| Targets |
BTR2000 is not a drug; its "target" is the Kelch domain of the E3 ubiquitin ligase adapter protein, KLHL20. It acts as a high-affinity ligand, binding with specificity to KLHL20. As a ligand for a component of the CUL3-KLHL20 E3 ubiquitin ligase complex, it serves as the "warhead" that recruits this enzyme to a target of interest. In the context of the BTR2004 PROTAC, a linker is attached to BTR2000, which is then connected to a BRD2/3/4 inhibitor. This multi-functional molecule brings the E3 ligase into close proximity with the target protein, leading to its ubiquitination and subsequent degradation by the proteasome. BTR2000 is a tool to study KLHL20-mediated targeted protein degradation.
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| ln Vitro |
The in vitro activity of BTR2000 is defined by its binding affinity for the KLHL20 Kelch domain. In a biophysical assay such as Surface Plasmon Resonance (SPR) or Isothermal Titration Calorimetry (ITC), the equilibrium dissociation constant (Kd) for the BTR2000-KLHL20 interaction is measured, revealing high-affinity binding in the low nanomolar range. In a cellular assay, the BTR2004 PROTAC is used, where its activity is measured by the degradation of BRD2/3/4. In a western blot of treated cells, BTR2004 will lead to a concentration-dependent decrease in BRD2/3/4 protein levels (e.g., DC50 < 100 nM). BTR2000 itself, lacking the target-binding moiety, will show no degradation activity.
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| ln Vivo |
In vivo, BTR2000 is not used as a therapeutic; it is used as a research tool to build a PROTAC. The in vivo activity is demonstrated with the BTR2004 PROTAC, which has been shown to degrade BRD2/3/4 in mouse tissues and exhibit anti-tumor activity in xenograft models. BTR2000 itself would have no in vivo activity. As a building block, its utility is in understanding the E3 ligase-ligand interaction and its use in creating chemical tools to degrade other proteins of interest.
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| Enzyme Assay |
General in vitro binding assay (Surface Plasmon Resonance, SPR): Recombinant KLHL20 Kelch domain protein is immobilized on a sensor chip. A series of concentrations of BTR2000 (0.1-100 nM) in running buffer are flowed over the chip. The association rate (ka) and dissociation rate (kd) are measured, and the equilibrium dissociation constant (Kd) is calculated as kd/ka. The Kd for BTR2000 will be in the low nanomolar range, confirming its high affinity. A control compound known not to bind KLHL20 is used to assess non-specific binding.
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| Cell Assay |
General in vitro PROTAC activity assay (Western Blot): HEK293T cells are seeded in 6-well plates (5×10⁵ cells/well) and treated with the BTR2004 PROTAC (0.1-1000 nM) for 6-24 hours. As a control, cells are treated with BTR2000 alone (1000 nM) or a vehicle (DMSO). After treatment, cells are lysed, and protein lysates are separated by SDS-PAGE. Membranes are probed with an anti-BRD2, anti-BRD3, or anti-BRD4 antibody and an anti-GAPDH loading control. The BTR2004 PROTAC will cause a dose-dependent reduction in the target protein levels, while BTR2000 alone will have no effect. The DC50 (concentration needed for 50% degradation) is calculated.
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| Animal Protocol |
General in vivo animal protocol for BTR2004 PROTAC (not BTR2000): BTR2004 PROTAC is dissolved in a suitable vehicle (e.g., 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline) and administered to female NCr nu/nu mice bearing xenografts of a cancer cell line (e.g., 5×10⁶ MV-4-11 cells) by intraperitoneal injection at 0, 10, 30, and 100 mg/kg every other day. Tumor volume is measured twice weekly. The PROTAC will cause dose-dependent tumor growth inhibition (TGI). For a 28-day repeat-dose toxicology study, the PROTAC would be administered to rats at 0, 5, 15, and 50 mg/kg/day to establish a NOAEL.
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| ADME/Pharmacokinetics |
BTR2000 is a small molecule (MW 765) used as a building block for a PROTAC. The pharmacokinetics of BTR2000 itself are not characterized; rather, the properties of the final PROTAC molecule, BTR2004, are determined. As a synthetic intermediate, it is not intended for in vivo administration. Therefore, no formal ADME studies are conducted on BTR2000. Its solubility and stability are characterized for use in synthesis.
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| Toxicity/Toxicokinetics |
As a research tool, BTR2000 has not undergone formal toxicity testing. The TFA salt form may contribute some low-level toxicity, but it is not considered a genotoxic impurity. The potential toxicity of the final PROTAC (BTR2004) is what is relevant for drug development. For use as a reference standard, BTR2000 is handled as a standard hazardous chemical.
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| References | |
| Additional Infomation |
BTR2000 is a specific ligand for KLHL20 (Kelch-like protein 20), a substrate adaptor protein for the Cullin3-RING ubiquitin ligase (CRL3) complex. KLHL20 is of significant interest because it is overexpressed in certain cancers and is involved in the degradation of tumor suppressors. The discovery of BTR2000 allowed for the development of BTR2004, a PROTAC degrader of the Bromodomain and Extra-Terminal (BET) family proteins (BRD2, BRD3, BRD4). This work represents an advance in targeted protein degradation, moving beyond commonly used ligands like CRBN and VHL to a new E3 ligase (KLHL20). This expands the toolbox for creating degraders for proteins that may not be effectively targeted by conventional approaches. BTR2000 TFA is stored as a powder at -20degC and is soluble in DMSO.
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| Related CAS # |
BTR2000
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| Appearance |
White to off-white 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 |
| 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 : ~62.5 mg/mL (~81.72 mM; with sonication)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (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.
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