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VY3-135 (VY-3135; VY3135) is a novel and potent ACSS2 (acetyl-CoA synthetase 2) inhibitor ( IC50 = 44 nM) with antitumor effects. Acetyl-CoA is a vitally important and versatile metabolite used for many cellular processes including fatty acid synthesis, ATP production, and protein acetylation. Recent studies have shown that cancer cells upregulate acetyl-CoA synthetase 2 (ACSS2), an enzyme that converts acetate to acetyl-CoA, in response to stresses such as low nutrient availability and hypoxia. Stressed cancer cells use ACSS2 as a means to exploit acetate as an alternative nutrient source. Genetic depletion of ACSS2 in tumors inhibits the growth of a wide variety of cancers. However, there are no studies on the use of an ACSS2 inhibitor to block tumor growth.
| Targets |
In ACSS2high Brpkp110 and ACSS2low A7C11 cells, VY-3-135 (0.1, 1 μM; for 24 hours) facilitates the relay labeling of qatar by 13C2-linkers [1].
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| ln Vitro |
In ACSS2high Brpkp110 and ACSS2low A7C11 cells, VY-3-135 (0.1, 1 μM; for 24 hours) facilitates the relay labeling of qatar by 13C2-linkers [1].
VY-3-135 potently inhibits ACSS2 activity in a biochemical assay with an IC50 of 44 ± 3.85 nM. It does not show inhibitory activity against recombinant human ACSS1 or ACSS3. In breast cancer cell lines (SKBr3, BT474, MDA-MB-468), VY-3-135 completely blocks acetate-dependent fatty acid synthesis from 13C2-acetate, as measured by palmitate labeling, particularly under hypoxic and low serum (H1) stress conditions. It also blocks the incorporation of 13C2-acetate into the cytosolic metabolite UDP-N-acetylglucosamine (UDP-GlcNAc). Treatment with VY-3-135 (10 µM) causes modest growth inhibition of BT474 and SKBr3 cells cultured under H1 conditions over 72 hours. It does not decrease 13C2-acetate labeling of citrate, indicating no off-target effect on mitochondrial ACSS1. Molecular docking simulations predict that VY-3-135 acts as a transition state mimetic, occupying the acetyl-AMP and part of the CoA binding sites of acetyl-CoA synthetase. Immunoblotting shows VY-3-135 does not affect ACSS1 protein levels.[1] |
| ln Vivo |
MDA-MB-468 (ACSS2high) tumor growth is inhibited by VY-3-135 (100 mg/kg/day; sidewall 30 days), whereas WHIM12 (ACSS2low) tumor growth in foam is largely unaffected [1].
In mouse triple-negative breast cancer (TNBC) models, VY-3-135 (100 mg/kg daily IP) significantly inhibits the growth of ACSS2-high Brpkp110 tumors but has a minimal effect on ACSS2-low A7C11 tumors. In human tumor xenograft models, VY-3-135 (100 mg/kg daily PO or IP) inhibits the growth of ACSS2-high tumors (MDA-MB-468 and BT474) but is ineffective against ACSS2-low WHIM12 tumors. Notably, VY-3-135 treatment completely abrogates BT474 tumor growth over two weeks, with one tumor showing full regression. Stable isotope tracer studies in tumor-bearing mice show that VY-3-135 treatment markedly decreases acetate-dependent labeling of palmitate and UDP-GlcNAc in tumors, confirming on-target inhibition of ACSS2 activity in vivo. No significant effect on acetate labeling of citrate is observed in tumors, confirming specificity for ACSS2 over ACSS1. QuantSeq 3' mRNA sequencing of tumors from VY-3-135-treated mice shows minimal effects on gene transcription, with no pathways significantly altered at FDR<5%. Immunohistochemistry of tumors shows decreased Ki67 staining in VY-3-135-treated groups, suggesting reduced proliferation.[1] |
| Enzyme Assay |
ACSS2 enzyme activity was measured using a TranScreener TRF AMP/GMP assay. Recombinant ACSS2 was incubated in assay buffer (containing HEPES, NaCl, MgCl2, sodium acetate, DTT, and detergent) with ATP and CoA. The reaction proceeded for 120 minutes. The production of AMP was detected by adding a terbium-conjugated AMP antibody and AMP tracer, followed by HTRF signal measurement. Test compounds were serially diluted in DMSO and added to the reaction. Data were normalized to percent inhibition relative to controls without enzyme and with DMSO.
Similar assays were performed for ACSS1 and ACSS3 using acetate or propionate as substrates to assess inhibitor specificity.[1] |
| Cell Assay |
For stable isotope tracing, cells were cultured in serum-like modified Eagle's medium (SMEM) under normoxic or hypoxic (1% O2) conditions, supplemented with uniformly labeled 13C2-acetate for 24 hours. For fatty acid analysis, cells were washed, and lipids were extracted with methanol. Fatty acids were saponified with potassium hydroxide in methanol, extracted with hexane, and analyzed by LC-MS to determine 13C enrichment.
For polar metabolite analysis (e.g., UDP-GlcNAc), cells were extracted with a methanol/acetonitrile/water mixture, and extracts were analyzed by LC-MS. Cell growth assays were performed by seeding cells in appropriate media under stress conditions (hypoxia, low serum) with or without VY-3-135. Cell numbers were assessed over 72 hours. Western blotting was used to analyze protein expression. Cells were lysed, proteins separated by SDS-PAGE, transferred to membranes, and probed with specific antibodies. Nuclear fractionation was performed to assess ACSS2 localization. Cells were lysed, nuclei were isolated and further fractionated into soluble and chromatin-bound fractions, which were then analyzed by western blotting.[1] |
| Animal Protocol |
For tumor xenograft studies, cancer cells were suspended in a PBS:Matrigel mixture and injected subcutaneously into immunodeficient (NSG) mice. After tumor establishment, mice were randomized into treatment groups.
VY-3-135 was dissolved in a vehicle containing 10% DMSO, 20% solutol, and 70% water with 0.5% Tween20. It was administered daily via intraperitoneal (IP) injection at 100 mg/kg or by oral gavage (PO) at 100 mg/kg. Control mice received vehicle alone. Tumor dimensions were measured regularly with calipers, and volume was calculated. At the endpoint, tumors were harvested for further analysis (e.g., metabolomics, immunohistochemistry). For stable isotope tracing in vivo, tumor-bearing mice were provided with 2% heavy-labeled acetate (D3- or 13C2-acetate) in drinking water for two days and received an IP bolus of heavy acetate 90 minutes before sacrifice. Tumors were then harvested and processed for LC-MS analysis.[1] |
| ADME/Pharmacokinetics |
The water solubility of VY-3-135 in PBS buffer was 21.7 µM. Microsomal stability tests showed that VY-3-135 had better stability in mouse and human liver microsomes compared with the related compound VY-3-249. Pharmacokinetic analysis in CD1 mice showed that VY-3-135 was completely absorbed after gavage (30 mg/kg), intraperitoneal injection (10 mg/kg), or intravenous injection (2 mg/kg), indicating good exposure and pharmacokinetic characteristics. Specific pharmacokinetic parameters (Cmax, Tmax, AUC, t1/2) have been calculated but are not listed in the text. [1]
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| References | |
| Additional Infomation |
VY-3-135 is a small molecule inhibitor of ACSS2, an enzyme that converts acetate into acetyl-CoA. Cancer cells, especially under metabolic stresses such as hypoxia and nutrient deprivation, highly express ACSS2 to utilize acetate as an alternative nutrient source. This inhibitor is thought to act as a transition state mimic, blocking the binding sites of ACSS2 to acetylAMP and coenzyme A. High expression of ACSS2 in tumors (ACSS2-high) is associated with sensitivity to VY-3-135, suggesting that it may be a biomarker for patient screening. This study proposes that inhibiting ACSS2 represents a novel therapeutic strategy for acetate-dependent cancers and suggests the use of 11C-acetate PET imaging to identify patients with acetate-affinity tumors. [1]
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| Molecular Formula |
C26H27N3O3
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|---|---|
| Molecular Weight |
429.51
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| Exact Mass |
429.205
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| Elemental Analysis |
C, 72.71; H, 6.34; N, 9.78; O, 11.17
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| CAS # |
1824637-41-3
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| PubChem CID |
92131155
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| Appearance |
White to light yellow solid powder
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| LogP |
3.1
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
32
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| Complexity |
599
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| Defined Atom Stereocenter Count |
1
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| SMILES |
CCN1C2=C(C=CC(=C2)C(=O)NC[C@@H](C)O)N=C1C(C3=CC=CC=C3)(C4=CC=CC=C4)O
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| InChi Key |
KTPYOTKTDCLZHR-GOSISDBHSA-N
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| InChi Code |
InChI=1S/C26H27N3O3/c1-3-29-23-16-19(24(31)27-17-18(2)30)14-15-22(23)28-25(29)26(32,20-10-6-4-7-11-20)21-12-8-5-9-13-21/h4-16,18,30,32H,3,17H2,1-2H3,(H,27,31)/t18-/m1/s1
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| Chemical Name |
3-ethyl-2-[hydroxy(diphenyl)methyl]-N-[(2R)-2-hydroxypropyl]benzimidazole-5-carboxamide
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| Synonyms |
VY3-135VY3135 VY-3135VY 3135 VY-3-135 VY 3-135
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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 Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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 : ~50 mg/mL (~116.41 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.82 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 (5.82 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.82 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 | 2.3282 mL | 11.6412 mL | 23.2823 mL | |
| 5 mM | 0.4656 mL | 2.3282 mL | 4.6565 mL | |
| 10 mM | 0.2328 mL | 1.1641 mL | 2.3282 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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