| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| Other Sizes |
| Targets |
0.79 µM (ASH1L histone methyltransferase)[1]
AS-99 TFA targets ASH1L (also known as KMT2H), a histone methyltransferase responsible for catalyzing the methylation of histone H3 at lysine 36 (H3K36me2). This enzyme plays a crucial role in transcriptional activation and is involved in MLL-rearranged leukemia pathogenesis. AS-99 TFA exhibits >100-fold selectivity for ASH1L over 20 other histone methyltransferases, including NSD1, NSD2, NSD3, and SETD2. |
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| ln Vitro |
AS-99 TFA is measured against a panel comprising NSD1, NSD2, NSD3, and SETD2, among other 20 histone methyltransferases. At 50 µM of AS-99 TFA, no discernible inhibition is seen on any of the examined histone methyltransferases, suggesting a selectivity of over 100 times for ASH1L[1]. At 10 µM and greater concentrations, AS-99 exhibits little or very little influence on the growth of leukemia cells without MLL1 translocations, such as SET2 and K562[1]. (1–8 µM; 7 days) According to the quantification of Annexin V positive cells, TFA also causes apoptosis in MLL leukemia cells but not in K562 cells[1]. MLL fusion-driven transcriptional pathways are suppressed by AS-99 TFA[1]. Compared to the cells treated with DMSO, AS-99 produces less H3K36me2 peaks[1].
In vitro, AS-99 TFA shows no significant inhibition on any of 20 tested histone methyltransferases (including NSD1, NSD2, NSD3, and SETD2) at 50 microM, indicating >100-fold selectivity for ASH1L. It affects MLL leukemia cell growth (MV4;11, MOLM13, KOPN8, RS4;11) with GI50 values of 1.8-3.6 microM. At 1-8 microM for 7 days, it induces apoptosis in MLL leukemia cells but not in K562 cells, as confirmed by Annexin V staining, and suppresses MLL fusion-driven transcriptional programs. |
| ln Vivo |
In mice, leukemia burden is decreased by AS-99 (30 mg/kg; ip; qd, administered for 14 consecutive days) TFA[1]. When administered intravenously (IV) and intraperitoneally (IP) to mice, AS-99 TFA exhibits good exposure in plasma (AUC = 9701 hr* ng/mL and 10,699 hr* ng/mL, respectively), a reasonable half-life (~5–6 h), and a Cmax >10 µM[1].
In vivo, AS-99 TFA (30 mg/kg; i.p.; once daily for 14 consecutive days) reduces leukemia burden in mice bearing MV4;11 MLL leukemia xenografts. It decreases the leukemia burden in this xenotransplantation mouse model without affecting blood counts in normal mice. The compound shows favorable plasma exposure and a suitable pharmacokinetic profile for in vivo efficacy studies in mice. |
| Enzyme Assay |
For AS-99 TFA, ASH1L inhibition is measured using biochemical methyltransferase assays with purified recombinant ASH1L enzyme. Various concentrations of the compound are incubated with the enzyme and its substrate (histone H3 peptide) in the presence of 3H-labeled S-adenosyl methionine (SAM). Methylation activity is quantified by measuring incorporation of 3H into the peptide substrate, allowing calculation of IC50 (0.79 microM) and Kd (0.89 microM) values.
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| Cell Assay |
RT-PCR[1]
Cell Types: MOLM13 cells Tested Concentrations: 2-6 µM Incubation Duration: 7 days Experimental Results: Led to a dose-dependent downregulation of canonical MLL fusion target genes required for leukemogenesis including MEF2C, DLX2, FLT3, and HOXA9. For AS-99 TFA, MLL leukemia cells (MV4;11, MOLM13, KOPN8, RS4;11) and control cells (K562, SET2) are cultured and treated with AS-99 (1-8 microM for 7 days). Apoptosis is measured by flow cytometry using Annexin V/propidium iodide staining. Proliferation is assessed using CellTiter-Glo viability assays. Downregulation of MLL fusion target genes (MEF2C, DLX2, FLT3, HOXA9) is measured by RT-PCR after treatment with 2-6 microM AS-99 for 7 days. |
| Animal Protocol |
Animal/Disease Models: 8- to 10 -week old female NSG mice (bearing MV4;11 cells)[1]
Doses: 30 mg/kg Route of Administration: Ip; qd, treated for 14 days Experimental Results: decreased the leukemia burden in the xenotransplantation mouse model of MLL leukemia without affecting blood counts in normal mice. For AS-99 TFA, in vivo efficacy is assessed in 8-10 week old female NSG mice bearing MV4;11 cell xenografts. AS-99 TFA (30 mg/kg) is administered intraperitoneally once daily for 14 consecutive days. Leukemia burden is measured by analyzing bioluminescence or human CD45+ cells in peripheral blood. Normal mouse blood counts are also monitored to assess myelosuppression. Pharmacokinetic parameters are measured in separate cohorts after IV and IP administration. |
| ADME/Pharmacokinetics |
AS-99 TFA shows favorable PK properties in mice, with plasma exposure upon intravenous administration showing an AUC of 9701 hr*ng/mL and intraperitoneal administration showing an AUC of 10,699 hr*ng/mL. It has a suitable half-life of approximately 5-6 hours and a Cmax greater than 10 microM. These parameters support once-daily dosing in mouse efficacy studies. Formulation for in vivo use should be optimized based on solubility properties.
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| Toxicity/Toxicokinetics |
Detailed toxicological data for AS-99 TFA are not extensively reported in standard product documentation. In mouse efficacy studies at 30 mg/kg IP for 14 days, the compound reduces leukemia burden without affecting blood counts in normal mice, suggesting a degree of selectivity. Standard safety pharmacology and genotoxicity studies would be required for therapeutic development. For laboratory research, standard chemical safety precautions for histone methyltransferase inhibitors are recommended.
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| References | |
| Additional Infomation |
AS-99 TFA is a first-in-class ASH1L histone methyltransferase inhibitor discovered by Rogawski et al. and published in Nature Communications (2021). The free base (CAS 2323623-93-2) has a molecular weight of 593.68. It blocks cell proliferation, induces apoptosis and differentiation, downregulates MLL fusion target genes (including MEF2C, DLX2, FLT3, HOXA9), and reduces leukemia burden in MLL-rearranged leukemia xenograft models. The compound shows >100-fold selectivity for ASH1L over 20 other histone methyltransferases, making it a valuable chemical probe for epigenetic research. No clinical approval has been obtained.
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| Molecular Formula |
C29H31F6N5O5S2
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| Molecular Weight |
707.71
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| Related CAS # |
AS-99 free base;2323623-93-2;AS-99
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| Appearance |
Light yellow to yellow solid powder
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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: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 :~100 mg/mL (~141.30 mM)
H2O :~12.5 mg/mL (~17.66 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (3.53 mM) 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; with sonication.
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. Solubility in Formulation 2: ≥ 2.08 mg/mL (2.94 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.4130 mL | 7.0650 mL | 14.1301 mL | |
| 5 mM | 0.2826 mL | 1.4130 mL | 2.8260 mL | |
| 10 mM | 0.1413 mL | 0.7065 mL | 1.4130 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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