BCL-XL

DT2216 is a Von Hippel-Lindau (VHL) E3 ligase-targeted BCL-XL proteolysis targeting chimera (PROTAC) that directs BCL-XL for degradation. Because VHL is poorly expressed in platelets, DT2216 is significantly less toxic to platelets than ABT263, making it more effective against a variety of BCL-XL-dependent leukemia and cancer cells. [1]

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DT2216 selectively kills Bcl-xL-dependent TCL cell lines by degrading Bcl-xL in a VHL-dependent manner. DT2216 can synergistically kill TCL PDX cells in combination with ABT199 in vitro. [2]

As a single agent or in combination with other chemotherapeutic drugs, DT2216 effectively slows the growth of several xenograft tumors in vivo without significantly increasing thrombocytopenia.[1]
DT2216 is a more potent antitumor agent than ABT263 with reduced platelet toxicity in vivo.[1]
Increased antitumor activity of DT2216 in combination with other BCL-2 family protein inhibitors.[1]
Synergy between DT2216 and conventional chemotherapy.[1]

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In vivo, DT2216 alone was highly effective against MyLa TCL xenografts in mice without causing significant thrombocytopenia or other toxicity. Furthermore, DT2216 combined with ABT199 (a selective Bcl-2 inhibitor) synergistically reduced disease burden and improved survival in a TCL PDX mouse model dependent on both Bcl-2 and Bcl-xL.[2]

AlphaScreen for the determination of DT2216-BCL-XL, BCL-2 and BCL-W binding affinity[1]
To evaluate the binding affinities of DT2216 and ABT263 towards BCL-XL, BCL-2 and BCL-W the AlphaScreen competitive binding assays were performed. The assay was performed at room temperature and reagents were diluted in a buffer containing 250 mM HEPES pH 7.5, 1 M NaCl, 1% BSA, and 0.05% Tween-20. Purified recombinant His-tagged BCL-XL (0.1 nM), BCL-2 (0.2 nM) or BCL-W (0.2 nM) were incubated with increasing concentrations of DT2216 or ABT263 and 15 nM biotin-tagged BAD (Biotin-LWAAQRYGRELRRMSDEFEGSFKGL N-terminal) or 30 nM BIM peptides (Biotin-MRPEIWIAQELRRIGDEFNA N-terminal) to a final volume of 40 μL in 96-well PCR plate. BAD peptide was used to assess the binding affinity of compounds towards BCL-XL whereas BIM peptide was used for BCL-2 and BCL-W. After 24 h incubation, 5 μL 6X His-acceptor beads were added to each well at 20 μg/mL final concentration and incubated for 1 h. Thereafter, 5 μL streptavidin-donor beads were added to each well at 20 μg/mL final concentration and incubated for 30 min. At the end of the incubation period, 17 μL of each sample was transferred in adjacent wells of 384-well proxy plate. The plate was scanned using Alpha program on Biotek’s Synergy Neo2 multi-mode plate reader. The inhibition constant (Ki) was calculated using non-linear regression, one site, competitive binding, Fit Ki function on GraphPad Prism 7 software based on experimentally determined Kd for each protein/peptide pair.[1]

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AlphaLISA assay for ternary complex formation[1]
We used AlphaLISA assay to monitor ternary complex formation between target protein, PROTAC and E3 ligase, as described previously44, 58. To validate the formation of the ternary complex of BCL-XL/BCL-2, DT2216 and the VHL E3 ligase, a fixed concentration of His-tagged recombinant proteins (100 nM BCL-XL and10 nM BCL-2) and recombinant active GST-tagged VHL/Elongin B/C complex (50 nM for BCL-XL, 5 nM for BCL-2) was incubated with varying concentrations of test compounds in 4-fold serial dilutions to a final volume of 40 μL in 96-well PCR plate. After 30 min incubation at room temperature, 5 μL Alpha Glutathione-donor beads were added to each well at 20 μg/mL final concentration and incubated for 15 min. Thereafter, 5 μL 6X His-acceptor beads were added to each well at 20 μg/mL final concentration and incubated for an additional 45 min at room temperature. Thereafter, 17 μL of each sample was transferred in adjacent wells of 384-well proxy plate and the plate was scanned using Alpha program on Biotek’s Synergy Neo2 multi-mode plate reader. The data were expressed as average AlphaLISA signal and plotted against different concentrations of compounds.

The indicated concentrations of DT2216 or ABT263 are then applied to MOLT-4 cells for 24 hours after they have been seeded in 60 mm dishes (2.5 × 106 cells in 5 mL complete cell culture medium/dish). A freeze-thaw cycle in an ice-ethanol bath or a 30-minute incubation on ice are both effective ways to lyse cells in 1X cell lysis buffer.[1]

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Cellular thermal shift assay (CETSA)[1]
CETSA assay was adapted from Smith et al. Briefly, 2.5 × 107 MOLT-4 or RS4 cells were treated with 1 μM DT2216 or DMSO for 6 h, then harvested, washed with PBS, and resuspended in PBS containing protease and phosphatase inhibitors. For MOLT-4 cells, 10 μM MG132 was added in to prevent the degradation of BCL-XL. The resuspended cells were freeze–thawed four times with liquid nitrogen. After each freeze–thaw cycle, lysate was vortexed briefly to ensure homogenous thawing. The soluble fraction was separated from cell debris by centrifugation at 20,000 ×g for 30 min at 4 °C and then heated at gradient temperature from 42 °C to 69.5 °C for 3 min and cooled down to 25 °C for another 3 min. The treated samples were centrifugated at 20,000 ×g for 30 min at 4 °C to remove the denatured proteins and then analyzed by immunoblotting.

Pharmacodynamics of BCL-XL degradation by DT2216[1]
MOLT-4 xenografts were established in female CB-17 SCID-beige mice as described in following methods. Tumor-bearing mice were treated with single injection of vehicle or DT2216 (15 mpk/i.p.) when the tumors were ~600 mm3. Two mice each from vehicle and DT2216-treated groups were euthanized at each time point as indicated in the figure legend of Fig. 4b and tumors were harvested. The proteins were extracted from tumors and used for BCL-XL degradation by immunoblot analysis. Some portion of these tumors were used for DT2216 analysis as described in the previous method.

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In vivo platelet toxicity assays[1]
Single dosing with DT2216 or ABT263[1]
Female 5–6 weeks old CB-17 SCID-beige mice were treated with single i.p. doses of DT2216 (7.5, 15 and 25 mpk) or single p.o. doses of ABT263 (25, 50 and 100 mpk). Approximately 50 μL of blood was collected at different time points from each mouse as mentioned in Fig. 4c legend via submandibular plexus route in EDTA-tubes and platelets were enumerated using an automated hematology analyzer HEMAVET 950FS.[1]

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Daily dosing with ABT263 or once a week DT2216[1]
Female CB-17 SCID-beige mice were treated with ABT263 (50 mpk/day/p.o.) or DT2216 (15 mpk/week/i.p.). Approximately 50 μL of blood was collected from each mouse 6 h (0.25 d) after each dose of ABT263 or collected from each mouse after 0.25 d, 1.25 d, 2.25 d, 3.25d, 4.25 d, 5.25 d, 6.25 d, 7.25 d, 8.25 d, 9.25 d, 10.25 d, 11.25 d, 12.25 d, 17.25 d and 24.25 d of once a week dosing with DT2216. Platelets were enumerated using HEMAVET 950FS. MOLT-4 T-ALL xenograft mouse model[1]
To test the effect of DT2216 on tumor growth in MOLT-4 T-ALL xenografts, MOLT-4 T-ALL cells were harvested and suspended in regular RPMI medium and mixed with Matrigel (1:1). The cells (5 × 106 cells) suspended in 100 μL of RPMI medium-Matrigel mixture were subcutaneously (s.c.) implanted in the right flank of CB-17 SCID mice. Tumor growth was monitored daily and tumors were measured twice a week using Vernier caliper or digital calipers. Tumor volume was determined using the formula; [(L × W2) × 0.5], where L is length/long dimension in millimeter (mm) and W is the width/short dimension in mm. The treatment started once the average tumor volume reached 150–200 mm3. The animals were randomly assigned into separate groups (n = 6–8) in a way that each group had nearly equal starting average tumor volume. Mice were weighed twice a week and the treatments were given according to average mouse weight within each group before initiation of treatment. DT2216 and ABT263 for i.p. administration were formulated in 50% PHOSAL 50 PG, 45% MIGLYOL® 810 N and 5% Polysorbate 80. DT2216 and ABT263 were administered via i.p. injection at 15 mpk/week in 100 μL vehicle (Extended Data Fig. 8). ABT263 for oral administration was formulated in 10% ethanol, 30% PEG 400 and 60% PHOSAL 50 PG (Fig. 4). Control mice received 100 μL vehicle via i.p. injection. The mice were euthanized when the maximum tumor size in a mouse reached the humane endpoint according to institutional policy concerning tumor endpoints in rodents. In addition, to prevent excessive pain or distress, the mice were euthanized if the tumors became ulcerated or the mice showed any signs of ill health. Mice were euthanized by CO2 suffocation followed by cervical dislocation and various tissues including tumors were harvested for further analyses.

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H146 SCLC xenograft model[1]
To test the effect of DT2216 alone or in combination with ABT199 on tumor growth in H146 SCLC xenografts, 5 × 106 H146 SCLC cells were suspended in regular RPMI medium, mixed with Matrigel, and s.c. implanted in the right flank of female CB-17 SCID mice as described in the previous method. Tumor growth was monitored, tumors were measured, and the tumor volume was determined as mentioned in the above method. The treatment started after four weeks of cell implantation as shown in Fig. 5c. The animals were randomly assigned into groups treated with vehicle, DT2216 15 mpk/week (i.p. injection), ABT263 15 mpk/week (i.p. injection), ABT199 50 mpk/day (oral administration), and DT2216 + ABT199. DT2216 and ABT263 were formulated as described above. ABT199 was formulated for oral dosing in 60% PHOSAL 50 PG, 30% polyethylene glycol (PEG) 400 and 10% ethanol. All the animals were euthanized in accordance with the institutional policy and various tissues were harvested. Tumors were weighed and used for BCL-XL, BCL-2 and MCL-1 expression by immunoblotting.

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MDA-MB-231 BC xenograft model[1]
To test the effect of DT2216 in combination with Docetaxel on tumor growth in MDA-MB-231 breast cancer xenografts, 5 × 106 MDA-MB-231 cells were suspended in regular RPMI medium, mixed with Matrigel, and s.c. implanted in the right flank of female NOD-SCID mice. The animals were randomly assigned to vehicle, DT2216 (15 mpk/q4d/i.p.), Docetaxel (7.5 mpk/q14d/i.v.) and DT2216 + Docetaxel groups when the average tumor volume reached ~ 130 mm3. Docetaxel was dissolved in 5% DMSO + 30% PEG 300 + 5% Tween 80 + 60% dH2O. The solution was filter sterilized to obtain clear solution for intravenous administration. DT2216 was administered two days before starting dosing with Docetaxel, as also mentioned in Fig. 6c.

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T-ALL PDX models[1]
To establish T-ALL PDX mouse models, 8 weeks old NSG mice were sublethally irradiated (0.25 Gy) 24 h prior to cell inoculation. PDX cells (1 × 106) were suspended in PBS and injected into mice through the tail vein. Tumor engraftment was determined by co-staining for human and murine anti-CD45 in bone marrow aspiration samples (CUL76 and D115 T-ALL) or in peripheral blood (332X-Luci) at 10 days post injection. Mice were randomized into different groups. CUL76 PDX were treated with DT2216 (15 mpk/q4d/i.p.), ABT199 (100 mpk/day/p.o.), chemotherapy (vincristine 0.15 mpk + dexamethasone 5 mpk + L-asparaginase 1000 Units/kg, i.p., weekly), combination of DT2216 and ABT199 or chemotherapy, or ABT199 plus chemotherapy as shown in Fig. 6f. D115 and 332X-Luci PDXs were treated with DT2216, chemotherapy, or combination of DT2216 and chemotherapy as shown in Extended Data Fig. 10. Mice were monitored for disease progression by weekly assessing leukemic (hCD45+) cells in the peripheral blood or bone marrow aspirates and followed for survival.

[1]. A selective BCL-XL PROTAC degrader achieves safe and potent antitumor activity. Nat Med. 2019 Dec;25(12):1938-1947.

[2]. DT2216—a Bcl-xL-specific degrader is highly active against Bcl-xL-dependent T cell lymphomas. J Hematol Oncol. 2020; 13: 95.

Bcl-XL Proteolysis Targeting Chimera DT2216 is an anti-apoptotic protein B-cell lymphoma-extra large (Bcl-XL) targeted protein degrader, using the proteolysis targeting chimera (PROTAC) technology, with potential pro-apoptotic, immunomodulating and antineoplastic activities. DT2216 is composed of a Bcl-XL ligand attached to a Von Hippel-Lindau (VHL) E3 ligase ligand. Upon administration of DT2216, the Bcl-XL binding moiety specifically targets and binds to Bcl-XL which is expressed on tumor-infiltrating regulatory T-cells (Tregs) in the tumor microenvironment (TME) and cancer cells that are dependent on Bcl-XL for their survival, such as certain Bcl-XL-dependent T-cell malignancies. In turn, the VHL E3 ligase ligand is recruited to the endoplasmic reticulum (ER) and Bcl-XL is tagged by ubiquitin. This causes ubiquitination and proteasome-mediated degradation of Bcl-XL. The degradation of Bcl-XL leads to an inhibition of the anti-apoptotic activity of Bcl-XL and restores apoptotic processes in and causes depletion of Bcl-XL-expressing Tregs and Bcl-XL-dependent cancer cells. Reduction of Tregs may activate anti-tumor CD8-positive-mediated immune responses. This leads to the inhibition of tumor growth. Bcl-XL, a protein belonging to the Bcl-2 family, plays an important role in the negative regulation of apoptosis. Their expression in tumors is associated with increased Tregs survival. Tregs play a key role in cancer progression and tumor immunosuppression. Compared to other Bcl-XL inhibitors, DT2216 does not cause platelet toxicity as the VHL E3 ligase is not highly expressed in platelets.

C77H96CLF3N10O10S4

1542.3554

1540.58

C, 59.96; H, 6.27; Cl, 2.30; F, 3.70; N, 9.08; O, 10.37; S, 8.31

2365172-42-3

2365172-42-3;DT2216 HCl; DT2216NC; DT2216-isomer;

139331475

White to light yellow solid powder

12.8

5

20

29

105

3130

5

CC1=C(SC=N1)C2=CC=C(C=C2)[C@H](C)NC(=O)[C@@H]3C[C@H](CN3C(=O)[C@H](C(C)(C)C)NC(=O)CCCCCC(=O)N4CCN(CC4)CC[C@H](CSC5=CC=CC=C5)NC6=C(C=C(C=C6)S(=O)(=O)NC(=O)C7=CC=C(C=C7)N8CCN(CC8)CC9=C(CCC(C9)(C)C)C1=CC=C(C=C1)Cl)S(=O)(=O)C(F)(F)F)O

PXVFFBGSTYQHRO-REQIQPEASA-N

InChI=1S/C77H96ClF3N10O10S4/c1-51(53-18-20-55(21-19-53)70-52(2)82-50-103-70)83-73(96)66-44-61(92)48-91(66)74(97)71(75(3,4)5)85-68(93)16-12-9-13-17-69(94)90-42-36-87(37-43-90)35-33-59(49-102-62-14-10-8-11-15-62)84-65-31-30-63(45-67(65)104(98,99)77(79,80)81)105(100,101)86-72(95)56-24-28-60(29-25-56)89-40-38-88(39-41-89)47-57-46-76(6,7)34-32-64(57)54-22-26-58(78)27-23-54/h8,10-11,14-15,18-31,45,50-51,59,61,66,71,84,92H,9,12-13,16-17,32-44,46-49H2,1-7H3,(H,83,96)(H,85,93)(H,86,95)/t51-,59+,61+,66-,71+/m0/s1

(2S,4R)-1-[(2S)-2-[[7-[4-[(3R)-3-[4-[[4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]benzoyl]sulfamoyl]-2-(trifluoromethylsulfonyl)anilino]-4-phenylsulfanylbutyl]piperazin-1-yl]-7-oxoheptanoyl]amino]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

DT2216 HCl; DT-2216; DT 2216; DT2216; 2365172-42-3; DT-2216; (2s,4r)-1-((s)-2-(7-(4-((r)-3-((4-(N-(4-(4-((4'-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-4-(phenylthio)butyl)piperazin-1-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((s)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide; Y8JQ9V7JAJ; (2S,4R)-1-[(2S)-2-[[7-[4-[(3R)-3-[4-[[4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]benzoyl]sulfamoyl]-2-(trifluoromethylsulfonyl)anilino]-4-phenylsulfanylbutyl]piperazin-1-yl]-7-oxoheptanoyl]amino]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide; UNII-Y8JQ9V7JAJ; CHEMBL4745523; DT2216

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~100 mg/mL (~64.8 mM)
Ethanol: ~100 mg/mL (~64.8 mM)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)