PROTAC MLKL Degrader-1 (Compound 36): Human MLKL (dissociation constant \(K_{d}<10 nM\)) and Cereblon (CRBN); no detectable binding to human RIPK1 and RIPK3 up to 10 μM [1]

1. PROTAC MLKL Degrader-1 (Compound 36) exhibits potent dose-dependent MLKL degradation activity in HT29 cells: the degradation half-maximal concentration (DC₅₀) is 2.4 ± 0.5 μM, and the maximum degradation rate (Dmax) exceeds 90% at 10 μM, with only 7% ± 3% of MLKL remaining [1]
2. In the TSZ-induced necroptosis model of HT29 cells, PROTAC MLKL Degrader-1 (Compound 36) shows dose-dependent anti-necroptotic activity, achieving complete cell protection at 10 μM with an EC₅₀ of 1.2 μM [1]
3. PROTAC MLKL Degrader-1 (Compound 36) has MLKL degradation activity in multiple human cell lines: it exhibits comparable degradation efficacy in stomach cancer MKN74 cells and kidney epithelial HK-2 cells to that in HT29 cells, but significantly attenuated activity in primary keratinocyte HEK-a cells and mouse fibroblast NIH-3T3 cells [1]
4. The MLKL degradation by PROTAC MLKL Degrader-1 (Compound 36) is time-dependent in HT29 cells: within 5 hours of treatment, less than 10% of MLKL remains, maximal degradation is reached before 8 hours, and the low MLKL level is sustained for 24 hours; the degradation effect can last up to 72 hours [1]
5. PROTAC MLKL Degrader-1 (Compound 36) does not affect the levels of RIPK1 and RIPK3 in HT29 cells, nor does it modulate the phosphorylation of RIPK1 and RIPK3 induced by TSZ [1]
6. The MLKL degradation activity of PROTAC MLKL Degrader-1 (Compound 36) is proteasome-dependent: preincubation with the proteasome inhibitor MG-132 completely inhibits its degradation of MLKL [1]
7. The anti-necroptotic activity of PROTAC MLKL Degrader-1 (Compound 36) can be antagonized by MLKL ligand 1 and CRBN ligand lenalidomide: co-incubation with these ligands reduces the MLKL degradation ability and anti-necroptotic activity of Compound 36, while the ligands alone have no effect on MLKL levels or cell protection [1]
8. There is an approximately linear correlation between the extent of MLKL degradation by PROTAC MLKL Degrader-1 (Compound 36) and its anti-necroptotic efficacy [1]

1. Kinase binding assay for PROTAC MLKL Degrader-1 (Compound 36): The dissociation constants (\(K_{d}\)) of the compound against MLKL, RIPK1, and RIPK3 are determined through a kinase binding assay. The assay is conducted to evaluate the binding affinity of the compound to the target kinases, with the result showing potent binding to MLKL (\(K_{d}<10 nM\)) and no detectable binding to RIPK1 and RIPK3 at concentrations up to 10 μM [1]
2. Docking assay for PROTAC MLKL Degrader-1 (Compound 36): The crystal structure of MLKL (PDB: 5KNJ) is imported and prepared, and the compound is imported as a SMILES string, minimized, and docked using a "very accurate" calculation method. A docking constraint is applied to the carbonyl oxygen of the hinge binding residue Cys-286 to prioritize poses incorporating this key hydrogen bond interaction, and the top 10 poses are ranked based on LF Rank Score and LF dG [1]

1. MLKL degradation and protein level detection assay for PROTAC MLKL Degrader-1 (Compound 36): HT29, MKN74, HK-2, and HEK-a cells are seeded in appropriate well plates 24 hours before treatment. The compound is diluted in starvation media and incubated with cells at 37℃ and 5% CO₂ for 24 hours (or specified time points for time-course experiments). After treatment, cells are lysed with lysis buffer containing phosphatase and protease inhibitor cocktails for 30 minutes, and centrifuged to remove insoluble fractions. The protein concentration of the lysate is determined by a BCA assay, and samples are diluted to equal concentrations. MLKL, RIPK1, and RIPK3 levels are analyzed using a capillary immunoassay (JESS system) with specific antibodies, and the results are normalized to total protein signal and reported as a percentage of the vehicle control [1]
2. TSZ-induced necroptosis inhibition assay for PROTAC MLKL Degrader-1 (Compound 36): HT29 cells are seeded in 96-well plates at 2×10⁴ cells/well in 100 μL media and incubated at 37℃ and 5% CO₂ for 24 hours. The compound is diluted to indicated concentrations in starvation media containing TNF-α (20 ng/mL), SMAC mimetic LCL-161 (1 μM), and ZVAD-FMK (20 μM). The original media is aspirated, and replaced with the compound-containing media or DMSO vehicle (as high and low controls). The plate is incubated for 24 hours, then cell viability is determined by ATP quantification using a cell viability assay reagent. Luminescence is detected with a microplate reader, and the efficacy (\(E_{TSZ}\)) is calculated by normalizing to control wells [1]
3. Proteasome dependence verification assay for PROTAC MLKL Degrader-1 (Compound 36): HT29 cells are preincubated with MG-132 (3 μM) or DMSO vehicle for 30 minutes, then treated with Compound 36 (10 μM) alone or in combination with MG-132. After 8 hours of incubation, cells are lysed, and MLKL levels are detected by capillary immunoassay to verify if the degradation effect is dependent on the proteasome [1]
4. Competition assay for PROTAC MLKL Degrader-1 (Compound 36): HT29 cells are treated with Compound 36 alone or in combination with MLKL ligand 1 or CRBN ligand lenalidomide. After 24 hours of incubation, MLKL levels are detected by capillary immunoassay, and TSZ-induced necroptosis assay is performed to evaluate the anti-necroptotic activity, so as to verify the targeting specificity of Compound 36 [1]

[1]. Rathje OH, Perryman L, Payne RJ, Hamprecht DW. PROTACs Targeting MLKL Protect Cells from Necroptosis. J Med Chem. 2023 Aug 24;66(16):11216-11236.

1. PROTAC MLKL Degrader-1 (Compound 36) is a bifunctional PROTAC molecule composed of a pyrazole carboxamide-based MLKL ligand, a CRBN ligand (phenyl dihydrouracil derivative), and a linker with a rigid spiro-piperidine motif [1]
2. The chemical structure of PROTAC MLKL Degrader-1 (Compound 36) has a molecular formula of \(C_{46}H_{55}F_{2}N_{9}O_{9}S\), a molecular weight of 948.30 (m/z [M+H]⁺), and a purity of ≥95% as determined by UHPLC (retention time \(R_{t}=5.73\) min under conditions A) [1]
3. PROTAC MLKL Degrader-1 (Compound 36) exerts its anti-necroptotic effect by recruiting CRBN E3 ligase to MLKL, inducing ubiquitination and subsequent proteasomal degradation of MLKL, thereby blocking the necroptotic pathway [1]
4. The design of PROTAC MLKL Degrader-1 (Compound 36) is based on a high-affinity pyrazole carboxamide-based MLKL ligand, which has nanomolar binding affinity to MLKL and more than 1000-fold selectivity over RIPK1 and RIPK3 [1]
5. PROTAC MLKL Degrader-1 (Compound 36) shows no significant impact on cell viability in the absence of TSZ challenge, even when combined with lenalidomide or MLKL ligand 1 [1]

C46H55F2N9O9S

948.045616388321

947.38115186

3032600-90-8

168476062

Off-white to light yellow solid powder

5.5

5

14

19

67

1800

0

COC1C(N2C(=O)NC(=O)CC2)=CC(C(N2CCC3(CCN(C(CCCCCCCOC4C=CC=C(NC5NN=C(C6C=CC(NS(C(F)F)(=O)=O)=CC=6)C=5C(=O)N)C=4)=O)CC3)CC2)=O)=CC=1

XDPRKJSOJYMCMZ-UHFFFAOYSA-N

InChI=1S/C46H55F2N9O9S/c1-65-36-16-13-31(28-35(36)57-22-17-37(58)51-45(57)62)43(61)56-25-20-46(21-26-56)18-23-55(24-19-46)38(59)10-5-3-2-4-6-27-66-34-9-7-8-33(29-34)50-42-39(41(49)60)40(52-53-42)30-11-14-32(15-12-30)54-67(63,64)44(47)48/h7-9,11-16,28-29,44,54H,2-6,10,17-27H2,1H3,(H2,49,60)(H2,50,52,53)(H,51,58,62)

5-[4-(difluoromethylsulfonylamino)phenyl]-3-[3-[8-[3-[3-(2,4-dioxo-1,3-diazinan-1-yl)-4-methoxybenzoyl]-3,9-diazaspiro[5.5]undecan-9-yl]-8-oxooctoxy]anilino]-1H-pyrazole-4-carboxamide

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 (105.5 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.

---

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).

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)]
*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)

---

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
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

---

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.

---

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