BRD2 (Kd = 4.7~34 nM); BRD3 (Kd = 8.3 nM); BRD4 (Kd = 9.6 nM)
ARV-771 targets bromodomain and extra-terminal (BET) family proteins (BRD2, BRD3, BRD4) and von Hippel-Lindau (VHL) E3 ubiquitin ligase. It also modulates androgen receptor (AR) signaling and AR levels [1]

In comparison to BET inhibition, ARV-771, a small-molecule pan-BET degrader based on proteolysis-targeting chimera (PROTAC) technology, shows markedly increased efficacy in CRPC cellular models. With a DC50 of less than 5 nM, ARV-771 potently destroys BRD2/3/4 in 22Rv1 cells. One of BET proteins' downstream effectors is the c-MYC protein. When c-MYC is treated with ARV-771, its levels are reduced with an IC50 of less than 1 nM. ARV-771 demonstrates a potent antiproliferative effect on cell lines that are 22Rv1, VCaP, and LnCaP95. Treatment with ARV-771 significantly alters cell shape in a way that is consistent with apoptosis. Treatment with 10 nM ARV-771 causes a down-regulation of FL-AR and AR-V7 mRNA in VCaP cells. In VCaP cells, ARV-771 exhibits antiandrogenic activity on several AR-regulated genes[1].

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1. ARV-771 is a potent pan-BET degrader: Incubation of castration-resistant prostate cancer (CRPC) cell lines (22Rv1, VCaP, LnCaP95) with ARV-771 for 16 hours results in depletion of BRD2, BRD3, and BRD4 (Western blot, n=3 independent experiments). [1]

2. ARV-771 suppresses c-MYC expression: Treatment for 16 hours reduces cellular c-MYC levels at the protein level (ELISA, n=3 triplicates) and mRNA level (qPCR, two biological replicates with triplicate measurements each, n=3). At 1 μM, it shows more potent c-MYC suppression than the inactive diastereomer ARV-766 (1 μM) or BET inhibitor OTX015 (1 μM). [1]

3. ARV-771 attenuates AR signaling and reduces AR levels: After 16-hour treatment, full-length AR (FL-AR) protein levels are lowered (ELISA, n=3 triplicates; Western blot, n=2 independent experiments). In VCaP cells, it decreases mRNA levels of FL-AR and AR-V7 (qPCR, two biological replicates with triplicate measurements each, n=3) and suppresses androgen-responsive gene expression. Pretreatment with ARV-771 for 1 hour blocks R1881-induced ERG induction in VCaP cells. [1]

4. ARV-771 induces CRPC cell death: Treatment for 72 hours exerts antiproliferative effects (n=3 triplicates); treatment for 24 hours activates caspases (n=3 triplicates) and induces PARP cleavage in 22Rv1 cells (Western blot). [1]

5. ARV-771-mediated BRD4 degradation is VHL- and proteasome-dependent: Pretreatment of cells with VHL ligand ARV-056 (10 μM) or proteasome inhibitor carfilzomib (1 μM) for 30 minutes blocks BRD4 degradation induced by 8-hour ARV-771 treatment (Western blot, n=2 independent experiments). [1]

6. ARV-771 alters 22Rv1 cellular morphology consistent with apoptosis (20× magnification). [1]

When non-castrated male Nu/Nu mice with tumor xenografts carrying AR-V7+ 22Rv1 are given daily subcutaneous injections of ARV-771 at a dose of 10 mg/kg for three days, the levels of BRD4 and c-MYC in the tumor tissue are down-regulated by 37% and 76%, respectively. Following ARV-771 treatment, a notable down-regulation in AR-V7 levels is seen in the 22Rv1 tumors[1].

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1. ARV-771 downregulates BRD4 and suppresses c-MYC in vivo: Daily subcutaneous administration of ARV-771 (10 mg/kg) for 3 days in Nu/Nu mice bearing 22Rv1 tumor xenografts reduces BRD4 levels and c-MYC expression (n=9 animals per cohort). [1]

2. ARV-771 exerts dose-dependent tumor growth inhibition (TGI) and regression: In a 14-day 22Rv1 xenograft study (n=9 animals per cohort), subcutaneous dosing of ARV-771 (3 mg/kg, 10 mg/kg) downregulates BRD4 and c-MYC (Western blot); 30 mg/kg daily subcutaneous dosing induces tumor regression (n=10 animals per cohort). [1]

3. ARV-771 lowers AR-V7 levels in tumors: Daily subcutaneous injection of 10 mg/kg ARV-771 for 14 days reduces AR-V7 levels in 22Rv1 xenografts (n=9 animals per cohort). [1]

4. ARV-771 is effective in VCaP xenograft models: Intermittent dosing schedules of ARV-771 induce TGI in CB17 SCID mice bearing VCaP tumor xenografts (n=10 animals per cohort). It depletes BRD4 and suppresses c-MYC in VCaP tumors (Western blot) and reduces serum prostate-specific antigen (PSA) levels (ELISA, n=8, 5, 8, 8 replicates for respective treatments). [1]

5. ARV-771 outperforms BET inhibitor OTX015: In 22Rv1 xenografts, ARV-771 shows more potent BRD4 downregulation and c-MYC suppression than OTX015 (Western blot, n=9 animals per cohort). [1]

c-MYC ELISA. [1]
22Rv1 cells (30,000 cells per well) were dosed with compounds serially diluted at 1:3 ratio for an eight-point dose curve. The medium was aspirated, and cells were washed once with PBS. RIPA buffer (50 μL) supplemented with protease and phosphatase inhibitors was used to lyse cells. Lysates were centrifuged and transferred to a 96-well c-MYC ELISA plate.

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AR ELISA. [1]
VCaP cells (40,000 cells per well) were dosed with compounds serially diluted at 1:3 ratio for an eight-point dose curve. Medium was aspirated, and cells were lysed in cell lysis buffer supplemented with protease and phosphatase inhibitors. Lysates were centrifuged and transferred to a 96-well Androgen Receptor ELISA plate

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1. BET protein binding assay: Kd values of ARV-771 against BRD1 and BRD2 are determined using a binding assay (specific method not detailed), with ARV-766 as an inactive control (unable to bind VHL). [1]

2. ELISA for protein level detection: CRPC cells are treated with ARV-771 for 16 hours, and cellular c-MYC or FL-AR protein levels are quantified by ELISA. The assay is performed in triplicate to ensure reproducibility. [1]

3. qPCR for mRNA level detection: After 16-hour treatment with ARV-771 (or controls ARV-766, OTX015) at specified concentrations, total RNA is extracted from CRPC cells, reverse-transcribed into cDNA, and qPCR is performed with specific primers for c-MYC, FL-AR, AR-V7, and androgen-responsive genes. GAPDH serves as an internal control; experiments include two biological replicates with triplicate measurements each. [1]

ARV-771 is dissolved in DMSO. 22Rv1 cells (5,000 cells per well) are dosed with ARV-771 serially diluted 1:3 for a 10-point dose curve for 72 h. CellTiter-Glo Luminescent Cell Viability Assay is added, and the plate is read on a luminometer. Data are analyzed and plotted using GraphPad Prism software[1].

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1. Western blot for protein degradation/cleavage: CRPC cells (22Rv1, VCaP, LnCaP95) are treated with ARV-771 for 16 hours (BET/AR detection) or 8 hours (BRD4 degradation mechanism) or 24 hours (PARP cleavage). Cell lysates are prepared, and Western blot is performed to detect BRD2/3/4, FL-AR, PARP, and loading controls. For mechanism validation, cells are pretreated with ARV-056 (10 μM) or carfilzomib (1 μM) for 30 minutes before ARV-771 treatment. [1]

2. Cell proliferation assay: CRPC cells are seeded in 96-well plates and treated with ARV-771 for 72 hours. Proliferation is quantified using standard methods, with experiments performed in triplicate. [1]

3. Caspase activation assay: CRPC cells are treated with ARV-771 for 24 hours, and caspase activity is measured to assess apoptosis induction (n=3 triplicates). [1]

4. Cellular morphology observation: 22Rv1 cells are treated with ARV-771, and morphological changes consistent with apoptosis are observed under a microscope at 20× magnification. [1]

5. ERG induction inhibition assay: VCaP cells are pretreated with ARV-771 for 1 hour, followed by R1881 treatment. ERG induction is detected to evaluate AR signaling inhibition. [1]

Mice were implanted subcutaneously with 5 × 106 22Rv1 or VCaP cells in Matrigel. Dosing was carried out for up to 3 wk, depending on the experiment. Mice were sacrificed 8 h after the final dose. Plasma and tissues were harvested and flash frozen for further analysis. All PK analysis was carried out at Drumetix Laboratories. Plasma PSA was analyzed by the PathScan Total PSA/KLK3 Sandwich ELISA Kit following the manufacturer’s protocol.[1]

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1. 22Rv1 xenograft model (Nu/Nu mice): Tumor xenografts are established by implanting 22Rv1 cells into Nu/Nu mice. Mice are divided into treatment cohorts (n=9–10 per cohort) and administered ARV-771 via subcutaneous injection at doses of 3 mg/kg, 10 mg/kg, or 30 mg/kg. Dosing is performed once daily for 14 days (or 3 days for short-term studies). Tumors and plasma are harvested 8 hours after the last dose for Western blot, ELISA, and qPCR analysis. [1]

2. VCaP xenograft model (CB17 SCID mice): VCaP tumor xenografts are established in CB17 SCID mice. Mice are treated with ARV-771 via subcutaneous injection using intermittent dosing schedules (n=10 per cohort). The study duration is 14 days, with tumors, plasma, and serum collected 8 hours after the last dose for BRD4/c-MYC detection (Western blot) and PSA quantification (ELISA). [1]

1. ARV-771 has a well-defined pharmacokinetic (PK) profile: ARV-771 was administered to mice via different routes and at different concentrations, and its plasma concentration was monitored over time (Figure S4A). Specific PK parameters (e.g., half-life, oral bioavailability) were not provided. [1]

1. ARV-771 did not show obvious toxicity in mice: In the 22Rv1 and VCaP xenograft studies, the weight of mice did not show obvious abnormalities (Figures S5A and S5B), indicating that it was well tolerated. [1]

[1]. PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer. Proc Natl Acad Sci U S A. 2016 Jun 28;113(26):7124-9.

This study demonstrates that the BET PROTAC inhibitor ARV-771, based on the von Hippel-Landau (VHL) E3 ligase, exhibits high activity against a castration-resistant prostate cancer (CRPC) cell model. ARV-771 induces rapid degradation of BET protein in these cells, with a DC50 value (drug concentration resulting in 50% protein degradation) <1 nM. Notably, ARV-771-mediated BET degradation leads to decreased transcriptional levels of FL-AR and AR-V7. In contrast, treatment of CRPC cells with BET inhibitors suppresses AR-V7 expression but does not affect FL-AR expression. Furthermore, ARV-771-induced apoptosis is significantly higher than that induced by BET inhibitors. Finally, subcutaneous injection of ARV-771 is effective in two different CRPC mouse models and induces regression of enzalutamide-resistant 22Rv1 xenografts. Therefore, this study confirms that BET protein degradation is a promising clinical strategy for treating metastatic castration-resistant prostate cancer (CRPC) and demonstrates the feasibility of using PROTACs-mediated small molecule protein degradation to treat solid tumors. [1] 1. ARV-771 is a protein hydrolysis-targeting chimera (PROTAC) based on pan-BET degradation technology. [1] 2. Its inactive diastereomer is ARV-766, which cannot bind to VHL and cannot induce BET degradation. [1] 3. ARV-771 was developed for the treatment of castration-resistant prostate cancer (CRPC), a disease that depends on the androgen receptor (AR) signaling pathway and has a poor prognosis after anti-androgen resistance. [1] 4. Unlike BET inhibitors, ARV-771 can simultaneously inhibit AR signaling and AR levels, showing significantly improved efficacy in CRPC models. [1]
5. This study is the first to demonstrate the efficacy of small molecule BET degraders in solid tumors. [1]

C49H60CLN9O7S2

986.6398

985.374

C, 59.65; H, 6.13; Cl, 3.59; N, 12.78; O, 11.35; S, 6.50

1949837-12-0

126619980

Typically exists as White to light yellow solids at room temperature

1.4±0.1 g/cm3

1.674

3.84

4

13

19

68

1750

5

ClC1C([H])=C([H])C(=C([H])C=1[H])C1C2C(C([H])([H])[H])=C(C([H])([H])[H])SC=2N2C(C([H])([H])[H])=NN=C2[C@]([H])(C([H])([H])C(N([H])C([H])([H])C([H])([H])OC([H])([H])C([H])([H])C([H])([H])OC([H])([H])C(N([H])[C@]([H])(C(N2C([H])([H])[C@@]([H])(C([H])([H])[C@@]2([H])C(N([H])[C@@]([H])(C([H])([H])[H])C2C([H])=C([H])C(C3=C(C([H])([H])[H])N=C([H])S3)=C([H])C=2[H])=O)O[H])=O)C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])=O)=O)N=1

HJGNHEQIOZDQRW-VZRXUJQISA-N

InChI=1S/C50H63N9O7S2/c1-28-11-13-35(14-12-28)43-42-29(2)32(5)68-49(42)59-33(6)56-57-46(59)38(54-43)24-40(61)51-19-22-65-20-10-21-66-26-41(62)55-45(50(7,8)9)48(64)58-25-37(60)23-39(58)47(63)53-30(3)34-15-17-36(18-16-34)44-31(4)52-27-67-44/h11-18,27,30,37-39,45,60H,10,19-26H2,1-9H3,(H,51,61)(H,53,63)(H,55,62)/t30-,37+,38-,39-,45+/m0/s1

(2S,4R)-1-((S)-2-(tert-butyl)-4,14-dioxo-15-((S)-2,3,9-trimethyl-4-(p-tolyl)-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-6,10-dioxa-3,13-diazapentadecanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

ARV771; ARV 771; CHEMBL4215078; (2S,4R)-1-[(2S)-2-[[2-[3-[2-[[2-[(9S)-7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaen-9-yl]acetyl]amino]ethoxy]propoxy]acetyl]amino]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide; (2S,4R)-1-((2S)-2-(tert-butyl)-15-((6S)-4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-4,14-dioxo-6,10-dioxa-3,13-diazapentadecan-1-oyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide; (2S,4R)-1-((S)-2-(tert-butyl)-15-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-4,14-dioxo-6,10-dioxa-3,13-diazapentadecanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide; ARV771; SCHEMBL18551355; ARV-771.

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 : ≥ 50 mg/mL (~50.68 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.53 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (2.53 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (2.53 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.

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