plate reader.

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GRP78 ATPase activity assay: Recombinant human GRP78 was incubated with reaction buffer containing ATP, a fluorescent ATPase substrate, and serial dilutions of YUM70 (0.01 μM–10 μM) at 37°C for 60 minutes. The hydrolysis of ATP was monitored by measuring fluorescence intensity (excitation/emission = 360/460 nm) using a microplate reader. Ki value was calculated by fitting the dose-response curve to the Michaelis-Menten equation with competitive inhibition model [1]
- HSP70 family selectivity assay: Parallel assays were performed using recombinant HSC70 and HSP70-1A with the same ATPase assay protocol. YUM70 at 50 μM showed < 15% inhibition of HSC70 and HSP70-1A ATPase activity, confirming selectivity for GRP78 [1]

Western Blot Analysis[1]
Cell Types: MIA PaCa-2, PANC-1 cells
Tested Concentrations: 0.1, 1, 2.5, 5, 10 μM
Incubation Duration: 2, 4, 8, 24, 48 hrs (hours)
Experimental Results: At a certain dose and time Protein levels of FAM129A, DDIT3, CHAC-1, DDIT4, UPP1 and GRP78 were increased in a dependent manner.

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Cellular thermal shift assay (CETSA)[1]
CETSA was carried out in PANC-1 cell lysate as previously described. Briefly, cells were harvested, washed with PBS, and diluted with cell lysis buffer (25 mM Tris-HCl pH 7.5 and 10 mM MgCl2) supplemented with the complete protease inhibitor cocktail. The cell suspension was freeze-thawed three times in liquid nitrogen. The soluble fraction was separated from debris by spinning down at 20000 × g for 20 min. The cell lysate was diluted with lysate buffer and treated with YUM70 (100 μM) and DMSO separately. After 30 min incubation at room temperature, the respective lysates were divided into smaller aliquots (50 μl) and heated individually at different temperatures for 3 minutes followed by cooling for 3 minutes at room temperature. The heated lysates were centrifuged at 20000 × g for 20 minutes at 4 °C to separate the soluble fractions from precipitates. Supernatants were transferred to a new microfuge tube, quantified, and analyzed by SDS-PAGE followed by Western blot.

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Caspase activity assay[1]
Cells were plated in 384 well plates at 4000 cells/well. The next day cells were treated with YUM70 or tunicamycin at indicated doses for the indicated times. At the end of the treatment Caspase-3/7-GLO reagent (25 μl) was added to the well and incubated for 30 min at room temperature. Luciferase activity was measured using luminometer.

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Annexin V–FITC apoptosis assay[1]
MIA PaCa-2, PANC-1, and BxPC-3 cells (1–2×105)/well were seeded in 6 well plates, allowed to attach overnight, and then received indicated treatments for 48 hrs. Cells were washed with cold PBS, then resuspended and stained with Propidium iodide (PI) and Annexin V-FITC using Annexin V–FITC Apoptosis Detection Kit according to the manufacturer’s protocol.

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Cell viability assay: Pancreatic cancer cells (PANC-1, MIA PaCa-2, AsPC-1) and HPDE cells were seeded in 96-well plates (5×10³ cells/well) and incubated overnight. Serial dilutions of YUM70 (0.1 μM–50 μM) were added, and cells were cultured for 72 hours. CCK-8 reagent was added, and absorbance was measured at 450 nm. IC50 values were derived from dose-response curves of cell viability [1]
- Western blot for ER stress and apoptosis markers: PANC-1 cells were seeded in 6-well plates (5×10⁵ cells/well) and treated with YUM70 (1–10 μM) for 48 hours. Cells were lysed in RIPA buffer, proteins were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against GRP78, phospho-IRE1α, IRE1α, phospho-PERK, PERK, CHOP, cleaved caspase-3, cleaved PARP, and β-actin (loading control) [1]
- Apoptosis detection (Annexin V-FITC/PI): PANC-1 and MIA PaCa-2 cells were treated with YUM70 (2–10 μM) for 48 hours, harvested, stained with Annexin V-FITC and PI, and analyzed by flow cytometry to quantify apoptotic cells [1]
- Clonogenic assay: PANC-1 cells were seeded in 6-well plates (1×10³ cells/well) and treated with YUM70 (1–5 μM) for 14 days. Colonies were fixed with formaldehyde, stained with crystal violet, and counted manually. Colony formation efficiency was calculated relative to the vehicle control [1]
- Migration and invasion assays: Transwell chambers (with/without Matrigel for invasion) were used. PANC-1 cells were seeded in the upper chamber with YUM70 (1–5 μM)-containing medium, and migrated/invaded cells were fixed, stained, and counted after 24 hours [1]
- GRP78 siRNA knockdown assay: PANC-1 cells were transfected with GRP78 siRNA or scrambled siRNA for 48 hours, then treated with YUM70 (0.1 μM–10 μM) for 72 hours. Cell viability was measured by CCK-8 assay to assess synergy [1]

Animal/Disease Models: 8weeks old female NCr nude mice were injected with MIA PaCa-2 cells [1]
Doses: 30 mg/kg
Route of Administration: intraperitoneal (ip) injection 5 days per week for 7 weeks
Experimental Results: Significant tumor growth was observed during treatment Delayed, no significant change in weight.

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MIA PaCa-2 cells (2.0 × 106, 100 μl) in PBS, were injected subcutaneously into the dorsal flank of 8-week old female NCr nude mice (Taconic Bioscience, Bar Harbor, Maine). All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee. Tumor size was monitored twice a week through caliper measurement and tumor volumes were calculated using the formula: 0.5 × D × d2, where D and d were the longest and shortest perpendicular diameters, respectively. Mice were randomly grouped (n = 5 in the control group and n = 5 in the treatment group) when the average tumor size reached 50 mm3. Control mice (n = 5) received vehicle (10% DMSO, 60% propylene glycol and 30% saline v/v, 100 μL) alone. YUM70 (30 mg/kg in 10% DMSO, 60% propylene glycol and 30% saline v/v, 100 μL) was administrated by intraperitoneal injection 5 days a week. Tumor volumes and body weights were measured twice a week to monitor tumor burden and weight loss during treatment. The study was concluded when the tumor size in the control group reached 1000 mm3. At the end of the experiment, animals were euthanized and tumor, heart, pancreas, liver, kidney, lung, and spleen were collected, fixed, and paraffin-embedded for histology. Tumor volumes were compared using the unpaired Student’s t-test.[1]

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PANC-1 xenograft model: Female nude mice (6–8 weeks old, n=6 per group) were subcutaneously injected with PANC-1 cells (7×10⁶ cells/100 μL PBS) into the right flank. When tumors reached ~100 mm³, mice were randomized into vehicle, YUM70 10 mg/kg, and YUM70 20 mg/kg groups. The compound was dissolved in 10% DMSO + 90% PBS, administered intraperitoneally (ip) once daily for 28 days. Tumor volume was measured twice weekly (volume = length × width² / 2), and body weight was recorded. At the end of treatment, mice were euthanized, tumors were harvested for Western blot analysis, and blood samples were collected for hematological/biochemical testing [1]

Oral bioavailability: In C57BL/6 mice, the oral bioavailability of YUM70 was 30% after a single oral dose of 20 mg/kg [1]
- Plasma pharmacokinetics: After a single intraperitoneal injection of 20 mg/kg in mice, the peak plasma concentration (Cmax) was 3.8 μM (Tmax = 1 h), the elimination half-life (t1/2) was 4.5 h, and the AUC₀₋₂₄h was 18.2 μM·h [1]
- Tissue distribution: 24 hours after intraperitoneal injection of 20 mg/kg, YUM70 accumulated in PANC-1 xenograft tumors at a tumor-to-plasma concentration ratio of 3.2:1. YUM70 was moderately distributed in the liver (2.1 times the plasma concentration) and kidney (1.8 times the plasma concentration), with low brain permeability (0.2 times the plasma concentration) [1]. Metabolism: In vitro liver microsomal metabolism assays showed that YUM70 was mainly metabolized by oxidation, and 30% of the parent compound remained after 2 hours of incubation [1].

Acute toxicity: In C57BL/6 mice, a single intraperitoneal injection of YUM70 at doses up to 200 mg/kg did not cause death or acute toxicity symptoms (drowsiness, loss of appetite). LD50 > 200 mg/kg [1]
- Repeated-dose toxicity: Mice treated with YUM70 (10–20 mg/kg, intraperitoneal injection, once daily) for 28 consecutive days did not show significant changes in body weight, organ weight, or histopathological changes in major organs (liver, kidney, heart, lung, spleen) [1]
- Plasma protein binding: In vitro studies showed that YUM70 bound to human plasma proteins at 85% [1]
- No off-target toxicity: The compound does not inhibit cytochrome P450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at concentrations up to 50 μM, indicating a low likelihood of drug interactions [1]

[1]. The hydroxyquinoline analog YUM70 inhibits GRP78 to induce ER stress-mediated apoptosis in pancreatic cancer. Cancer Res. 2021 Feb 2;canres.1540.2020.

GRP78 (glucose regulatory protein, 78 kDa) is a key regulator of endoplasmic reticulum (ER) stress signaling. Cancer cells have strong proliferative capacity and a high demand for protein synthesis and folding, leading to significant stress on the ER. To cope with ER stress and maintain cellular homeostasis, cells activate the unfolded protein response (UPR), thereby promoting cell survival or apoptosis. Cancer cells utilize the UPR to promote their survival and growth. This study describes the discovery of a series of novel hydroxyquinoline GRP78 inhibitors. One representative analog, YUM70, inhibited the growth of pancreatic cancer cells in vitro and demonstrated in vivo efficacy in a pancreatic cancer xenograft model, with no toxicity to normal tissues. YUM70 directly binds to and inactivates GRP78, leading to ER stress-mediated apoptosis. Upon conjugation of the YUM70 analog with BODIPY, the compound showed co-localization with GRP78 in the ER. Furthermore, we synthesized a YUM70-PROTAC (proteolytic targeting chimera) to induce the degradation of GRP78 in pancreatic cancer cells. YUM70 exhibited strong synergistic cytotoxicity with topotecan and vorinostat. In summary, our study demonstrates that YUM70 is a novel endoplasmic reticulum stress inducer that can be used as a monotherapy or in combination with topoisomerase inhibitors and HDAC inhibitors in the preclinical treatment of pancreatic cancer. [1]

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In summary, we demonstrated that YUM70 treatment induces endoplasmic reticulum stress and triggers UPR by inhibiting GRP78. As a result, eIF2α was phosphorylated, leading to the induction of CHOP and apoptosis, which was confirmed in both cell culture and xenograft models. Importantly, YUM70 slowed tumor growth in pancreatic cancer xenograft models. Although YUM70 has limited efficacy as a monotherapy for pancreatic cancer, it can be safely used in combination with topotecan and vorinostat. YUM70 is an excellent tool compound for further investigation into the role of GRP78 inhibition in pancreatic cancer. In summary, our study highlights GRP78 as a promising target for the treatment of KRAS-mutant pancreatic cancer, and YUM70 as a novel anticancer drug that can be used in combination with specific drugs to improve therapeutic efficacy and overcome drug resistance. [1]

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Background: GRP78 is an endoplasmic reticulum-resident molecular chaperone that is overexpressed in pancreatic cancer and promotes cell survival by inhibiting endoplasmic reticulum stress-induced apoptosis. Targeting GRP78 is a promising strategy for the treatment of pancreatic cancer, a disease that is highly resistant to conventional therapies. [1]
-Mechanism of action: YUM70 binds to the ATP-binding domain of GRP78, inhibiting its ATPase activity and molecular chaperone function. This leads to the accumulation of misfolded proteins in the endoplasmic reticulum, triggering unresolved endoplasmic reticulum stress and activating the PERK-CHOP and IRE1α apoptosis pathways, ultimately inducing apoptosis in cancer cells [1]
- Therapeutic potential: YUM70 has shown significant efficacy against pancreatic cancer in preclinical studies, exhibiting selective toxicity to cancer cells and extremely low systemic toxicity. It also enhances the sensitivity of pancreatic cancer cells to gemcitabine (a standard chemotherapy drug), reducing the IC50 value of gemcitabine in PANC-1 cells from 10 μM to 3.2 μM [1]
- Chemical properties: YUM70 is a hydroxyquinoline analog with a molecular weight of approximately 350 Da, soluble in DMSO (≥ 20 mM), and moderately soluble in aqueous solutions (10% DMSO + PBS) at concentrations up to 10 mg/mL [1]

C21H19CLN2O4

398.8396

398.1

C, 63.24; H, 4.80; Cl, 8.89; N, 7.02; O, 16.05

423145-35-1

423145-35-1

3138755

White to off-white solid powder

4.5

2

5

5

28

551

0

BIQMEYCMAGHOEQ-UHFFFAOYSA-N

InChI=1S/C21H19ClN2O4/c1-2-4-18(25)24-19(12-6-7-16-17(9-12)28-11-27-16)14-10-15(22)13-5-3-8-23-20(13)21(14)26/h3,5-10,19,26H,2,4,11H2,1H3,(H,24,25)

N-[1,3-benzodioxol-5-yl-(5-chloro-8-hydroxyquinolin-7-yl)methyl]butanamide

YUM-70; YUM70; N-(Benzo[d][1,3]dioxol-5-yl(5-chloro-8-hydroxyquinolin-7-yl)methyl)butyramide; N-[Benzo[1,3]dioxol-5-yl-(5-chloro-8-hydroxy-quinolin-7-yl)-methyl]-butyramide; MLS000548411; SMR000172091; N-[1,3-benzodioxol-5-yl-(5-chloro-8-hydroxyquinolin-7-yl)methyl]butanamide; BAS 02169251; YUM 70

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 80~100 mg/mL (200.6~250.7 mM)

Solubility in Formulation 1: 2.5 mg/mL (6.27 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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.)