ARV-825 targets bromodomain and extraterminal domain (BET) family member BRD4 and cereblon (CRBN, a component of the E3 ubiquitin ligase complex). [1]

Transient chimera ARV-825 is a heterobifunctional protein that brings BRD4 to the brain of the E3 ubiquitin ligase. BRD4 is actively recruited by ARV-825 to the brain, which leads to the quick and effective proteosome-mediated destruction of both the cereblon-binding moieties of ARV-825 and BRD4. The Kd values for ARV-825's targets range from 28-90 nM and approximately 3 μM, respectively. These values indicate that ARV-825 mediates the substoichiometric conditioning process when BRD4 is introduced. On the other hand, ARV-825 therapy inhibited downstream signaling and extended BRD4 transcription [1].

---

1. ARV-825 is a heterobifunctional PROTAC that mediates fast, efficient, and prolonged degradation of BRD4 in all tested Burkitt's lymphoma (BL) cell lines (Namalwa, Ramos, CA-46). Concentration-dependent BRD4 degradation is observed after overnight treatment with increasing doses of ARV-825, and time-dependent degradation is confirmed in Namalwa and Ramos cells treated with 0.1 µM ARV-825 for indicated time points (detected by immunoblot, actin as loading control). [1]

2. ARV-825-induced BRD4 degradation is CRBN-dependent: co-treatment with pomalidomide (10 µM) abrogates the degradation effect of ARV-825 in Namalwa and Ramos cells (overnight treatment, immunoblot confirmation). [1]

3. ARV-825-induced BRD4 degradation is proteasome-dependent: pre-treatment with proteasome inhibitors MG132 (5 µM) or carfilzomib (5 µM) blocks BRD4 degradation in Namalwa cells treated with ARV-825 (0.01 µM or 0.1 µM, overnight treatment, immunoblot confirmation). [1]

4. ARV-825 more effectively suppresses c-MYC levels and downstream signaling than small-molecule BRD4 inhibitors (JQ1, OTX015). Overnight treatment with increasing doses of ARV-825 (up to 1.0 µM) results in more significant c-MYC reduction than JQ1 (up to 10.0 µM) or OTX015 (up to 10.0 µM) in Namalwa and Ramos cells (immunoblot detection). [1]

5. The c-MYC suppression by ARV-825 is longer lasting: Namalwa cells treated with 0.1 µM ARV-825 for 24 hours show sustained c-MYC downregulation after compound withdrawal, while c-MYC levels recover quickly in cells treated with JQ1 (1.0 µM) or OTX015 (1.0 µM) (immunoblot and qPCR confirmation, GAPDH as internal control for qPCR). [1]

6. ARV-825 exerts superior antiproliferative activity in BL cell lines: various BL cells seeded at 50,000 cells/100 µl in 96-well plates and treated with increasing doses of ARV-825, JQ1, or OTX015 show greater proliferation inhibition with ARV-825 (CellTiter-Glow (CTG) assay, 72 hours post-treatment). The antiproliferative effect of ARV-825 is longer lasting: Namalwa cells treated with 0.1 µM ARV-825 for 24 hours maintain suppressed proliferation 24 and 48 hours after compound withdrawal, unlike JQ1 or OTX015. [1]

7. ARV-825 induces stronger apoptosis in BL cells than BRD4 inhibitors: treatment with 0.1 µM ARV-825 for 24 hours increases caspase 3/7 activity (Caspase 3/7-Glow assay) in various BL cell lines, compared to JQ1 (1.0 µM) or OTX015 (1.0 µM). Overnight treatment with increasing doses of ARV-825 (up to 1.0 µM) induces more significant PARP cleavage (a marker of apoptosis) than JQ1 or OTX015 (up to 10.0 µM) in Ramos and CA-46 cells (immunoblot detection, actin as loading control). [1]

8. Pomalidomide partially rescues the antiproliferative effect of ARV-825: BL cell lines treated with ARV-825 (0.01 µM or 0.1 µM) alone or in combination with pomalidomide (1.0 µM or 10.0 µM) for 72 hours show reduced proliferation inhibition in the combined group (CTG assay). Pomalidomide alone has no significant effect on BL cell proliferation at tested doses. [1]

1. BRD4 and c-MYC expression detection by immunoblot: BL cell lines (Namalwa, Ramos, CA-46) are treated with ARV-825, JQ1, or OTX015 at indicated concentrations (increasing doses or fixed concentrations) for specified times (overnight, 24 hours, or time-course). Cell lysates are prepared, and immunoblot analysis is performed to detect BRD4, c-MYC, and PARP cleavage (apoptosis marker), with actin as the loading control. [1]

2. CRBN dependence validation assay: Namalwa and Ramos cells are treated overnight with ARV-825 (various concentrations), pomalidomide (10 µM), or their combination. Cell lysates are analyzed by immunoblot to detect BRD4 levels, verifying whether pomalidomide blocks ARV-825-mediated BRD4 degradation. [1]

3. Proteasome dependence validation assay: Namalwa cells are treated overnight with ARV-825 (0.01 µM or 0.1 µM) alone, MG132 (5 µM) or carfilzomib (5 µM) alone, or combinations of ARV-825 with either inhibitor. Immunoblot analysis of BRD4 levels is performed to confirm proteasome involvement. [1]

4. Cell proliferation assay (CTG assay): BL cell lines are seeded in 96-well plates at 50,000 cells/100 µl. Cells are treated with increasing doses of ARV-825, JQ1, OTX015, or pomalidomide (alone or in combination) for 72 hours. For long-lasting effect testing, cells are treated with ARV-825, JQ1, or OTX015 for 24 hours, washed to remove compounds, and re-seeded; proliferation is measured 24 and 48 hours later. Relative proliferation is quantified using the CTG assay. [1]

5. Apoptosis assay: BL cell lines are treated with ARV-825, JQ1, OTX015, or puromycin (positive control, 10 mg/ml) for 24 hours. Caspase 3/7 activity is detected using the Caspase 3/7-Glow assay to assess apoptosis induction. [1]

6. qPCR analysis: Namalwa cells are treated with ARV-825, JQ1, or OTX015 for 24 hours, washed to remove compounds, and re-seeded. RNA is extracted at 0, 6, and 24 hours post-compound withdrawal, reverse-transcribed into cDNA, and quantified by qPCR using SLC19A1-specific primers (GAPDH as internal control) to assess sustained c-MYC suppression at the transcriptional level. [1]

[1]. Hijacking the E3 Ubiquitin Ligase Cereblon to Efficiently Target BRD4. Chem Biol. 2015 Jun 18;22(6):755-63.

ARV-825 is a PROTAC linked by a Cerebro-BRD4 ligand. ARV-825 binds to the BD1 and BD2 domains of BRD4. 1. ARV-825 is a heterobifunctional PROTAC designed to hijack the E3 ubiquitin ligase CRBN to target and degrade BRD4. [1] 2. BRD4 is a member of the BET family and is involved in transcriptional regulation. It is an attractive target for cancer therapy, especially in MYC-driven malignancies such as Burkitt lymphoma (BL). [1] 3. Traditional small molecule inhibitors of BRD4 (such as JQ1 and OTX015) lead to a large accumulation of BRD4 protein, resulting in limited c-MYC inhibition, weak anti-proliferative activity, and lack of apoptosis induction—while ARV-825 overcomes these limitations. [1]
4. The mechanism of action of ARV-825 includes recruiting CRBN to BRD4, promoting BRD4 ubiquitination, and degradation via the proteasome pathway. [1]
5. Compared with traditional small molecule inhibitors, ARV-825 provides a more effective strategy for targeting BRD4, and its superior efficacy in inhibiting c-MYC, inhibiting proliferation, and inducing BL cell apoptosis has been demonstrated. [1]

C46H47CLN8O9S

923.4316

922.287

1818885-28-7

92044400

Light yellow to yellow solid powder

1.5±0.1 g/cm3

1.701

2.72

3

14

19

65

1720

1

CC1=C(SC2=C1C(=N[C@H](C3=NN=C(N32)C)CC(=O)NC4=CC=C(C=C4)OCCOCCOCCOCCNC5=CC=CC6=C5C(=O)N(C6=O)C7CCC(=O)NC7=O)C8=CC=C(C=C8)Cl)C

RWLOGRLTDKDANT-TYIYNAFKSA-N

InChI=1S/C46H47ClN8O9S/c1-26-27(2)65-46-39(26)41(29-7-9-30(47)10-8-29)50-35(42-53-52-28(3)54(42)46)25-38(57)49-31-11-13-32(14-12-31)64-24-23-63-22-21-62-20-19-61-18-17-48-34-6-4-5-33-40(34)45(60)55(44(33)59)36-15-16-37(56)51-43(36)58/h4-14,35-36,48H,15-25H2,1-3H3,(H,49,57)(H,51,56,58)/t35-,36?/m0/s1

2-((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)-N-(4-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethoxy)phenyl)acetamide

ARV825; ARV-825; ARV 825.

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 (~54.15 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.71 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 (2.71 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.)