Structure Guided Design of Potent and Selective Ponatinib-Based Hybrid Inhibitors for RIPK1

Abstract

Summary 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 TNFα 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 RIPK1/3-driven inflammatory pathologies.

Necroptosis-independent signaling by the RIP kinases in inflammation

“…In addition to benefiting basic science, a holistic understanding of the biology and mechanism of RIP kinase activation is important for potential therapeutic targeting of these molecules in inflammatory diseases. In this regard, RIPK1 and RIPK3 inhibitors have been developed [56, 95, 98–101]. Developing inhibitors that can target additional inflammatory pathways beyond necroptosis may magnify therapeutic potential of RIP kinase-targeted therapies.…”

Complex Pathologic Roles of RIPK1 and RIPK3: Moving Beyond Necroptosis

“…This was explained by an increased flexibility of the activation loop of human kinase, which was necessary to accommodate the major conformational changes in the activation segment, the C-helix, and the glycine-rich loop upon compound binding. We have previously reported that the DLG motif of RIPK1 (as opposed to DFG present in the majority of the kinases) was also essential in providing flexibility to the allosteric pocket of RIPK1 [125]. These data reveal that the allosteric back pocket of RIPK1 possesses unusual flexibility, which allows development of small molecule inhibitors with unprecedented specificity.…”

“…This includes synthesis of TNFα, and the generation of an auto-feedback loop promoting apoptotic cell death and expression of multiple cytokines and chemokines [74, 123, 124]. These responses required kinase activity of RIPK1 and RIPK3, acting in a kinase and MLKL-independent manner [74, 125]. SMAC mimetics were also shown to promote IL-1β processing by both NLRP3 and caspase-8 inflammasomes, which was abolished in Ripk3 -/- cells [89].…”

“…SMAC mimetics were also shown to promote IL-1β processing by both NLRP3 and caspase-8 inflammasomes, which was abolished in Ripk3 -/- cells [89]. Conversely, addition of zVAD.fmk converted RIPK1 kinase-dependent apoptosis into RIPK3 kinase-dependent necroptosis [125]. Notably, not all cancer cells produce TNFα in response to SMAC mimetics.…”

“…5c). Notably, structural analysis indicated that this inhibitor and Nec-1s, which is also highly specific for RIPK1 [71, 157], target the same allosteric αC-Glu-out/DLG-out back pocket [125, 160] that, thus far, has only been reported for RIPK1. Another unusual property of these molecules was a high degree of selectivity (>200 fold) towards human and primate RIPK1 versus rodent homologs.…”