Discovery of a cooperative mode of inhibiting RIPK1 kinase

RIPK1, a death domain-containing kinase, has been recognized as an important therapeutic target for inhibiting apoptosis, necroptosis, and inflammation under pathological conditions. RIPK1 kinase inhibitors have been advanced into clinical studies for the treatment of various human diseases. One of the current bottlenecks in developing RIPK1 inhibitors is to discover new approaches to inhibit this kinase as only limited chemotypes have been developed. Here we describe Necrostatin-34 (Nec-34), a small molecule that inhibits RIPK1 kinase with a mechanism distinct from known RIPK1 inhibitors such as Nec-1s. Mechanistic studies suggest that Nec-34 stabilizes RIPK1 kinase in an inactive conformation by occupying a distinct binding pocket in the kinase domain. Furthermore, we show that Nec-34 series of compounds can synergize with Nec-1s to inhibit RIPK1 in vitro and in vivo. Thus, Nec-34 defines a new strategy to target RIPK1 kinase and provides a potential option of combinatorial therapy for RIPK1-mediated diseases.

A solution of 13 (4.24 g, 16.3 mmol) and hydroxylamine hydrochloride (2.24 g, 32.61 mmol) in pyridine/EtOH (30 mL/10 mL) was heated at 65 ℃ for 3 h. The reaction mixture was then concentrated under reduced pressure, and the residue was dissolved in DCM (100 mL), washed with water (50 mL) and brine (50 mL). The DCM phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure to afford the crude title compound 14 which 10 was used for next step without further purification. LC-MS (ESI, +ve) m/z: [M + H] + calcd for C12H12F3NO3 275.08; found, 276.1 .
Meldrum's acid (0.014 g, 0.97 mmol) and 4-methylmorpholine (0.10 g, 0.99 mmol) were then added to the mixture. After stirring at r.t. for 1 h, the mixture was heated to reflux for 4 h under N2. The reaction mixture was cooled to r.t. and concentrated under reduced pressure to afford crude compound 19 which was used for next step without further purification. LC-MS (ESI,ve) m/z: [M -H]calcd for C14H8F3N4OS -337.04 ; found, 337.0 .
A solution of 19 (0.021 g, 0.47 mmol), 10 (0.094 g, 0.47 mmol) and tetrabutylammonium iodide (0.017 g, 0.05 mmol) in DMA (1 mL) was stirred at r.t. for 10 min. Water (10 mL) was added to the reaction mixture and the mixture was extracted with EtOAc (2 x 10 mL). The scanning calorimetry by real-time PCR and the melting temperatures were calculated by Protein Thermal ShiftÔ Software. Three replicates for each reaction were performed. d Cellular thermal shift assay. MEFs were treated with 10 μM Nec-34 or Nec-1s for 2 h and then harvested and resuspended with PBS. The cells were incubated at 37, 46, 48, 50, 52, 54, 56 • C for 3min, and then frozen in liquid nitrogen quickly. The cells were subject to repeated freezethaw three times, and then centrifuged at 20,000g at 4 • C for 10min. The soluble part was lysed with 2%SDS buffer and analyzed by western blotting analysis of RIPK1 and actin antibodies as indicated. e Nec-34 was measured at a concentration of 10 μM against RIP kinase family members using a radiometric HotSpotSM enzymatic assay by Reaction Biology Corporation.
f Flag-tagged RIPK3 was overexpressed in 293T cells for 24h, cells were then treatment with 10 μM Nec-34 or GSK872 for 12 h after transfection and lysed with Nonidet P-40 buffer 24 h after transfection (Left). Flag-tagged RIPK1 (residues 1-330) was overexpressed in 293T cells for 24h, and cells were then treated with 10 μM Nec-1s or Nec-34 for 12h after transfection and lysed with Nonidet P-40 buffer 24 h after transfection (Right). The lysates were analyzed by western blotting with indicated antibodies. g Cellular thermal shift assay. WT-RIPK1 or S161A-RIPK1 reconstituted RIPK1 -/-MEFs were treated with 10 μM Nec-34 or Nec-1s for 2 h and then harvested and resuspended with PBS. The cells were incubated at 37, 46, 48, 50, 52, 54 • C for 3min, and then frozen in liquid nitrogen quickly. The cells were subject to repeated freeze-thaw three times, and then centrifuged at 20,000g at 4 • C for 10min. The soluble part was lysed with 2%SDS buffer and analyzed by western blotting analysis of RIPK1 and actin antibodies as indicated.  WinNonlin (CERTARA, USA). Cmax was determined as the maximum plasma concentration, and Tmax was the time to reach the maximum concentration.
Supplementary Table. S3 The concentration of 484 in plasma and brain after p.o.

administration.
Compound 484 (30 mg/kg, 10 mL/kg) was dissolved in PEG400 and H2O (1/1, v/v), and was given to CD-1 mice (male, 18-22 g, n = 3 for each time point) by p.o. administration. Mice were sacrificed by carbon dioxide at 1 h, 4 h and 12 h post dosing. Plasma and brain samples were collected and the compound concentration in plasma was measured as described above, and the brain samples were prepared via homogenization with 5 times volume of acetonitrile/methanol (1 : 1, v/v). Samples were analyzed by 6500 triple quadrupole Mass Spectrometer (Sciex, USA). An Acquity UPLC BEH C18 column (1.7 μm, 50 mm × 2.1 mm, Waters, USA) was used for the analysis. Gradient elution was performed with a mobile phase composed of solvent A (water containing 0.1% formic acid and 5 mM NH4OAc) and solvent B (acetonitrile containing 0.1% formic acid).