IRAK4 (DC50=6 nM); CRBN; Interleukin-1 receptor-associated kinase 4 (IRAK4), Ikaros (IKZF1), Aiolos (IKZF3) [1]

Compound KT-413 (32) demonstrated potent degradation of IRAK4, Ikaros, and Aiolos in OCI-Ly10 cells, with DC50 values of 6 nM, 1 nM, and 1 nM, respectively. The maximal degradation (DMax) was >95% for IRAK4 and >95% for Ikaros and Aiolos. [1]
In a four-day CellTiter-Glo (CTG) assay in OCI-Ly10 cells, KT-413 exhibited a potent antiproliferative effect with an IC50 of 11 nM. [1]
A time-course experiment in OCI-Ly10 cells (41 nM) showed that degradation of Ikaros and Aiolos occurred with faster kinetics, reaching DMax within 2 hours, whereas maximal degradation of IRAK4 occurred after approximately 24 hours, indicating hierarchical substrate degradation with Ikaros and Aiolos being preferentially degraded. [1]
Global proteomic profiling in human PBMCs treated with KT-413 for 24 hours showed selective reduction of IRAK4 and five known IMiD substrates (IKZF1, IKZF3, ZFP276, ZFP91, WIZ) exceeding the significance cutoff. Two other proteins containing zinc finger domains, LPXN and FGD2, were also significantly downregulated. [1]
Proteomic analysis in OCI-Ly10 cells treated with KT-413 for 2 or 8 hours revealed significant upregulation of type I interferon response, a known downstream effect of IKZF1 and IKZF3 degradation. GSPT1, a pan-essential IMiD substrate, remained unaffected in both cell types. [1]
Compound KT-413 (32) was found to be highly selective across 468 kinases evaluated in the KINOMEscan screening platform with an S(10) at 1 µM = 0.005. [1]

In an OCI-Ly10 (MYD88 mutant) xenograft model, KT-413 (32) administered orally once daily at doses of 3, 10, and 30 mg/kg for 38 days resulted in tumor growth regression at all doses, with complete responses observed at the 10 and 30 mg/kg dose levels. [1]
In a patient-derived xenograft (PDX) model (LY140019) bearing a MYD88 L265P mutation alongside a CD79 mutation, intermittent oral dosing of KT-413 at 20 mg/kg (3 days on, 5 days off) achieved complete tumor regression. [1]

The affinity of the IRAK4 ligand (compound 4) used in the development of KT-413 was evaluated. It showed very potent affinity for IRAK4 with a dissociation constant (Kd) of 0.32 nM. [1]
The affinity of heterobifunctional degraders for CRBN was measured using a homogeneous time-resolved fluorescence (HTRF) assay. For KT-413 (32), the CRBN IC50 was 130 nM. The affinity of the (S)- and (R)- enantiomers of 32 (33 and 34) for CRBN was also assessed, with compound 33 showing >30-fold higher affinity (IC50 = 6.3 nM) than compound 34 (IC50 = 199 nM). [1]
The binding affinity of various heterobifunctional degraders for IRAK4 was assessed using a DiscoverX Kd Elect assay. KT-413 (32) had an IRAK4 Kd of 0.17 nM. [1]

IRAK4 degradation was measured in OCI-Ly10 cells at 24 hours using a Mesoscale Discovery (MSD) assay. For KT-413 (32), the IRAK4 DC50 was 6 nM (DMax >95%). [1]
Ikaros and Aiolos degradation were measured in OCI-Ly10 cells at 6 hours using an MSD assay. For KT-413 (32), the Ikaros DC50 was 1 nM (DMax >95%) and the Aiolos DC50 was 1 nM (DMax >95%). [1]
A four-day CellTiter-Glo (CTG) assay was utilized to measure cell viability in OCI-Ly10 cells. KT-413 (32) showed an IC50 of 11 nM. [1]
A time-course degradation experiment was performed in OCI-Ly10 cells using 41 nM of KT-413 (32). Protein levels of IRAK4, Ikaros, and Aiolos were measured at various time points to determine degradation kinetics. [1]
Global proteomic profiling was performed on unstimulated human PBMCs treated with KT-413 (32) or DMSO for 24 hours. Cells were lysed, tryptic peptides were desalted, labeled with TMTpro reagents, and analyzed by tandem mass tag (TMT)-based quantitative proteomics. [1]
For proteomic analysis in OCI-Ly10 cells, cells were treated with KT-413 (32) for 2 or 8 hours and analyzed using the same TMT-based quantitative proteomics workflow to identify degradation targets and downstream biology-associated changes. [1]
Selectivity across 468 kinases was evaluated using the KINOMEscan screening platform at a concentration of 1 µM. The S(10) score for KT-413 (32) was reported as 0.005. [1]
CRBN affinity (IC50) was measured using an HTRF assay. [1]

OCI-Ly10 xenograft model: Human OCI-Ly10 cells were implanted subcutaneously (10 × 10⁶ cells/mouse in 0.2 mL of 1:1 PBS:Matrigel) into the right flanks of 6-8 week old female CB-17 SCID mice. Mice bearing OCI-Ly10 tumors were administered KT-413 (32) orally once daily (po, qd) at doses of 3, 10, and 30 mg/kg for 38 days. Tumor volumes were measured manually by caliper, and tumor growth inhibition (TGI) was assessed. [1]
Patient-derived xenograft (PDX) model LY140019: This model, bearing a MYD88 L265P mutation alongside a CD79 mutation, was passaged by subcutaneous implant in NPG mice. Mice bearing tumors were dosed orally with KT-413 (32) at 20 mg/kg using an intermittent schedule (3 days on, 5 days off). Tumor volumes were measured at least twice weekly. [1]
In vivo PK studies: For intravenous (iv) pharmacokinetic studies, compounds were formulated in 10% DMSO/40% PEG400/50% water. For oral (po) pharmacokinetic studies, compounds were formulated in 20% HP-β-CD/80% water. Plasma samples were collected and processed by protein precipitation using acetonitrile, followed by LC-MS/MS analysis. [1]

Passive permeability (A→B) of compounds was measured in a wild-type MDCK cell line (units = ×10⁻⁶ cm/s). For KT-413 (32), the permeability was 1.3 ×10⁻⁶ cm/s. [1]
Intrinsic clearance (CLint) was obtained from isolated liver microsomes with NADPH (units = µL/min/mg). For KT-413 (32), the human liver microsome CLint was 1.4 µL/min/mg, and the rat liver microsome CLint was 1.2 µL/min/mg. [1]
Intrinsic clearance (CLint) from isolated hepatocytes (units = µL/min/10⁻⁶ cells) for KT-413 (32) was 4.0 µL/min/10⁻⁶ cells in mouse, 0.8 µL/min/10⁻⁶ cells in rat, 2.3 µL/min/10⁻⁶ cells in dog, 2.7 µL/min/10⁻⁶ cells in non-human primate (NHP), and 3.5 µL/min/10⁻⁶ cells in human. [1]
Plasma protein binding (PPB) for KT-413 (32) was measured using ultracentrifugation, showing the fraction unbound (Fu) was 0.024 in rat, 0.020 in dog, 0.018 in NHP, and 0.033 in human. [1]
In vivo pharmacokinetic parameters for KT-413 (32) after intravenous (iv) dosing at 2 mg/kg (mouse, rat) or 0.2 mg/kg (dog, NHP) and oral (po) dosing at 10 mg/kg (mouse, rat) or 2.1 mg/kg (dog, NHP) were reported: Mouse (iv: CL 4.0 mL/min/kg, Vss 8.6 L/kg, t1/2 2.8 h; po: AUC 17.5 µM·h, F 41%). Rat (iv: CL 1.2 mL/min/kg, Vss 9.1 L/kg, t1/2 2.7 h; po: AUC 15.2 µM·h, F 17%). Dog (iv: CL 4.2 mL/min/kg, Vss 28 L/kg, t1/2 0.46 h; po: AUC 21.6 µM·h, F 21%). NHP (iv: CL 8.6 mL/min/kg, Vss 5.8 L/kg, t1/2 3.6 h; po: AUC 16.4 µM·h, F 42%). [1]
For the enantiomers of 32 (33 and 34), racemization half-life (t1/2) was evaluated in PBS buffer and found to be approximately 24 hours. In rat, dog, NHP, and human plasma, the t1/2 for racemization of 33 was 129, 155, 201, and 242 minutes, respectively; for 34, it was 143, 184, 182, and 175 minutes, respectively. [1]
In vivo, following iv dosing of 33 in rat, exposure of 34 accounted for 57% of the total exposure, and following iv dosing of 34, 48% of the observed exposure was 33. Similar results were observed in NHP. [1]

The abstract mentions a favorable preclinical safety package for KT-413 (32) that supported its selection as a development candidate, but no specific toxicity data (e.g., LD50, organ toxicity) are detailed in the provided text. [1]

[1]. Discovery of KT-413, a Targeted Protein Degrader of IRAK4 and IMiD Substrates Targeting MYD88 Mutant Diffuse Large B-Cell Lymphoma. J Med Chem. 2024 Jul 11;67(13):10548-10566.

[2]. Preparation of benzothiazole derivatives as IRAK degraders and uses thereof. World Intellectual Property Organization, WO2021127190 A1. 2021-06-24.

KT-413 (32) is a first-in-class dual-functioning molecule, referred to as an IRAKIMiD, that concurrently degrades IRAK4 (via a heterobifunctional PROTAC mechanism) and the IMiD substrates Ikaros (IKZF1) and Aiolos (IKZF3) (via a molecular glue mechanism). This approach was designed to maximize NF-κB inhibition while upregulating the type I interferon response to effectively treat MYD88 mutant (MYD88MT) diffuse large B-cell lymphoma (DLBCL). [1]
It was discovered through iterative medicinal chemistry SAR aimed at optimizing the degradation of both IRAK4 and IMiD neosubstrates. The structure of KT-413 was solved via microcrystal electron diffraction, revealing an extended conformation with three intramolecular hydrogen bonds. [1]
KT-413 is currently being evaluated in a first-in-human, Phase 1 dose escalation study in patients with relapsed and/or refractory B-cell non-Hodgkin lymphoma. [1]

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Zomiradomide is a small-molecule protein degrader that targets interleukin-1 receptor-associated kinase 4 (IRAK4) as well as the immunomodulatory imide drug (IMiD) substrates Ikaros (IKZF1) and Aiolos (IKZF3), exhibiting both immunomodulatory and antineoplastic activities. Upon administration, zomiradomide engages the E3 ubiquitin ligase complex, promoting the ubiquitination and subsequent proteasome-mediated degradation of IRAK4, Ikaros, and Aiolos. Degradation of IRAK4 disrupts IRAK4-dependent signaling, thereby preventing activation of the nuclear factor-kappa B (NF-κB) pathway and reducing the expression of inflammatory cytokines and pro-survival factors. This results in inhibited proliferation of tumor cells characterized by MYD88-activating mutations or hyperactivated toll-like receptor (TLR) pathways. Meanwhile, degradation of the transcription factors Ikaros and Aiolos leads to downregulation of downstream targets such as interferon regulatory factor 4 (IRF4), which enhances type I interferon signaling and further suppresses NF-κB activity. Collectively, these actions induce apoptosis and suppress tumor cell proliferation. IRAK4 is a serine/threonine kinase that functions downstream of the adaptor protein MYD88, serving as a critical node linking TLR and interleukin-1 receptor (IL-1R) signaling to the NF-κB pathway.

C45H48F3N7O6S

871.97

871.333

C, 61.98; H, 5.55; F, 6.54; N, 11.24; O, 11.01; S, 3.68

2655656-99-6

156506667

Light yellow to yellow solid powder

6.7

4

14

11

62

1730

0

S1C2C=C(C(C(C)(C)O)=CC=2N=C1C1CCC(CC1)CN1CC2(C1)CC(CCNC1=CC=CC3C(N(C4C(NC(CC4)=O)=O)C(C1=3)=O)=O)C2)NC(C1C=CC=C(C(F)(F)F)N=1)=O

WDRJGMSUTPVXDH-UHFFFAOYSA-N

InChI=1S/C45H48F3N7O6S/c1-43(2,61)28-17-32-34(18-31(28)51-38(57)30-7-4-8-35(50-30)45(46,47)48)62-40(52-32)26-11-9-24(10-12-26)21-54-22-44(23-54)19-25(20-44)15-16-49-29-6-3-5-27-37(29)42(60)55(41(27)59)33-13-14-36(56)53-39(33)58/h3-8,17-18,24-26,33,49,61H,9-16,19-23H2,1-2H3,(H,51,57)(H,53,56,58)

N-[2-[4-[[6-[2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]amino]ethyl]-2-azaspiro[3.3]heptan-2-yl]methyl]cyclohexyl]-5-(2-hydroxypropan-2-yl)-1,3-benzothiazol-6-yl]-6-(trifluoromethyl)pyridine-2-carboxamide

zomiradomide; KT-413; KT413; 2655656-99-6; IRAK degrader-1; N-[2-[4-[[6-[2-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]amino]ethyl]-2-azaspiro[3.3]heptan-2-yl]methyl]cyclohexyl]-5-(2-hydroxypropan-2-yl)-1,3-benzothiazol-6-yl]-6-(trifluoromethyl)pyridine-2-carboxamide; 2655660-58-3;

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)

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

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.)