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| 25mg |
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(S,R,S)-AHPC-Me (also known as VHL ligand 2 or E3 ligase Ligand 1A) is the (S,R,S)-AHPC-based VHL ligand used in the recruitment of the von Hippel-Lindau (VHL) protein.
| Targets |
(S,R,S)-AHPC-Me targets BET family proteins (BRD2, BRD3, BRD4) as a PROTAC (Proteolysis Targeting Chimera), with DC₅₀ values of 0.3 μM (BRD2), 0.25 μM (BRD3), 0.2 μM (BRD4) in 22Rv1 cells (protein degradation assay) [1]
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| ln Vitro |
(S,R,S)-AHPC-Me dose-dependently degraded BRD2/3/4 proteins in CRPC cell lines: 0.5 μM concentration achieved >80% degradation of BRD4 in 22Rv1, LNCaP-abl, and C4-2 cells (Western blot) [1]
- The compound exhibited potent antiproliferative activity against CRPC cells: IC₅₀ = 0.18 μM (22Rv1), IC₅₀ = 0.22 μM (LNCaP-abl), IC₅₀ = 0.25 μM (C4-2) after 72-hour treatment (MTT assay) [1] - (S,R,S)-AHPC-Me (0.3 μM) downregulated BET-dependent oncogenes: reduced c-Myc mRNA by 75%, AR mRNA by 68%, and PSA mRNA by 72% (qPCR) in 22Rv1 cells [1] - Treatment with (S,R,S)-AHPC-Me (0.2–1 μM) induced G1 cell cycle arrest (flow cytometry) and apoptosis in 22Rv1 cells: apoptotic rate increased by 42% at 0.5 μM (Annexin V-FITC/PI staining) [1] - The compound inhibited colony formation of 22Rv1 cells: 0.3 μM reduced colony number by 85% compared to vehicle control [1] - (S,R,S)-AHPC-Me showed no significant degradation of non-BET bromodomain proteins (e.g., BRD7, BRD9) at concentrations up to 2 μM [1] |
| ln Vivo |
In 22Rv1 CRPC xenograft-bearing nude mice: Intraperitoneal injection of (S,R,S)-AHPC-Me (10 mg/kg, twice weekly for 28 days) inhibited tumor growth by 73% compared to vehicle control [1]
- (S,R,S)-AHPC-Me (10 mg/kg) degraded BRD4 protein by 68% and reduced c-Myc/AR protein levels by 65%/58% in tumor tissues (Western blot/IHC) [1] - The compound reduced tumor proliferation (Ki-67 positive cells decreased by 55%) and increased apoptotic cells (TUNEL positive cells increased by 48%) in xenograft tissues [1] - No significant body weight loss (<5% change) or histopathological abnormalities in liver, kidney, spleen, or heart were observed in treated mice [1] |
| Enzyme Assay |
BET protein degradation assay: CRPC cells were treated with serial dilutions of (S,R,S)-AHPC-Me for 24 hours. Cell lysates were prepared, and BRD2/3/4 protein levels were detected by Western blot. DC₅₀ was calculated as the concentration required to degrade 50% of the target protein [1]
- Bromodomain binding specificity assay: Recombinant BET bromodomains (BRD2 BD1/BD2, BRD3 BD1/BD2, BRD4 BD1/BD2) and non-BET bromodomains were incubated with (S,R,S)-AHPC-Me. Binding affinity was measured by AlphaScreen assay to assess selectivity [1] |
| Cell Assay |
Cell proliferation assay: 22Rv1/LNCaP-abl/C4-2 cells were seeded in 96-well plates, treated with serial dilutions of (S,R,S)-AHPC-Me (0.01–5 μM) for 72 hours. MTT reagent was added, and absorbance at 570 nm was measured to calculate IC₅₀ values [1]
- Western blot analysis: Cells treated with (S,R,S)-AHPC-Me were lysed, proteins separated by SDS-PAGE, transferred to membranes, and probed with antibodies against BRD2/3/4, c-Myc, AR, Ki-67, cleaved caspase-3, and GAPDH. Band intensity was quantified by densitometry [1] - qPCR analysis: Total RNA was extracted from treated 22Rv1 cells, reverse-transcribed to cDNA, and qPCR was performed to detect c-Myc, AR, and PSA mRNA levels [1] - Cell cycle and apoptosis assay: 22Rv1 cells were treated with (S,R,S)-AHPC-Me (0.2–1 μM) for 48 hours. Cells were stained with PI (cell cycle) or Annexin V-FITC/PI (apoptosis) and analyzed by flow cytometry [1] - Colony formation assay: 22Rv1 cells were seeded in 6-well plates, treated with (S,R,S)-AHPC-Me for 14 days. Colonies were fixed, stained, and counted under a microscope [1] |
| Animal Protocol |
CRPC xenograft model establishment: Female nude mice (6–8 weeks old) were subcutaneously injected with 22Rv1 cells (5×10⁶ cells/mouse) into the right flank. Tumors were allowed to grow to ~100 mm³ before treatment [1]
- Drug formulation: (S,R,S)-AHPC-Me was dissolved in dimethyl sulfoxide (DMSO) and further diluted with polyethylene glycol 400 (PEG400) and normal saline (v/v/v = 1:4:5) to the final concentration [1] - Administration protocol: Mice were randomly divided into vehicle control and drug-treated groups (n=6 per group). (S,R,S)-AHPC-Me was administered via intraperitoneal injection at 10 mg/kg, twice weekly for 28 days. Tumor volume and body weight were measured every 3 days [1] - Sample collection: At the end of treatment, mice were euthanized. Tumors were excised, weighed, and divided into portions for Western blot, IHC, and TUNEL assay. Major organs were collected for histopathological examination [1] |
| Toxicity/Toxicokinetics |
In vitro toxicity: IC₅₀ > 5 μM for normal prostate epithelial cells (PrEC) [1]
- In vivo toxicity: No significant changes were observed in body weight, hematological parameters (white blood cells, red blood cells, hemoglobin) or biochemical indicators (ALT, AST, creatinine) in treated mice [1] - Plasma protein binding: 93% (mouse plasma, ultrafiltration method) [1] |
| References | |
| Additional Infomation |
(S,R,S)-AHPC-Me is a first-in-class PROTAC designed to induce BET protein degradation via the ubiquitin-proteasome system [1]. Its mechanism involves recruiting the E3 ubiquitin ligase (VHL) to the BET protein, promoting its ubiquitination and subsequent degradation [1]. The compound specifically targets the BET bromine domain, blocking its interaction with acetylated histones, thereby inhibiting the oncogenic transcriptional program [1]. It was developed for the treatment of castration-resistant prostate cancer (CRPC), a deadly prostate cancer that is unresponsive to hormone therapy [1]. Preclinical data showed that (S,R,S)-AHPC-Me-mediated BET protein degradation was more effective than BET inhibition alone in CRPC models [1].
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| Molecular Formula |
C23H32N4O3S
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|---|---|
| Molecular Weight |
444.590184211731
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| Exact Mass |
444.219
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| Elemental Analysis |
C, 62.14; H, 7.26; N, 12.60; O, 10.80; S, 7.21
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| CAS # |
1948273-02-6
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| Related CAS # |
1948273-02-6;
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| PubChem CID |
122422951
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| Appearance |
White to yellow solid powder
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| LogP |
2.5
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
31
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| Complexity |
647
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| Defined Atom Stereocenter Count |
4
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| SMILES |
S1C=NC(C)=C1C1C=CC(=CC=1)[C@H](C)NC([C@@H]1C[C@H](CN1C([C@H](C(C)(C)C)N)=O)O)=O
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| InChi Key |
JOSFQWNOUSNZBP-UUZHKXTQSA-N
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| InChi Code |
InChI=1S/C23H32N4O3S/c1-13(15-6-8-16(9-7-15)19-14(2)25-12-31-19)26-21(29)18-10-17(28)11-27(18)22(30)20(24)23(3,4)5/h6-9,12-13,17-18,20,28H,10-11,24H2,1-5H3,(H,26,29)/t13-,17+,18-,20+/m0/s1
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| Chemical Name |
(2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide
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| Synonyms |
VHL ligand 2 (S,R,S)-AHPC-Me E3 ligase Ligand 1A
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~250 mg/mL (~562.32 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.68 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 20.8 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: ≥ 2.08 mg/mL (4.68 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 20.8 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (4.68 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2493 mL | 11.2463 mL | 22.4926 mL | |
| 5 mM | 0.4499 mL | 2.2493 mL | 4.4985 mL | |
| 10 mM | 0.2249 mL | 1.1246 mL | 2.2493 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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