Characterization of GSK'963: a structurally distinct, potent and selective inhibitor of RIP1 kinase

Abstract

Necroptosis and signaling regulated by RIP1 kinase activity is emerging as a key driver of inflammation in a variety of disease settings. A significant amount has been learned about how RIP1 regulates necrotic cell death through the use of the RIP1 kinase inhibitor Necrostatin-1 (Nec-1). Nec-1 has been a transformational tool for exploring the function of RIP1 kinase activity; however, its utility is somewhat limited by moderate potency, off-target activity against indoleamine-2,3-dioxygenase (IDO), and poor pharmacokinetic properties. These limitations of Nec-1 have driven an effort to identify next-generation tools to study RIP1 function, and have led to the identification of 7-Cl-O-Nec-1 (Nec-1s), which has improved pharmacokinetic properties and lacks IDO inhibitory activity. Here we describe the characterization of GSK'963, a chiral small-molecule inhibitor of RIP1 kinase that is chemically distinct from both Nec-1 and Nec-1s. GSK'963 is significantly more potent than Nec-1 in both biochemical and cellular assays, inhibiting RIP1-dependent cell death with an IC50 of between 1 and 4 nM in human and murine cells. GSK'963 is >10 000-fold selective for RIP1 over 339 other kinases, lacks measurable activity against IDO and has an inactive enantiomer, GSK'962, which can be used to confirm on-target effects. The increased in vitro potency of GSK'963 also translates in vivo, where GSK'963 provides much greater protection from hypothermia at matched doses to Nec-1, in a model of TNF-induced sterile shock. Together, we believe GSK'963 represents a next-generation tool for examining the function of RIP1 in vitro and in vivo, and should help to clarify our current understanding of the role of RIP1 in contributing to disease pathogenesis.

Figure 1

GSK′963A is a potent and selective inhibitor of RIP1 kinase. (a) Chemical structures of GSK′963A (active analog), GSK′962A (inactive analog) and Necrostatin-1. (b) Dose–response curves for GSK′963, GSK′962 and Nec-1 in the FP binding assay evaluating the affinity of compounds for RIP1 (ATP-binding pocket). Graphs represents n=52 for Nec-1, n=4 for GSK′962A and n=7 for GSK′963A. Error bars indicate S.D. (c) Dose–response curves for GSK′963, GSK′962 and Nec-1 in ADP-Glo kinase assay measuring autophosphorylation of RIP1 kinase domain in vitro. Graph represents n=20 for Nec-1, n=2 for GSK′962A and n=2 for GSK′963A. Error bars indicate S.D. (d) GSK′963 does not block the activity of any of the tested 339 human kinases at 10μM concentration, as evaluated at the Reaction Biology Corporation. Each dot represents an individual kinase with the color indicating the level of inhibition (red>90%, yellow 70–90%, orange 50–70% and green <50%) (e) Effect of GSK′963, GSK′962 and Nec-1 on indoleamine 2,3-dioxygenase (IDO) activity evaluated by an in vitro enzymatic assay. Menadione was used as a positive control for IDO inhibition. The results are representative of three independent experiments. =Nec-1, =GSK′962, =GSK′963 and =Menadione.

Figure 2

GSK′963A is highly potent in human and mouse cell-based assays and selective for inhibition of necroptosis. (a–d) Dose–response curves for GSK′963, GSK′962 and Nec-1 in cell-based assays. Necroptosis induced with TNF and zVAD in (a) mouse fibrosarcoma L929 cells, (b) human monocytic U937 cells and (c) primary murine bone marrow-derived macrophages was evaluated by measuring cell viability using CellTiter-Glo assay. (d) Primary human neutrophils were stimulated with TNF, zVAD and SMAC mimetic to induce necroptosis. Cell viability was evaluated as in a. The graphs represent combined data from at least three independent experiments. Error bars represent S.D. (e) Cell viability and Caspase 3/7 activity were measured in BMDM treated with TNF and cycloheximide. Cell viability was measured using the CellTiter-Glo assay at 20 h, Caspase 3/7 activity using the Caspase-Glo 3/7 assay at 3 h. GSK′963 and GSK′962 were used at 100 nM and Nec-1 was used at 10 μM. Similar data were generated in four independent experiments. Error bars represent S.D. between two experiments measured on the same plate. (f) Western blot analysis of IκB phosphorylation and degradation in BMDM stimulated with TNF. IκB phosphorylation was evaluated at 5 min and IκB degradation at 15 min. Tubulin was used as a loading control. GSK′963 and GSK′962 were used at 100 nM and Nec-1 was used at 10 μM. Data are representative of experiments from four different animals. =Nec-1, =GSK′962, and =GSK′963. C, control; CHX, cycloheximide.

Figure 3

GSK′963A protects mice from TNF+zVAD-induced hypothermia. (a) Pharmacokinetic profile of GSK′963A dosed i.p. at 10 mg/kg in C57BL/6 mice. The data represent the combined results of the three independent animals. (b) Modeling of predicted % inhibition against RIP1 using the observed pharmacokinetic profile of GSK′963 in conjunction with the potency in inhibiting TNF+zVAD necroptosis in mouse L929 cells. =2 mg/kg and =0.2 mg/kg (c–e) The effect of GSK′963, GSK′962 and Nec-1 on temperature loss in the TNF+zVAD-induced shock model. C57BL/6 mice were pretreated i.p. with (c) GSK′963, (d) GSK′962A or (e) Nec-1 15 min prior to i.v. injection of TNF+zVAD. Temperature was monitored over the course of 3 h by rectal probe. The results are representative of three independent experiments, each containing seven animals per group. Error bars indicate the S.D. from the seven animals for each group. =control mice, =TNF+zVAD-treated mice, =TNF+zVAD-treated mice dosed with GSK′963 at 0.2 mg/kg, =TNF+zVAD-treated mice dosed with GSK′963 at 2 mg/kg, =TNF+zVAD-treated mice dosed with GSK′962 at 20 mg/kg, =TNF+zVAD with Nec-1 at 2 mg/kg and =TNF+zVAD with Nec-1 at 0.2 mg/kg.