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Purity: ≥98%
BAY-299 (BAY299) is a novel, highly potent, and selective dual inhibitor of BRD1 and TAF1 with important biological activity. It inhibits BRPF2 bromodomains (BD), TAF1 BD2 and TAF1L BD2 with IC50s of 67 nM, 8 nM, and 106 nM, respectively. Bromodomains (BD) are readers of lysine acetylation marks present in numerous proteins associated with chromatin. The substituted benzoisoquinolinedione series was identified by high-throughput screening, and subsequent structure-activity relationship optimization allowed generation of low nanomolar BRPF2 BD inhibitors with strong selectivity against BRPF1 and BRPF3 BDs. In addition, a strong inhibition of TAF1/TAF1L BD2 was measured for most derivatives. The best compound of the series was BAY-299, which is a very potent, dual inhibitor with an IC50 of 67 nM for BRPF2 BD, 8 nM for TAF1 BD2, and 106 nM for TAF1L BD2. Importantly, no activity was measured for BRD4 BDs. Furthermore, cellular activity was evidenced using a BRPF2- or TAF1-histone H3.3 or H4 interaction assay.
| Targets |
BAY-299 functions as a dual inhibitor, targeting both the TATA box-binding protein-associated factors TAF1 and TAF1L as well as the bromodomain and PHD finger (BRPF) family member BRPF2. With an IC50 of 67 nM and a selectivity that is 47 and 83 times greater than that of BRPF1 and BRPF3 BD, respectively, the TR-FRET test demonstrated that BAY-299 is a strong inhibitor of BRPF2 BD. The AlphaScreen test provided additional confirmation of BAY-299's properties. An IC50 of 97 nM was found, suggesting 23- and 25-fold selectivity over BRPF1 and BRPF3 BD, respectively. With IC50 values of 575 and 825 nM, respectively, BAY-299 inhibits the interaction of BRPF2 BD with histones H4 and H3.3, according to NanoBRET studies. The IC50 values for TAF1 BD2 were 970 and 1400 nM, in that order. At doses up to 10 μM, BAY-299 showed no inhibitory effect on the interaction between BRPF1 or BRD4 and histone H4. The MOLM-13, MV4-11, 769-P, Jurkat, NCI-H526, CHL-1, and 5637 cell growth is inhibited by BAY-299, with corresponding GI50 values of 1060, 2630, 3210, 3900, 6860, 7400, and 7980 nM [1].
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| ln Vitro |
BAY-299 functions as a dual inhibitor, targeting both the TATA box-binding protein-associated factors TAF1 and TAF1L as well as the bromodomain and PHD finger (BRPF) family member BRPF2. With an IC50 of 67 nM and a selectivity that is 47 and 83 times greater than that of BRPF1 and BRPF3 BD, respectively, the TR-FRET test demonstrated that BAY-299 is a strong inhibitor of BRPF2 BD. The AlphaScreen test provided additional confirmation of BAY-299's properties. An IC50 of 97 nM was found, suggesting 23- and 25-fold selectivity over BRPF1 and BRPF3 BD, respectively. With IC50 values of 575 and 825 nM, respectively, BAY-299 inhibits the interaction of BRPF2 BD with histones H4 and H3.3, according to NanoBRET studies. The IC50 values for TAF1 BD2 were 970 and 1400 nM, in that order. At doses up to 10 μM, BAY-299 showed no inhibitory effect on the interaction between BRPF1 or BRD4 and histone H4. The MOLM-13, MV4-11, 769-P, Jurkat, NCI-H526, CHL-1, and 5637 cell growth is inhibited by BAY-299, with corresponding GI50 values of 1060, 2630, 3210, 3900, 6860, 7400, and 7980 nM [1].
BAY-299 demonstrated potent and selective cellular target engagement. - In NanoBRET assays, it disrupted the interaction between BRPF2 BD and histone H4 with an IC₅₀ of 575 nM, and with histone H3.3 with an IC₅₀ of 825 nM. - It also disrupted the interaction between TAF1 BD2 and histone H4 (IC₅₀ = 970 nM) and histone H3.3 (IC₅₀ = 1400 nM). - No inhibitory effect was observed on the interactions of BRPF1 BD or BRD4 BD with histone H4 up to 10 µM. - Antiproliferative activity was generally low across a panel of cell lines. Weak inhibition was observed in some leukemia cell lines. [1] |
| ln Vivo |
Research on the pharmacokinetic characteristics of BAY-299 in rats reveals that the drug has a large steady-state volume of distribution, a long to very long terminal half-life (t1/2=10 hours), a low blood clearance rate (about 17% of hepatic blood flow), and a high bioavailability (F=73%). Although in vivo blood clearance was less than anticipated based on hepatocyte data, it was in line with estimates based on rat liver microsomal values [1].
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| Enzyme Assay |
Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding competition assays were performed.
Compounds were serially diluted in DMSO and dispensed into assay plates. His-tagged or GST-tagged bromodomain proteins (e.g., BRPF2, TAF1 BD2) in assay buffer were added and incubated. Subsequently, biotinylated acetylated histone H4 peptides and detection reagents (e.g., anti-His-XL665 and streptavidin-Eu, or anti-GST-Tb cryptate and streptavidin-XL665) were added. After incubation, TR-FRET signals were measured using a microplate reader. IC₅₀ values were calculated from the dose-response curves. [1] Isothermal Titration Calorimetry (ITC) was used to determine binding affinity (KD). Protein and compound solutions were prepared in buffer. The compound in the cell was titrated with the protein from the syringe (or vice versa). Heat changes upon binding were measured, and data were fitted to a single-site binding model to obtain thermodynamic parameters. [1] Thermal Shift Assays (TSA) were conducted. Proteins were mixed with the compound and a fluorescent dye (SYPRO Orange) in buffer. The mixture was subjected to a temperature gradient in a real-time PCR instrument. The shift in protein melting temperature (ΔTm) in the presence of the compound indicated binding. [1] |
| Cell Assay |
Cellular target engagement was assessed using the NanoBRET assay.
Expression vectors encoding Nanoluc luciferase fused to the bromodomain of interest (e.g., BRPF2 BD, TAF1 BD2) and HaloTag fused to full-length histone H4 or H3.3 were transfected into HCT116 cells. Cells were treated with the HaloTag ligand and then incubated with serially diluted compounds for 4 or 24 hours. BRET signals were measured, and IC₅₀ values for inhibition of the protein-histone interaction were calculated. [1] Cell proliferation assays were performed. Various cell lines were treated with the compound during logarithmic growth. Cell viability was determined after a defined period using a metabolic dye (AlamarBlue). [1] Permeability was assessed using Caco-2 cell monolayers. Cells were grown on transwell inserts for 15 days. The test compound was added to either the apical or basolateral compartment. Samples were taken from both compartments before and after a 2-hour incubation. Compound concentrations were analyzed by LC-MS/MS, and apparent permeability (Papp) was calculated. [1] |
| Animal Protocol |
The in vivo pharmacokinetic properties of BAY-299 were evaluated in male Wistar rats.
For intravenous (i.v.) administration, the compound was formulated as a solution and administered as a bolus. Blood samples were collected at multiple time points post-dose (e.g., 2 min to 24 h). For intragastric (p.o.) administration, the compound was given to fasted rats, and blood samples were collected similarly. Blood was centrifuged to obtain plasma. Plasma samples were processed (protein precipitation with acetonitrile) and analyzed using a validated LC-MS/MS method. Pharmacokinetic parameters were calculated by non-compartmental analysis. [1] |
| ADME/Pharmacokinetics |
BAY-299 exhibited high metabolic stability in liver microsomes of mice, rats, dogs, and humans (Clint < 0.19 L/h/kg). However, its stability in rat and dog hepatocytes was lower (Clint 3.8 and 1.5 L/h/kg, respectively), suggesting possible phase II metabolism. It showed high permeability in Caco-2 cell monolayers (Papp(AB) = 106 nm/s) with no significant efflux (BA/AB ratio 1.9). Plasma protein binding was low in mice (free fraction = 0.58%) and moderate in humans (free fraction = 4.3%). In rats, after intravenous administration, plasma clearance was low (0.73 L/h/kg), with a high steady-state volume of distribution (1.5 L/kg) and a large terminal volume of distribution. It has a long half-life (10 hours) and high oral bioavailability (73%). [1]
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| References | |
| Additional Infomation |
BAY-299 was discovered through high-throughput screening and subsequent optimization of the benzisoquinoline dione series of compounds. It is the first reported inhibitor of selective BRPF2 BD with superior inhibitory activity to paralogs such as BRPF1 and BRPF3.
The cocrystal structure of the closely related analogues to the BRPF2 BD domain revealed key interactions, including a hydrogen bond with Ser592, which is crucial for its selectivity for BRPF1 (Pro at the corresponding position). The compound exhibits a transisomerism due to rotational restriction; the racemic version was used as a chemical probe. A negative control compound (BAY-364) with very low activity was synthesized for comparative studies. BAY-299 is considered a high-quality chemical probe for studying the physiological and pathological effects of BRPF2 and TAF1/TAF1L. [1] |
| Exact Mass |
429.168
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|---|---|
| CAS # |
2080306-23-4
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| PubChem CID |
122705990
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
688.7±55.0 °C at 760 mmHg
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| Flash Point |
370.3±31.5 °C
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| Vapour Pressure |
0.0±2.3 mmHg at 25°C
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| Index of Refraction |
1.697
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| LogP |
2.3
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
32
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| Complexity |
787
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
OFWWWKWUCDUISA-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H23N3O4/c1-14-12-20-21(27(3)25(32)26(20)2)13-19(14)28-23(30)17-8-4-7-16-15(6-5-11-29)9-10-18(22(16)17)24(28)31/h4,7-10,12-13,29H,5-6,11H2,1-3H3
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| Chemical Name |
6-(3-Hydroxypropyl)-2-(1,3,6-trimethyl-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
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| Synonyms |
BAY299; BAY 299; BAY-299.
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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 : ~25 mg/mL (~58.21 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% 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.) |
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.
omparison of5with previously described BRPF inhibitors.J Med Chem. 2017 May 11;60(9):4002-4022. |
Screening strategy for the discovery of6and identification of5.J Med Chem. 2017 May 11;60(9):4002-4022. |
Binding mode of7in BRPF2 BD and basis for selectivity. (A) Cartoon representation of BRPF2 BD domain (white) with key interacting side-chains shown in stick representation with C, N, and O atoms colored white, blue, and red, respectively.J Med Chem. 2017 May 11;60(9):4002-4022. |
Atropisomerism. (A) Atropisomers5aand5bof5.J Med Chem. 2017 May 11;60(9):4002-4022. |
Biophysical selectivity profile of5 |
Cellular activity of5and31in NanoBRET assays.J Med Chem. 2017 May 11;60(9):4002-4022. |
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