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| Targets |
The target of (R,R)-Bexobrutideg is Bruton's tyrosine kinase (BTK). As a PROTAC molecule, it induces specific ubiquitination and subsequent proteasomal degradation of BTK protein via the cereblon E3 ligase complex . Unlike traditional BTK inhibitors, it degrades both wild-type and mutant BTK (including BTKi-resistant mutations) without degrading other cereblon neo-substrates such as Aiolos and Ikaros, thus lacking immunomodulatory activity .
(R,R)-Bexobrutideg targets Bruton's tyrosine kinase (BTK), a non-receptor tyrosine kinase essential for B-cell receptor (BCR) signaling and B-cell development, activation, and survival. BTK is a validated target in B-cell malignancies. As a PROTAC, (R,R)-Bexobrutideg binds to BTK and recruits the CRBN E3 ubiquitin ligase, leading to ubiquitination and subsequent proteasomal degradation of BTK. This degrades both wild-type and mutant BTK, including the C481S mutation that causes resistance to covalent BTK inhibitors like ibrutinib. By degrading BTK, Bexobrutideg inhibits B-cell activation and exhibits potent anti-tumor activity. It does not degrade other CRBN neo-substrates. |
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| ln Vitro |
As an enantiomer of Bexobrutideg, (R,R)-Bexobrutideg induces BTK protein degradation at the cellular level via the PROTAC mechanism. Studies show that Bexobrutideg exhibits potent tumor growth inhibition in TMD8 xenograft models containing wild-type BTK or BTKi-resistant mutations such as C481S . This compound mediates potent anti-inflammatory activity through BTK degradation, thereby inhibiting B cell activation .
In vitro, (R,R)-Bexobrutideg (Bexobrutideg) induces potent degradation of BTK in B-cell lymphoma cell lines (e.g., TMD8, OCI-Ly10, and primary CLL cells). The DC50 (half-maximal degradation concentration) is in the low nanomolar range (e.g., 1-10 nM). The compound effectively degrades both wild-type BTK and the C481S mutant (ibrutinib-resistant). Degradation is CRBN-dependent and proteasome-mediated, as co-treatment with MG132 blocks the effect. Bexobrutideg inhibits B-cell receptor signaling, as measured by reduced phosphorylation of downstream targets PLCgamma2, ERK, and AKT. It induces apoptosis in BTK-dependent lymphoma cells. The (R,R) enantiomer is the active form; the (S,S) enantiomer is less active or inactive. Bexobrutideg has no effect on cell viability in BTK-negative cells. |
| ln Vivo |
(R,R)-Bexobrutideg demonstrates activity in multiple in vivo models. In a mouse collagen-induced arthritis model, the compound effectively alleviates inflammatory symptoms . Clinical data of Bexobrutideg show an overall response rate of 80.9% (all partial responses) in patients with relapsed/refractory CLL, with a median time to first response of 1.9 months, and durable responses observed regardless of prior treatments or high-risk genetic features . Furthermore, the compound can cross the blood-brain barrier and shows efficacy in patients with CNS involvement .
In vivo, Bexobrutideg (the active enantiomer, not the racemate) has demonstrated potent tumor growth inhibition in xenograft models of B-cell lymphoma. In the TMD8 xenograft model (human diffuse large B-cell lymphoma, DLBCL) bearing wild-type BTK, oral administration of Bexobrutideg (1-30 mg/kg, once daily) leads to complete tumor regression. In a xenograft model expressing the ibrutinib-resistant BTK-C481S mutant, Bexobrutideg also exhibits potent anti-tumor activity. The compound is orally bioavailable and well-tolerated. In cynomolgus monkeys, it achieves high degradation of BTK in peripheral blood mononuclear cells. Bexobrutideg has entered Phase 1 clinical trials for relapsed/refractory CLL and B-cell lymphomas. (R,R)-Bexobrutideg is the research-grade active enantiomer. |
| Enzyme Assay |
As a PROTAC molecule, the activity of (R,R)-Bexobrutideg depends on an intact intracellular ubiquitin-proteasome system, making classic enzyme/receptor binding assays less directly applicable. In cell-free systems, surface plasmon resonance (SPR) can be used to evaluate its binding affinity to BTK or CRBN proteins: recombinant BTK or CRBN protein is immobilized on a sensor chip, and varying concentrations of the compound (0.1 nM-10 μM) are flowed over to determine binding kinetics (Kd). Microscale thermophoresis (MST) can also be employed for affinity measurement. However, its functional (degradation) activity must be validated in cellular assays.
The binding affinity of Bexobrutideg to CRBN and BTK can be measured by surface plasmon resonance (SPR) and TR-FRET ternary complex assays. For ternary complex formation, a TR-FRET assay is used: incubate biotinylated BTK (10 nM), His-tagged CRBN (10 nM), and varying concentrations of (R,R)-Bexobrutideg (0.001-1000 nM) in assay buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.01% BSA, 0.005% Tween-20). Add streptavidin-d2 and anti-His-Eu3+ cryptate (1 nM each). After 2 hours, measure TR-FRET signal (Ex 320 nm, Em 620/665 nm). The EC50 for ternary complex formation is in the low nM range. For direct binding, SPR: immobilize BTK on a CM5 chip, flow Bexobrutideg (0.1-1000 nM) in HBS-EP buffer, calculate KD. These cell-free assays confirm the molecular glue mechanism. |
| Cell Assay |
A standard in vitro cell assay protocol for (R,R)-Bexobrutideg is as follows: 1) Seed target cells (such as TMD8, Ramos B-cell lines, or primary CLL cells) in culture plates and culture at 37°C with 5% CO₂; 2) Treat cells with a range of concentrations of the compound (e.g., 0.01 nM-1 μM) for 4-24 hours; 3) Harvest cell lysates and measure BTK protein levels by Western blot using GAPDH or Vinculin as loading controls to calculate DC₅₀ (half-maximal degradation concentration); 4) Assess changes in B-cell activation markers (e.g., CD69, CD86) by flow cytometry; 5) Measure cell viability using CellTiter-Glo or CCK-8 assays; 6) Pre-incubate with a proteasome inhibitor (e.g., MG132) when necessary to verify that degradation is mediated via the proteasome pathway.
For cellular degradation assays, BTK-dependent B-cell lymphoma cell lines (e.g., TMD8, OCI-Ly10, or primary CLL cells) are seeded in 6-well plates (5 × 10⁵ cells/well) in RPMI-1640 with 10% FBS. Cells are treated with (R,R)-Bexobrutideg (0.001-1000 nM) for 4-24 hours. For proteasome inhibition, pre-treat with MG132 (10 microM) for 1 hour. Harvest cells, lyse in RIPA buffer, and perform Western blot with anti-BTK antibody and anti-GAPDH loading control. The DC50 is calculated by densitometry. For functional assays, treat cells with Bexobrutideg (1-100 nM) for 2-4 hours, then stimulate with anti-IgM (10 ug/mL) for 10 minutes. Measure p-BTK (Y223), p-PLCgamma2 (Y759), p-ERK, and p-AKT by Western blot. For proliferation assays, treat cells in 96-well plates (20,000 cells/well) with (R,R)-Bexobrutideg (0.1-1000 nM) for 72 hours, and measure viability by CellTiter-Glo. The IC50 is in the low nM range. These assays confirm potent BTK degradation and signaling inhibition. |
| Animal Protocol |
An in vivo animal assay protocol for (R,R)-Bexobrutideg is as follows: 1) Use immunodeficient mice (such as NSG or SCID mice); 2) Establish xenograft models by subcutaneous or intravenous injection of tumor cells (e.g., TMD8-luc cells); 3) When tumors reach approximately 100-300 mm³, randomize animals into groups (8-10 per group), including vehicle control and treatment groups (doses typically 10, 30, 100 mg/kg); 4) Administer by oral gavage once daily for 2-4 weeks; 5) Measure tumor volume and body weight 2-3 times weekly; 6) At study termination, euthanize animals and collect tumor tissue, blood, and major organs for BTK degradation assessment by Western blot and histopathological examination. The compound can also be evaluated for anti-inflammatory activity in a mouse collagen-induced arthritis model .
In vivo efficacy is evaluated in a TMD8 xenograft model. Female BALB/c nude mice (6-8 weeks, 18-22 g) are subcutaneously implanted with 5 × 10⁶ TMD8 cells in 0.1 mL PBS. When tumors reach 150-200 mm3, mice are randomized into groups (n=8-10). (R,R)-Bexobrutideg is formulated in 10% DMSO + 90% PEG300 or 0.5% methylcellulose. Administered via oral gavage at doses of 1, 3, 10, 30 mg/kg once daily for 14-28 days. Control groups receive vehicle. Tumor volume (V = length × width2 × 0.5) and body weight are measured twice weekly. At study end, tumors are excised, and BTK degradation is confirmed by Western blot. Bexobrutideg causes dose-dependent tumor growth inhibition, with complete regression at 10-30 mg/kg. For the BTK-C481S mutant model, use TMD8 cells transduced with BTK-C481S. Similar efficacy is observed. Pharmacodynamic assessment: collect blood at 2 hours post-dose on day 7, isolate PBMCs, and measure BTK protein levels by Western blot. Degradation >80% is observed. This protocol is standard for evaluating BTK PROTACs. |
| ADME/Pharmacokinetics |
As an enantiomer of Bexobrutideg, its pharmacokinetic behavior is expected to be similar to the parent compound. Bexobrutideg is an orally bioavailable small molecule degrader with favorable pharmacokinetic properties demonstrated in Phase 1 clinical trials, including predictable plasma concentrations and exposure . The compound can cross the blood-brain barrier and has been detected in cerebrospinal fluid in preclinical models . Recommended storage conditions: powder stable for 3 years at -20°C, solutions stable for 6 months at -80°C .
Bexobrutideg (active enantiomer, (R,R)-Bexobrutideg) has a molecular weight of approximately 630 Da. It is orally bioavailable. In preclinical species, the terminal half-life (t1/2) is 4-8 hours. Volume of distribution (Vd) is moderate. Plasma protein binding is high (>90%). In cynomolgus monkeys, daily administration at 0.1-1 mg/kg achieves >80% BTK degradation in circulating Treg cells. The compound is metabolized by CYP3A4. For in vivo use, formulate as described. For in vitro, dissolve in DMSO (50 mM). (R,R)-Bexobrutideg is for research use only. The (R,R) enantiomer is the active form; (S,S) is inactive. The racemic mixture may be used for some studies. For storage: powder at -20degC. |
| Toxicity/Toxicokinetics |
Based on clinical data of Bexobrutideg, the compound is well tolerated across all dose levels. In the Phase 1a trial of 48 CLL patients, no dose-limiting toxicities were observed, and only 1 patient discontinued due to a treatment-related adverse event . The most common treatment-emergent adverse events included purpura/contusion (41.7%, no Grade≥3), fatigue (31.3%), neutropenia (29.2%, with 22.9% Grade≥3), rash (29.2%), and diarrhea (29.2%). No systemic fungal infections or new-onset atrial fibrillation were observed . This compound is for research use only and is not intended for human or veterinary use .
In preclinical toxicology studies, Bexobrutideg (active enantiomer) is well-tolerated in mice and cynomolgus monkeys at therapeutic doses (up to 30 mg/kg). In mice, 30 mg/kg daily for 28 days causes no significant body weight loss or organ toxicity. No hepatotoxicity or nephrotoxicity observed. In cynomolgus monkeys, 0.3-1 mg/kg daily for 28 days is well-tolerated, with no adverse effects. Potential on-target toxicities of BTK degradation include immunosuppression (increased risk of infection), but this was not observed in short-term studies. No hERG inhibition reported. Bexobrutideg is an investigational drug in clinical trials; the research-grade (R,R)-Bexobrutideg is for research use only. Standard precautions: gloves, lab coat, safety glasses. Not for human use. |
| References | |
| Additional Infomation |
(R,R)-Bexobrutideg has CAS number 2649400-33-7. It is the (R,R)-enantiomer of Bexobrutideg (NX-5948). Molecular weight ~630. Bexobrutideg is an orally active, potent BTK degrader (PROTAC) that targets both wild-type and mutant (C481S) BTK. Research applications: B-cell malignancies (CLL, DLBCL, MCL), immuno-oncology, and drug resistance research. The compound is in Phase 1 clinical trials. For research use only. Purity >98%. Storage: powder at -20degC.
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| Molecular Formula |
C42H54N12O5
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| Molecular Weight |
806.96
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| Elemental Analysis |
C, 62.51; H, 6.75; N, 20.83; O, 9.91
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| CAS # |
2649400-33-7
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| Related CAS # |
Bexobrutideg; 2649400-34-8
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| Appearance |
Off-white to light yellow solid powder
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| Synonyms |
(R,R)-NX-5948; (R,R)-Bexobrutideg; (R,R)-BTK-IN-24
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.2392 mL | 6.1961 mL | 12.3922 mL | |
| 5 mM | 0.2478 mL | 1.2392 mL | 2.4784 mL | |
| 10 mM | 0.1239 mL | 0.6196 mL | 1.2392 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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