Structure guided design of potent and selective ponatinib-based hybrid inhibitors for RIPK1

Abstract

RIPK1 and RIPK3, two closely related RIPK family members, have emerged as important regulators of pathologic cell death and inflammation. In the current work, we report that the Bcr-Abl inhibitor and anti-leukemia agent ponatinib is also a first-in-class dual inhibitor of RIPK1 and RIPK3. Ponatinib potently inhibited multiple paradigms of RIPK1- and RIPK3-dependent cell death and inflammatory tumor necrosis factor alpha (TNF-α) gene transcription. We further describe design strategies that utilize the ponatinib scaffold to develop two classes of inhibitors (CS and PN series), each with greatly improved selectivity for RIPK1. In particular, we detail the development of PN10, a highly potent and selective "hybrid" RIPK1 inhibitor, capturing the best properties of two different allosteric RIPK1 inhibitors, ponatinib and necrostatin-1. Finally, we show that RIPK1 inhibitors from both classes are powerful blockers of TNF-induced injury in vivo. Altogether, these findings outline promising candidate molecules and design approaches for targeting RIPK1- and RIPK3-driven inflammatory pathologies.

Figure 1. Inhibition of necroptosis and RIPK1 kinase by ponatinib

A) Structures of necrostatins. B) Comparison of Glu-in/DLG-out (red, PDB: 4NEU) and Glu-out/DLG-out (blue, PDB 4ITH) conformations of RIPK1 kinase reveals movement of αC helix. Movement of αC helix is indicated by black arrow. Position of Nec-1 in Glu-out/DLG-out structure is shown (Nec-1 - green). Ionic bond between Glu63 and Lys45 in Glu-in conformation is indicated. C) Ponatinib inhibits recombinant RIPK1 and RIPK3 kinases in vitro. 2 μM kinases were used in the in vitro ³²P autophosphorylation assay. Nec-1 only inhibited RIPK1, while Gleevec lacked activity against both kinases. D) Gleevec does not inhibit necroptosis. FADD-deficient Jurkat cells were treated with 10 ng/ml human TNFα in the presence of 11 point dose ranges of Ponatinib and Gleevec for 24 hr. E) Ponatinib inhibits TNF-induced cell death in the presence of 100 nM TAK1 inhibitor 5z-7-oxozeaenol. MEFs were stimulated with 10 ng/ml mouse TNFα with 5z-7 for 24 hr to induce RIPK1-dependent apoptosis. To induce RIPK3-dependent necroptosis, cells were additionally treated with 50 μM zVAD.fmk. Inhibition of cell death by indicated concentrations of ponatinib, Nec-1 and RIPK3 inhibitor GSK-872 was determined. Cell viability data are presented as mean ± SD. See also Figure S1.

Figure 2. Modeling interactions of ponatinib CS analogs with RIPK1 and RIPK2 kinases

A) Ring A of ponatinib inserts into the lipophilic pocket formed by aliphatic portions of side-chains of Ile43, Lys45, Leu90 and Met92. The backside of the molecule is aligned with the side chain of Leu157 of DLG motif. B) Alignment of Leu157 of RIPK1 DLG and Phe165 of RIPK2 DFG motifs, Phe165 is in close proximity with the ATP binding pocket moiety of ponatinib. C) Chemical structures of CS analogs of ponatinib. See also Figure S2.

Figure 3. Selectivity profiling of CS analogs of ponatinib. Inhibition of a diversity set of 97 kinases (90 wild type kinase and 7 mutants, ScanEDGE, DiscoveRx) by 1 μM inhibitors

A) Selectivity scores of CS analogs. Selectivity score values reflect number of kinases inhibited by >65% (S35), >90% (S10) or 99% (S1). B) TreeView maps of kinase inhibition by ponatinib and CS analogs. Red circles indicate kinases that were inhibited by the molecules >65%. The diameter of the red circle reversely corresponds to the percentage of kinase activity remaining in the presence of inhibitor (i.e. 0% indicates complete inhibition and corresponds to the largest size of the circle). Green circles indicate kinases that were tested but were inhibited <65%. Full data are presented in Table S1. C) MM-GBSA energy profile analysis reveals unfavorable interactions of CS6 with Abl. Energy changes between free and bound sates of CS6 and residues in the Abl and RIPK1 binding pockets were calculated as described in Methods section. Colors indicate energy changes upon small molecule binding from favorable (blue) to unfavorable (red). Side chains of gatekeeper and DXG residues are shown. D) CS6 poorly inhibits M92T/L157F mutant of human RIPK1 kinase. FLAG-tagged kinases (a.a. 1–327) were expressed in HEK293T cells, immunopricipitated using anti-FLAG beads and used in 32P autophopshosphorylation assays with indicated concentrations of ponatinib (Pon) and CS analogs. E) Inhibition of IFNγ-induced cell death by Ponatinib. RIPK1−/− MEFs were treated with 10 ng/ml IFNγ in the presence of indicated concentrations of Ponatinib, CS4 and CS6 for 24 hr. F) Ponatinib and GSK-872 inhibit poly(I:C)-induced cell death. MEFs were treated with inhibitors, 5 μg/ml poly(I:C) and 50 μM zVAD.fmk for 24 hr. Cell viability data are presented as mean ± SD. See also Figure S3, Table S1.

Figure 4. Development of hybrid PN RIPK1 inhibitors

A) General design of hybrid PN molecules, combining DLG-out Nec-1, Ring A-containing linker and hinge-binding fragment of ponatinib. B) Activities of PN series of compounds. IC₅₀ values against recombinant RIPK1 kinase were determined using ADPGlo assay using 6-point dose range (5 μM–20 nM) of each compound. IC₅₀ values against necroptosis were determined in in TNF-treated FADD-deficient cells. Experiments were performed independently from those presented in Table 2f. C) Comparison of the binding poses of Nec-1 (from X-ray structure, PDB: 4ITH), ponatinib (from ponatinib/RIPK1 model, Fig. 3) and PN10 (resulting from “unconstrained” Glide docking). Interaction diagrams were generated using Maestro software. PN10 forms three out of 4 targeted hydrogen bonds to Met95 of the hinge and Val76/Asp156 in the DLG-out pocket. D, E) Selectivity of PN10 towards RIPK1. PN10 was screened against ScanEDGE panel (DiscoveRx) of 97 kinases at 1 μM, described in Fig. 3. RIPK1 was the only kinase with >99%. Some inhibition of DYRK1b (79% inhibition) was also observed. No other kinase was inhibited >65% (other kinases in the panel indicated by green circles). Full data are presented in Table S1. F) PN inhibitors display increased inhibition of M92T RIPK1 mutant with Thr gatekeeper. Wild type and M92T mutant were expressed in 293T cells. Proteins were immunopricipitated using anti-FLAG (M2) magnetic beads and used in ³²P autophosphorylation assay at 10 μM. Comparable amounts of wild type and mutant kinases in kinase reactions were confirmed by Western blot. G) Strain penalties were calculated using modified MM-GBSA algorithm for select PN hybrids based on constrained Glide docking to RIPK1. Two different binding poses were observed for PN9 with different strain values. PN10 was the only inhibitor without strain penalty. Notably, M92T mutation eliminated strain penalty in case of PN13, consistent with increased inhibition of this mutant in F). See also Figure S4 and Table S1.

Figure 5. Inhibition of necroptosis and inflammation by ponatinib analogs

A–B) Inhibition of TCZ-induced necroptosis in MEFs by various RIPK inhibitors. Cells were treated with 50 ng/ml mouse TNFα, 200 ng/ml cycloheximide and 25 μM zVAD.fmk for 18 hr in the presence of indicated concentrations of inhibitors. C) Ponatinib analogs inhibit necrosome formation in TCZ-stimulated MEFs. Cells were treated with 50 ng/ml mouse TNFα, 200 ng/ml cycloheximide, and 25 μM zVAD.fmk for 6 hr in the presence of indicated concentrations of inhibitors, followed by RIPK3 immunopricipitation. D–E) Ponatinib inhibits RIPK1-dependent TNFα mRNA upregulation in FADD-deficient Jurkat (D) and iBMM (E) cells. Jurkat cells were stimulated with 10 ng/ml human TNFα for 8 hr in the presence of indicated concentrations of specific RIPK1 kinase inhibitor Nec-1, ponatinib and indicated PN and CS analogs of ponatinib. Changes in TNF mRNA relative to GAPDH were determined by qRT-PCR. iBMM cells were stimulated with 10 ng/ml LPS and 50 μM zVAD.fmk for 7 hr. F–G) Ponatinib analogs block TNFα toxicity in vivo. Survival curves are shown in F). Reduction in TNF-induced tissue injury and inflammatory response was confirmed by assessing the circulating levels of injury markers (AST, LDH, CK) and mouse IL-6 in G). n=3–5 mice per group. * p<0.05 for TNF/inhibitors vs. TNF group. H–I) Ponatinib and PN10 display higher activity than Nec-1 in vivo. Survival curves are shown in H). Reduction in TNF-induced tissue injury and inflammatory response was confirmed by assessing the circulating levels of injury markers (AST, LDH, CK) and mouse IL-6 in I). n= 3–6 mice per group. * p<0.05 for TNF/inhibitors vs. TNF group, ** p<0.015 for TNF/ponatinib and TNF/PN10 groups vs. TNF/Nec-1 group. Injury marker and IL6 levels data are presented as mean ± SD. See also Figure S5.