Target: Estrogen receptor α (ERα) – noncompetitive inhibitor [1]

In Vitro: BHPI at 100–1000 nM completely inhibited proliferation of all 15 ERα+ cancer cell lines tested (breast, endometrial, ovarian) including drug‑resistant lines: 4‑OHT‑resistant ZR‑75‑1, tamoxifen/fulvestrant‑resistant BT‑474, EGF‑stimulated T47D (resistant to 4‑OHT, ICI, raloxifene), cisplatin‑resistant Caov‑3, and multidrug‑resistant OVCAR‑3. At 10 μM, BHPI had no effect on proliferation in 12 ERα− cell lines [1].
BHPI blocked anchorage‑independent growth of MCF‑7 cells in soft agar [1].
BHPI greatly reduced protein synthesis in all 14 ERα+ cell lines but not in 12 ERα− cell lines; ERα was necessary and sufficient for this effect. In MCF7ERαHA cells with doxycycline‑inducible ERα, increasing ERα level progressively increased BHPI inhibition of protein synthesis [1].
BHPI rapidly increased eIF2α phosphorylation (within 30 min) and activated PERK, ATF6α, and IRE1α arms of the UPR (increased p‑PERK, cleaved p50‑ATF6α, spliced XBP1 mRNA). PERK knockdown or dominant‑negative eIF2α‑S51A mutation abolished rapid inhibition of protein synthesis [1].
BHPI induced a large and sustained increase in intracellular Ca2+ (Fluo‑4 AM imaging), depleting ER Ca2+ stores via opening of IP3R calcium channels. 2‑APB (IP3R blocker) or IP3R siRNA abolished the Ca2+ increase and protein synthesis inhibition [1].
BHPI rapidly activated PLCγ (increased PLCγ‑Tyr783 phosphorylation), producing a large increase in IP3 levels (much higher than E2). siRNA knockdown of PLCγ abolished Ca2+ increase and protein synthesis inhibition [1].
BHPI depletes intracellular ATP, activates AMPK (increased p‑AMPKα‑Thr172, p‑AMPKβ‑Ser108, p‑ACC‑Ser79). Thapsigargin (SERCA inhibitor) prevented ATP depletion and AMPK activation [1].
BHPI induces eEF2 phosphorylation (Thr56) after ~2 h, blocking translation elongation. It transiently activates ERK1/2 and mTORC1‑p70S6K, leading to eEF2K‑Ser366 phosphorylation which delays eEF2 phosphorylation initially. BHPI blocks induction of chaperones BiP and p58IPK protein (despite increased mRNA), resulting in sustained eIF2α phosphorylation and sustained protein synthesis inhibition [1].
BHPI inhibits E2‑ERα‑regulated gene expression: it suppresses induction of pS2, GREB1, XBP1, CXCL2 mRNA and ERE‑luciferase, and blocks down‑regulation of IL1‑R1 and EFNA1 mRNA. This inhibition is noncompetitive (1,000‑fold excess E2 does not reverse it). BHPI does not compete with E2 for ERα binding, does not alter ERα protein levels or nuclear localization, but inhibits E2‑stimulated recruitment of ERα and RNA polymerase II to pS2 and GREB1 promoters (ChIP) [1].
BHPI significantly altered the fluorescence emission spectrum of purified full‑length ERα, and induced a 15‑kDa proteolytic fragment of ERα LBD upon proteinase K digestion, indicating direct interaction [1].

In Vivo: In a mouse xenograft model using MCF‑7 cells (estrogen‑supplemented), daily intraperitoneal injection of BHPI at 15 mg/kg for 10 days induced rapid and substantial regression in 48/52 tumors (average starting cross‑sectional area ~45 mm², set to 0% change). Vehicle‑treated tumors showed continued robust growth [1].
BHPI at 10 mg/kg every other day ultimately stopped tumor growth and reduced final tumor weight by ~60% compared to controls [1].
BHPI was well tolerated: BHPI‑treated and control mice exhibited similar food intake and weight gain [1].

Cell Assay: Cell proliferation was assessed by counting cells after treatment with BHPI (100–1000 nM) for several days; data expressed as mean ± SEM (n=6) [1].
Protein synthesis rates were measured by incorporation of 35S‑methionine into newly synthesized protein [1].
Western blot analysis: Cells were lysed, proteins separated by SDS‑PAGE, transferred to membranes, and probed with antibodies against p‑PERK, PERK, p‑eIF2α, eIF2α, p‑AMPKα, AMPKα, p‑ACC, ACC, p‑eEF2, eEF2, p‑eEF2K, eEF2K, p‑ERK1/2, ERK1/2, BiP, p58IPK, ERα, and actin [1].
qRT‑PCR: Total RNA isolated, reverse transcribed, and mRNA levels of pS2, GREB1, XBP1, CXCL2, IL1‑R1, EFNA1, CHOP, GADD34, and p58IPK were quantified using specific primers [1].
Chromatin immunoprecipitation (ChIP): MCF‑7 cells treated with E2 ± BHPI, crosslinked, chromatin sonicated, immunoprecipitated with anti‑ERα or anti‑RNA polymerase II antibodies, and PCR performed on pS2 and GREB1 promoter regions [1].
Calcium imaging: Cells loaded with Fluo‑4 AM, treated with BHPI (1 μM) at 30 s, and fluorescence monitored. For Ca2+‑free conditions, extracellular Ca2+ was removed. Quantitation of cytosolic Ca2+ levels performed [1].
siRNA knockdown: Cells transfected with noncoding siRNA or SmartPools targeting ERα, PERK, pan‑IP3R (all three isoforms), PLCγ, followed by BHPI treatment; protein synthesis and Ca2+ levels measured [1].
IP3 measurement: Intracellular IP3 levels quantified after 10 min treatment with E2 or BHPI [1].
Luciferase assay: Cells transfected with ERE‑luciferase reporter, treated with E2 ± BHPI, and luciferase activity measured [1].
Soft agar assay: Anchorage‑independent growth of MCF‑7 cells in soft agar with BHPI [1].

Animal Protocol: Female athymic mice with MCF‑7 cell xenografts (estrogen supplemented) were used. When tumors reached ~45 mm² cross‑sectional area, mice received daily intraperitoneal (i.p.) injections of BHPI at 15 mg/kg (or vehicle control) for 10 days. Tumor cross‑sectional area was measured regularly. In another regimen, mice received 10 mg/kg every other day. Food intake and body weight were monitored. All procedures approved by the Institutional Animal Care Committee [1].

Toxicity/Toxicokinetics: BHPI was well tolerated in the mouse xenograft study; BHPI‑treated and control mice exhibited similar food intake and weight gain, with no evidence of gross toxicity [1].
In vitro, BHPI at 10 μM had no effect on proliferation of 12 ERα− cell lines, indicating selectivity and low nonspecific toxicity [1].

[1]. Estrogen receptor α inhibitor activates the unfolded protein response, blocks protein synthesis, and induces tumor regression. Proc Natl Acad Sci U S A. 2015 Apr 14;112(15):4737-42.

Additional Info: BHPI is a noncompetitive ERα biomodulator that distorts the normal weak and transient E2‑ERα‑mediated UPR activation into a massive, sustained, and cytotoxic UPR activation. Unlike conventional antiestrogens that are primarily cytostatic, BHPI often kills therapy‑resistant ERα+ cancer cells. Its mechanism involves: ERα‑dependent hyperactivation of plasma membrane PLCγ → increased IP3 → opening of IP3R Ca2+ channels → depletion of ER Ca2+ stores → activation of all three UPR arms (PERK/eIF2α, ATF6α, IRE1α/XBP1). Simultaneously, the futile cycle of Ca2+ pumping by SERCA while IP3R channels remain open depletes ATP, activating AMPK. At later times, BHPI inhibits eEF2 via eEF2K activation, blocking translation elongation. By suppressing synthesis of UPR chaperones BiP and p58IPK, BHPI prevents UPR resolution, leading to sustained protein synthesis inhibition and cell death. BHPI also inhibits nuclear E2‑ERα‑regulated gene expression without competing for E2 binding. It shows high efficacy in gynecological cancers (breast, ovarian, endometrial) that often do not respond to current endocrine therapies [1].

C21H17NO3

331.36458

331.121

56632-39-4

3860640

White to off-white solid powder

3.777

3

3

2

25

469

0

FABLAHMQSQFDHR-UHFFFAOYSA-N

InChI=1S/C21H17NO3/c1-13-3-2-4-18-19(13)22-20(25)21(18,14-5-9-16(23)10-6-14)15-7-11-17(24)12-8-15/h2-12,23-24H,1H3,(H,22,25)

3,3-bis(4-hydroxyphenyl)-7-methyl-1H-indol-2-one

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 : ~120 mg/mL (~362.14 mM)

Solubility in Formulation 1: ≥ 3 mg/mL (9.05 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 30.0 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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Solubility in Formulation 2: ≥ 3 mg/mL (9.05 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 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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