IRAK4

KT-474 forms a ternary complex with CRBN and IRAK4, leading to the ubiquitination and proteasomal degradation of IRAK4. The elimination of IRAK4 from the myddosome using a degrader has the potential to block all downstream signaling, thereby inhibiting TLR-mediated and IL-1R-mediated cellular activation and cytokine induction.[2]
KYM-001 led to potent E3 ligase-dependent degradation of IRAK4. Notably, KYM-001 more effectively inhibited TLR-activated Myddosome signaling compared to IRAK4 kinase inhibitors in human PBMC. Degradation was highly selective for IRAK4 vs >10,000 other detected proteins in the MYD88 L265P mutant ABC DLBCL line OCI-LY10. IRAK4 degradation by KYM-001 resulted in cell cycle inhibition and apoptosis within 48-72 h in ABC DLBCL, with preferential activity in MYD88-mutant vs MYD88-WT cell lines.[3]

Oral dosing of KYM-001 showed dose-dependent antitumor activity in several mouse xenograft models of human MYD88-mutant ABC DLBCL at tolerated doses and schedules. In the OCI-LY10 model, tumor regression was associated with >80% degradation of IRAK4, establishing the pharmacodynamic effect required for maximal efficacy. Since alterations in BCR signaling and MYD88 frequently co-occur in B-cell malignancies, we investigated the potential for combined activity of IRAK4 degradation and BTK inhibition. In the OCI-LY10 xenograft model, which has activating mutations in both CD79B and MYD88, BTK inhibition with ibrutinib had an additive effect on KYM-001 antitumor activity.[3]

IRAK4 detection by flow methods in PBMCs IRAK4 degradation was evaluated in PBMCs using flow methods. Frozen PBMCs were thawed into RPMI with 10% FBS, and 90 μl of the solution was plated per well. KT-474 was prepared at a 10 μM starting dose, followed by fivefold dilution and a 10-point dose curve and added at a final DMSO concentration of 0.1%. Cells and compound were incubated at 37 °C and 5% CO2 overnight (20 h). After incubation, cells were fixed with Cytofix Fixation Buffer from BD Biosciences and washed two times with PBS/2% FBS, and pellets were stored at −80 °C until further processing. On flow run day, cell pellets were thawed, and pre-permeabilization staining cocktail (CD3/CD14/CD56/CD19) was added. Samples were subsequently permeabilized with 60% methanol for 10 min at 4 °C, followed by incubation with post-permeabilization staining cocktail (CD16/IRAK4). Stained samples were run on the Attune NxT flow cytometer. Data were analyzed using FlowJo, and GraphPad Prism was used to generate 50% inhibitory concentrations (IC50s) using a four-parameter logistic regression curve, free-fit.
Percent IRAK4 signal was identified in B cells (CD3−/CD19+), monocytes (CD3−/CD19−CD14+) and lymphocytes (identified by side scatter size and CD14−).[2]

Human cell cytokine release assay[2]
Frozen PBMCs were thawed into RPMI with heat-inactivated 10% FBS/1% penicillin–streptomycin and the same day plated into 96-well flat-bottom plates at 200,000 cells per well in 190 μl of media. KT-474, PF-06550833 and DMSO controls were prepared in duplicate for each donor. All cells were dosed using the Tecan automated liquid handler, followed by incubation at 37 °C and 5% CO2 for 16 h, with final testing concentrations of 0.0064, 0.032, 0.16, 0.8, 4, 20, 100 and 500 nM. After 16 h of pre-treatment with the compounds, LPS (O55:B5) (Sigma-Aldrich, L2637) or R848 (Invivogen, tlrl-r848) were added at 100 ng ml−1 or 10 μg ml−1 final concentration, respectively. Cells were incubated an additional 5 h at 37 °C and 5% CO2. After assay completion, plates were centrifuged at 300g for 5 min. A total of 150 μl of supernatant was carefully removed and placed into a new 96-well V-bottom plate and stored at −80 °C until further analysis. Meso Scale Discovery (MSD) human U-plex or V-plex assays were used to measure cytokine levels. On the day of cytokine analysis, supernatant samples were thawed, diluted with MSD assay diluent and added to MSD plates. The assay was further completed per standard manufacturer’s protocol. Cytokine data were normalized to stimulated and unstimulated controls. The concentration of cytokines in supernatant was determined using MSD Discovery Workbench software. GraphPad Prism was used to generate IC50s using a four-parameter logistic regression curve, free-fit.[2]

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Human B cell phospho-flow assays[2]
CD19+ B cells were purchased from BioIVT or isolated in-house using a negative selection kit (STEMCELL Technologies, 17954). Frozen B cells from n = 5 donors were thawed and plated at 450,000 cells per well in 190 μl of media in 96-well U-bottom plates. KT-474, PF-06550833 and DMSO controls were prepared in duplicate for each donor. All cells were dosed using the Tecan automated liquid handler, followed by incubation at 37 °C and 5% CO2 for 16 h, with final testing concentrations of 0.12, 0.489, 1.95, 7.81, 31.2, 125, 500 and 2,000 nM. After 16 h of compound pre-treatment, CpG-B (InvivoGen, tlrl-2006) was added at 2.5 μM final concentration for 60 min. After incubation, an equal volume of BD Cytofix Fixation Buffer was added to wells. Cells were washed with PBS + 2% FBS before being permeabilized on ice for 30 min using cold BD Perm Buffer III (BD Biosciences, 558050). Cells were washed again before being stained with fluorescently tagged antibody (phycoerythrin (PE) phospho-p65, clone K10-895.12.50 (BD Biosciences, 558423)) for 30 min at room temperature in the dark. Cells were then washed twice before being acquired using the Attune NxT flow cytometer, 10,000 events per well. Data were analyzed using FlowJo, and GraphPad Prism was used to generate IC50s using a four-parameter logistic regression curve, free-fit.

Tumor xenograft studies were conducted by implanting human ABC DLBCL lines into immunocompromised mouse strains and assessing tumor volume.[3]

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IRAK4 in human PBMC, ABC DLBCL cell lines and xenografts was quantified by immunoassays or targeted MS/MS. Myddosome signaling was monitored by mRNA and phosphoprotein endpoints. Cell viability and cell cycle were monitored by flow cytometry. Tumor xenograft studies were conducted by implanting human ABC DLBCL lines into immunocompromised mouse strains and assessing tumor volume.[3]

[1]. Irak degraders and uses thereof. Patent WO2020113233A1.

[2]. IRAK4 degrader in hidradenitis suppurativa and atopic dermatitis: a phase 1 trial. Nat Med. 2023 Dec;29(12):3127-3136.

[3]. Abstract LB-272: KYM-001, a first-in-class oral IRAK4 protein degrader, induces tumor regression in xenograft models of MYD88-mutant ABC DLBCL alone and in combination with BTK inhibition. Cancer Res (2019) 79 (13_Supplement): LB-272.

Inflammation mediated by interleukin-1 (IL-1) receptor-associated kinase 4 (IRAK4) and driven by Toll-like receptors and IL-1 receptors is involved in the pathophysiology of hidradenitis suppurativa (HS) and atopic dermatitis (AD). The IRAK4 degrader KT-474 (SAR444656) was investigated in a randomized, double-blind, placebo-controlled phase I clinical trial with safety and tolerability as the primary objective. Secondary objectives included pharmacokinetics, pharmacodynamics, and clinical activity in patients with moderate to severe HS and moderate to severe AD. In 105 healthy volunteers (HV), KT-474 was administered daily for 14 days following a single dose; subsequently, in an open-label cohort of 21 patients, KT-474 was administered for 28 days. IRAK4 degradation was observed in the blood of healthy volunteers (HV), with a mean reduction of ≥93% after a single dose of 600–1600 mg and a mean reduction of ≥95% after daily doses of 50–200 mg for 14 consecutive days. Similar degradation of IRAK4 was observed in the blood of patients, and IRAK4 levels returned to normal in skin lesions where IRAK4 was overexpressed relative to healthy volunteers. Disease-associated inflammatory biomarkers were reduced in both blood and skin in patients with hidradenitis suppurativa (HS) and atopic dermatitis (AD), and were associated with improvement in skin lesions and symptoms. No drug-related infections occurred. To the best of our knowledge, this is the first published clinical trial using a heterobifunctional degrader, and these results provide preliminary proof of concept for the use of KT-474 in HS and AD, requiring further confirmation in larger-scale trials. ClinicalTrials.gov Registration No.: NCT04772885. [1]

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Objective: This study evaluated the antitumor activity of selective small molecule IRAK4 degraders in in vitro human ABC type diffuse large B-cell lymphoma (DLBCL) cell lines and in vivo tumor xenograft models, including use alone and in combination with BTK inhibitors. [2]
Introduction: ABC type DLBCL accounts for approximately 45% of DLBCL and has a poorer prognosis with R-CHOP chemotherapy compared to GCB type DLBCL. 30-40% of ABC type DLBCL have MYD88 activating mutations; L265P is the most common MYD88 mutation, which can lead to constitutive assembly and activation of Myddosome. IRAK4 kinase and scaffold function are essential for the complete signaling of Myddosome to NFκB and MAPK pathways. Kymera Therapeutics developed heterobifunctional small molecule IRAK4 degraders, such as KYM-001, using a chemical knockdown strategy for the treatment of MYD88-driven lymphoma. [2]
Methods: IRAK4 in human peripheral blood mononuclear cells (PBMCs), ABC diffuse large B-cell lymphoma (DLBCL) cell lines, and xenografts was quantified by immunoassay or targeted tandem mass spectrometry. Myddosome signaling pathway was monitored by mRNA and phosphorylated protein endpoints. Cell viability and cell cycle were monitored by flow cytometry. Tumor xenograft studies were conducted by transplanting human ABC DLBCL cell lines into immunodeficient mouse strains and assessing tumor volume. [2]
Key data: KYM-001 effectively degrades IRAK4, and this degradation is dependent on E3 ligase. Notably, KYM-001 more effectively inhibits the TLR-activated Myddosome signaling pathway in human peripheral blood mononuclear cells (PBMCs) compared to IRAK4 kinase inhibitors. In the MYD88 L265P mutant ABC DLBCL cell line OCI-LY10, KYM-001 exhibited high selectivity for IRAK4 degradation, while having no significant effect on over 10,000 other detected proteins. KYM-001-mediated IRAK4 degradation inhibited the cell cycle and induced apoptosis in ABC DLBCL cells within 48–72 hours, with higher activity against MYD88 mutant cell lines than against MYD88 wild-type cell lines. Oral administration of KYM-001 demonstrated dose-dependent antitumor activity in various human MYD88 mutant ABC DLBCL mouse xenograft models under tolerable doses and dosing regimens. In the OCI-LY10 model, tumor regression was associated with over 80% IRAK4 degradation, confirming the pharmacodynamic effect required to achieve maximum efficacy. Since alterations in the BCR signaling pathway and MYD88 often occur simultaneously in B-cell malignancies, we investigated the potential of the combined effects of IRAK4 degradation and BTK inhibition. In the OCI-LY10 xenograft model, which has both CD79B and MYD88 activating mutations, the use of ibrutinib to inhibit BTK enhances the antitumor activity of KYM-001. [2] Conclusion: KYM-001 is a first-in-class, highly effective, selective, and orally efficient IRAK4 degrader that leads to tumor regression in the ABC-DLBCL model. IRAK4 degradation eliminates the kinase and scaffold protein function of IRAK4, and its efficacy may be superior to that of kinase inhibitors alone. These data suggest that IRAK4 degraders are a promising new approach for the treatment of MYD88-driven lymphoma, which can be used alone or in combination with other targeted approaches, such as BTK inhibitors. [2]

C44H49F2N11O6

865.926775693893

865.383

C, 61.03; H, 5.70; F, 4.39; N, 17.79; O, 11.09

2432994-31-3

146599824

White to off-white solid powder

2.5

2

13

11

63

1790

2

CN1C2=C(C=CC=C2N(C1=O)C3CCC(=O)NC3=O)C#CCOC4CCN(CC4)CC5CCC(CC5)N6C=C(C(=N6)C(F)F)NC(=O)C7=C8N=C(C=CN8N=C7)N9C[C@H]1C[C@@H]9CO1

NQGKNAVUMAHSQN-PKIOHZLWSA-N

InChI=1S/C44H49F2N11O6/c1-52-39-27(4-2-6-34(39)57(44(52)61)35-11-12-37(58)50-43(35)60)5-3-19-62-30-13-16-53(17-14-30)22-26-7-9-28(10-8-26)56-24-33(38(51-56)40(45)46)48-42(59)32-21-47-55-18-15-36(49-41(32)55)54-23-31-20-29(54)25-63-31/h2,4,6,15,18,21,24,26,28-31,35,40H,7-14,16-17,19-20,22-23,25H2,1H3,(H,48,59)(H,50,58,60)/t26?,28?,29-,31-,35?/m1/s1

5-((1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)-N-(3-(difluoromethyl)-1-(4-((4-((3-(1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-4-yl)prop-2-yn-1-yl)oxy)piperidin-1-yl)methyl)cyclohexyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide

KT-474; KYM-001; KT474; PROTAC IRAK4 degrader-7; KT-474; KT474; N-[3-(Difluoromethyl)-1-[trans-4-[[4-[[3-[1-(2,6-dioxo-3-piperidinyl)-2,3-dihydro-3-methyl-2-oxo-1H-benzimidazol-4-yl]-2-propyn-1-yl]oxy]-1-piperidinyl]methyl]cyclohexyl]-1H-pyrazol-4-yl]-5-(1R,4R)-2-oxa-5-azabicyclo[2.2.1]hept-5-ylpyrazolo[1,5-a]pyrimidine-3-carboxamide; N-[3-(Difluoromethyl)-1-[4-[[4-[3-[1-(2,6-dioxopiperidin-3-yl)-3-methyl-2-oxobenzimidazol-4-yl]prop-2-ynoxy]piperidin-1-yl]methyl]cyclohexyl]pyrazol-4-yl]-5-[(1R,4R)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide; 2SXR65P7E3; SCHEMBL21998241; KYM001

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 (~115.48 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.89 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (2.89 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.
 (Please use freshly prepared in vivo formulations for optimal results.)