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.

Toll-like receptor-driven and interleukin-1 (IL-1) receptor-driven inflammation mediated by IL-1 receptor-associated kinase 4 (IRAK4) is involved in the pathophysiology of hidradenitis suppurativa (HS) and atopic dermatitis (AD). KT-474 (SAR444656), an IRAK4 degrader, was studied in a randomized, double-blind, placebo-controlled phase 1 trial where the primary objective was safety and tolerability. Secondary objectives included pharmacokinetics, pharmacodynamics and clinical activity in patients with moderate to severe HS and in patients with moderate to severe AD. KT-474 was administered as a single dose and then daily for 14 d in 105 healthy volunteers (HVs), followed by dosing for 28 d in an open-label cohort of 21 patients. Degradation of IRAK4 was observed in HV blood, with mean reductions after a single dose of ≥93% at 600-1,600 mg and after 14 daily doses of ≥95% at 50-200 mg. In patients, similar IRAK4 degradation was achieved in blood, and IRAK4 was normalized in skin lesions where it was overexpressed relative to HVs. Reduction of disease-relevant inflammatory biomarkers was demonstrated in the blood and skin of patients with HS and patients with AD and was associated with improvement in skin lesions and symptoms. There were no drug-related infections. These results, from what, to our knowledge, is the first published clinical trial using a heterobifunctional degrader, provide initial proof of concept for KT-474 in HS and AD to be further confirmed in larger trials. ClinicalTrials.gov identifier: NCT04772885 .[1]

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Purpose: This work assessed the antitumor activity of selective small molecule IRAK4 degraders in human ABC DLBCL cell lines in vitro and in tumor xenograft models in vivo, alone and in combination with BTK inhibition.[2]
Introduction: ABC DLBCL comprises approximately 45% of DLBCL and has a worse outcome with R-CHOP chemotherapy compared to GCB DLBCL. Activating mutations in MYD88 occur in 30-40% of ABC DLBCL; L265P, the most prevalent MYD88 mutation, causes constitutive assembly and activation of the Myddosome. IRAK4 kinase and scaffolding functions are essential for full signaling through the Myddosome to NFκB and MAPK pathways. Kymera Therapeutics is using a chemical knockdown strategy to develop heterobifunctional small molecule IRAK4 degraders, exemplified by KYM-001, for the treatment of MYD88-driven lymphomas. [2]
Methods: 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.[2]
Key data: 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. 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.[2]
Conclusions: KYM-001 is a first-in-class, potent, selective and orally active IRAK4 degrader that causes tumor regression in ABC-DLBCL models. Degradation of IRAK4 removes both the kinase and scaffolding functions of IRAK4, and may be superior to kinase inhibition alone. These data support IRAK4 degraders as a promising new therapeutic opportunity for MYD88-driven lymphoma, both alone and in combination with other targeted approaches such as BTK inhibition.[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.)