Limits...
Characterization of GSK ′ 963: a structurally distinct, potent and selective inhibitor of RIP1 kinase

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4979471&req=5

fig2: 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.

Mentions: We next assessed the ability of GSK′963, GSK′962 and Nec-1 to block necroptosis in murine and human cell lines in vitro. To this end, mouse L929 and human U937 cells were stimulated with TNF+zVAD to induce necroptosis, and the ability of compounds to block cell death was evaluated overnight. GSK′963 efficiently blocked necroptosis in both murine and human cells with IC50 values of 1 nM and 4 nM, respectively, whereas the inactive analog GSK′962 was at least 1000-fold less potent in these assays (Figures 2a and b). Nec-1 was significantly less potent as compared with GSK′963, displaying IC50 values of 1 μM and 2 μM in L929 and U937 cells, respectively. The potency of the compounds was confirmed in primary mouse bone marrow-derived macrophages (BMDMs) and primary human neutrophils stimulated to induce necroptosis. In good agreement with the cell line data, the IC50 values were determined to be 3 nM for the primary mouse BMDMs and 0.9 nM for primary human neutrophils, with Nec-1 and GSK′963 being significantly less potent or inactive in the assays (Figures 2c and d).


Characterization of GSK ′ 963: a structurally distinct, potent and selective inhibitor of RIP1 kinase
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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4979471&req=5

fig2: 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.
Mentions: We next assessed the ability of GSK′963, GSK′962 and Nec-1 to block necroptosis in murine and human cell lines in vitro. To this end, mouse L929 and human U937 cells were stimulated with TNF+zVAD to induce necroptosis, and the ability of compounds to block cell death was evaluated overnight. GSK′963 efficiently blocked necroptosis in both murine and human cells with IC50 values of 1 nM and 4 nM, respectively, whereas the inactive analog GSK′962 was at least 1000-fold less potent in these assays (Figures 2a and b). Nec-1 was significantly less potent as compared with GSK′963, displaying IC50 values of 1 μM and 2 μM in L929 and U937 cells, respectively. The potency of the compounds was confirmed in primary mouse bone marrow-derived macrophages (BMDMs) and primary human neutrophils stimulated to induce necroptosis. In good agreement with the cell line data, the IC50 values were determined to be 3 nM for the primary mouse BMDMs and 0.9 nM for primary human neutrophils, with Nec-1 and GSK′963 being significantly less potent or inactive in the assays (Figures 2c and d).

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus