BiP/GRP78/HSPA5
BiP (GRP78/HSPA5) – binds to and inhibits BiP ATPase activity; no IC50/Ki/EC50 values provided in these references. [1][2][3]

HA15 (10 μM; 1-24 hours)auses an early endoplasmic reticulum stress (ER Stress) [1].
HA15 (0-10μM; 24 hours) has an IC50 of 1-2.5 μM in A375 cells and reduces melanoma cell viability in a dose-dependent manner when compared to control conditions (DMSO)[1].
HA15 (1-10 μM; 24 hours) causes apoptosis in A375 cells[1].
HA15 (1-24 μM; 24 hours) induces autophagy[1].
HA15 (10 μM; 48 hours) exhibits a high degree of efficacy in causing ER stress and cell death in melanoma cells that are resistant to BRAF inhibitors. Furthermore, through apoptotic and autophagic pathways triggered by ER stress, HA15 prevents the growth of tumors[1].
HA15 at low or high doses did not negatively impact the viability of human fibroblasts or normal human melanocytes[1].

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HA15 (10 μM, 48 h) decreased viability of human melanoma cell lines (A375, SKMel28, Mel501 etc.) with IC50 between 1–2.5 μM; no cytotoxicity on normal human melanocytes or fibroblasts at up to 100 μM. [1]
HA15 (10 μM) induced rapid ER stress in melanoma cells (A375): phosphorylation of PERK and eIF2α within 1 h, increased ATF4 and CHOP, XBP1 splicing, and dilated ER cisternae (electron microscopy). Co‑localized with ER Tracker (82% overlap) and with BiP (64% overlap). Inhibited BiP ATPase activity in a dose‑dependent manner (0–100 μM) in a cell‑free assay. [1]
HA15 induced apoptosis (annexin V, cleaved caspase‑3/9, PARP) and autophagy (LC3B‑II conversion, Beclin1 increase, autophagic vacuoles) in A375 cells. CHOP knockdown blocked both apoptosis and autophagy, and rescued cell viability. [1]
HA15 (10 μM, 48 h) decreased viability of BRAF inhibitor‑resistant melanoma cells (A375‑R, SKMel28‑R, WM9‑R) and patient‑derived resistant cells, with increased CHOP expression. [1]
HA15 (10 μM, 48 h) killed various other cancer cell lines: MIA PaCa‑2 (pancreas, resistant to gemcitabine), ImarR (myeloid leukemia, resistant to imatinib), MDA‑MB‑453 (breast), HT29 (colon), HCT116, etc. Induced CHOP and PARP cleavage in all; LC3B conversion varied. [1]
HA15 (IC50 for proliferation 3.8 μM in H295R adrenocortical carcinoma cells) reduced cell viability and proliferation in a dose‑dependent manner. Synergized with mitotane (combination index <1). Induced DDIT3 mRNA, XBP1 splicing, CHOP protein, and ATF6 nuclear translocation. Reduced forskolin‑stimulated cortisol and DHEA‑S production via downregulation of steroidogenic enzymes (HSD3B2, CYP11A1, CYP21A2, CYP17A1). [2]
HA15 (IC50 ranging 5.71–20.51 μM in MPM cell lines) selectively suppressed viability of MPM cells (MESO‑1, MESO‑4, H28, JL‑1, MSTO‑211H, H2052, primary BE261T) compared to normal fibroblasts (hFb16Lu) and mesothelial cells (Met‑5A). [3]
HA15 (20 μM) induced ER stress/UPR (increased EIF2AK3, ERN1, ATF4, DDIT3 mRNA; p‑eIF2α, CHOP protein), autophagy (ATG5, BECN1, LC3B‑II), and apoptosis (BCL2L11, BBC3, cleaved caspase‑7, PARP) in MPM cells. CHOP knockdown attenuated these effects and rescued viability. [3]
HA15 (20 μM) effectively reduced viability of chemo‑resistant MPM cells (MESO‑1R, MESO‑4R, H28R) generated by chronic exposure to cisplatin/pemetrexed, and increased UPR and apoptotic markers similar to parental cells. [3]

HA15 (0.7 mg/mouse/day; i.h.; over 2 weeks) prevents the growth of melanoma tumors in mice, causes no discernible toxicity, and has no effect on their behavior, body mass, or liver mass, indicating the lack of hepatomegaly[1].
HA15 (0.7 mg/mouse; i.p.; 5 days/week) inhibits the growth of MPM tumors in vivo[3].
HA15 (0.7 mg/mouse/day i.p. for 14 days) significantly inhibited growth of A375 melanoma xenografts in nude mice, comparable to PLX4032 (0.7 mg). Tumor weight reduced. No change in body weight, liver weight, or serum AST/ALT. TUNEL, CHOP, and LC3B staining increased in tumors. [1]
HA15 (0.7 mg/mouse/day i.p., 5 days/week for 5 weeks) suppressed growth of BRAF inhibitor‑resistant A375 xenografts in nude mice, while PLX4032 had no effect. [1]
HA15 (0.7 mg/mouse i.p., 5 days/week for 5 weeks) inhibited growth of patient‑derived xenograft (BE261T) in NSG mice, with greater effect than cisplatin/pemetrexed (3.75/83 mg/kg i.p. weekly). No significant body weight loss. Normal liver histology and AST/ALT levels. Tumor tissues showed increased CHOP, Beclin‑1, LC3B‑II, cleaved PARP/caspase‑3/caspase‑7. [3]

BiP ATPase activity inhibition assay: Recombinant human BiP (0.25 μM) was incubated with HA15 (0–100 μM) in 20 mM Tris (pH 7.5), 50 mM KCl, 1.5 mM MgCl₂. The reaction was started by adding 100 μM ATP (30 min, 37 °C). Liberated free phosphate was measured by malachite green‑phosphate assay (absorbance 620–640 nm). HA15 dose‑dependently inhibited BiP ATPase activity. [1]

Cell Line: A375 cells
Concentration: 1 μM,2.5 μM,5 μM,7.5 μM,10 μM
Incubation Time: 24 hours
Result: Decreased melanoma cell viability in a dose-dependent manner compared with control conditions (DMSO) in A375 cells.

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Cell viability assays: MTT, acid phosphatase (APH), or trypan blue exclusion. Cells seeded in 96‑well plates, treated with HA15 for 24–72 h. IC50 calculated. [1][2][3]
Clonogenic assay: Cells seeded in 6‑well plates (1000/well), treated with HA15 for 14 days (refresh every 3 days), colonies stained with crystal violet and quantified. [1][3]
Apoptosis assay: Annexin V‑FITC/PI staining followed by flow cytometry; or detection of cleaved caspase‑3, ‑7, ‑9, PARP by Western blot. [1][2][3]
Western blot: Proteins extracted in RIPA buffer, separated by SDS‑PAGE, transferred to PVDF/nitrocellulose, probed with antibodies against BiP, PERK, p‑eIF2α, ATF4, CHOP, IRE1α, LC3B, Beclin‑1, cleaved caspase‑7, PARP, β‑actin, etc. [1][2][3]
qRT‑PCR: RNA extracted with RNeasy, cDNA synthesis, TaqMan or SYBR Green assays for UPR, autophagy, and apoptosis genes. Normalized to GAPDH or TBP. [1][2][3]
XBP1 splicing assay: RT‑PCR using primers flanking the splice site, products resolved on 4% agarose gel. [1][2]
Immunofluorescence: Cells fixed, permeabilized, stained with anti‑BiP, anti‑LC3B, anti‑ATF6, or ER Tracker, and DAPI; confocal microscopy. [1][2]
Electron microscopy: Cells fixed with glutaraldehyde/osmium tetroxide, embedded, sectioned, examined for ER dilation and autophagic vacuoles. [1]

6-weeks female BALB/c nu/nu (nude) mice with A375 melanoma cells xenograft[1]
0.7 mg/mouse/day
Subcutaneous injection; over a period of 2 weeks

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Mouse xenograft (melanoma, reference 1): Female athymic nude mice (6 weeks). A375 cells (1.5×10⁶) injected subcutaneously. After 8 days (tumor ~50 mm³), treated with vehicle (Labrafil), PLX4032 (0.7 mg/mouse/day), or HA15 (0.7 mg/mouse/day) i.p. once daily for 14 days. Tumor volume measured every 3–4 days. At endpoint, tumors weighed, blood collected for AST/ALT, liver fixed for H&E. [1]
Mouse xenograft (BRAF‑resistant melanoma): Similar protocol with A375‑R cells (1.5×10⁶), treatment started at day 7. [1]
Mouse PDX (MPM, reference 3): NSG mice (NOD‑scid IL2Rγnull). BE261T primary MPM cells (1×10⁶) mixed with Matrigel injected subcutaneously. When tumors palpable, mice randomized to: vehicle (control), cisplatin (3.75 mg/kg) + pemetrexed (83 mg/kg) i.p. once weekly for 5 weeks, or HA15 (0.7 mg/mouse) i.p. 5 days/week for 5 weeks. Tumor volume measured every 3 days. At endpoint, blood for AST/ALT, liver and tumor histology, IHC for caspase‑3. [3]

HA15 showed no apparent toxicity in mouse models: no body weight loss, no behavioral changes, normal liver histology, and normal serum AST/ALT levels at the administered doses (0.7 mg/mouse/day for 2–5 weeks). [1][3]
In vitro, HA15 up to 100 μM had no effect on viability of normal human melanocytes, fibroblasts (hFb16Lu), or mesothelial cells (Met‑5A). [1][3]
No significant changes in liver enzymes or histology in treated mice. [1][3]

[1]. Compounds Triggering ER Stress Exert Anti-Melanoma Effects and Overcome BRAF Inhibitor Resistance. Cancer Cell. 2016 Jun 13;29(6):805-19.

[2]. The GRP78/BiP inhibitor HA15 synergizes with mitotane action against adrenocortical carcinoma cells through convergent activation of ER stress pathways. Mol Cell Endocrinol. 2018 Oct 15;474:57-64.

[3]. Endoplasmic Reticulum Stress Signaling as a Therapeutic Target in Malignant Pleural Mesothelioma. Cancers (Basel). 2019 Oct; 11(10): 1502.

HA15 is a sulfonamide formed by the condensation of the sulfonic acid group of 5-(dimethylamino)naphthalene-1-sulfonic acid with the aniline nitrogen of 3-(2-acetamido-1,3-thiazolyl)aniline. It has been reported that HA15 can reduce the viability of melanoma cells by triggering endoplasmic reticulum (ER) stress response, without toxicity to normal cells. It possesses antitumor activity. HA15 is a sulfonamide compound belonging to the 1,3-thiazole, acetamide, tertiary amine, aminonaphthalene, and biaryl groups.

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HA15 is a thiazole benzenesulfonamide that targets BiP/GRP78, an ER chaperone. It induces ER stress by dissociating BiP from PERK, IRE1α, and ATF6, leading to persistent UPR activation. This triggers both apoptosis and autophagy in cancer cells. HA15 is effective against melanoma (including BRAF inhibitor‑resistant), adrenocortical carcinoma (synergizes with mitotane), and malignant pleural mesothelioma (including chemo‑resistant). It has low toxicity to normal cells and in vivo. These studies support further clinical investigation. [1][2][3]

C23H22N4O3S2

466.58

466.113

C, 59.21; H, 4.75; N, 12.01; O, 10.29; S, 13.74

1609402-14-3

73890923

Light yellow to green yellow solid powder

1.4±0.1 g/cm3

1.701

4.56

2

7

6

32

757

0

S(C1=C([H])C([H])=C([H])C2C(=C([H])C([H])=C([H])C=21)N(C([H])([H])[H])C([H])([H])[H])(N([H])C1=C([H])C([H])=C([H])C(C2=C([H])SC(N([H])C(C([H])([H])[H])=O)=N2)=C1[H])(=O)=O

LBSMEKVVMYSTIH-UHFFFAOYSA-N

InChI=1S/C23H22N4O3S2/c1-15(28)24-23-25-20(14-31-23)16-7-4-8-17(13-16)26-32(29,30)22-12-6-9-18-19(22)10-5-11-21(18)27(2)3/h4-14,26H,1-3H3,(H,24,25,28)

N-[4-[3-[[5-(dimethylamino)naphthalen-1-yl]sulfonylamino]phenyl]-1,3-thiazol-2-yl]acetamide

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 (5.36 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.)