Activity and specificity of necrostatin-1, small-molecule
inhibitor of RIP1 kinase
Cell Death and Differentiation (2013) 20, 366; doi:10.1038/cdd.2012.133; published online 30 November 2012
Necroptosis has emerged as an important form of regulated
necrotic cell death, which can occur during mouse develop-
ment and is also an important contributor to necrotic tissue
injury in a variety of mouse models of human pathologies,
including brain trauma, ischemia-reperfusion injuries, viral
infections and acute inflammatory responses.1Necrostatin-1
(Nec-1; methyl-thiohydantoin-tryptophan (MTH-Trp)), identi-
fied by us in a cell-based small-molecule necroptosis screen,2
was found to selectively target the kinase activity of RIP1, a
key mediator of necroptosis.3Nec-1 is commercially available
and has been used extensively both in vitro and in vivo by
multiple groups to elucidate the role of necroptosis. However,
because this molecule is also known to inhibit indoleamine-
2,3-dioxygenase (IDO),4we feel that it is important to revisit
and summarize some of the key published and new data
regarding activity and specificity of Nec-1.
We assessed inhibition of RIP1 and IDO by Necs, including
Nec-1, original molecule identified in our screen,27-Cl-O-
Nec-1, optimized analog of Nec-1,3,5inactive analogs of both
Necs (denoted by ‘I’) and an IDO inhibitor, 1-methyl-D,
to RIP1 inhibition, our data confirmed excellent correlation of
Nec’s activity against RIP1 kinase in vitro and necroptosis in
Jurkat cells (Supplemantary Figure S1B). Notably, 1-MT failed
to inhibit necroptosis, confirming the specific role of RIP1
kinase, rather than IDO, in necroptosis. Conversely, only the
(Supplemantary Figure S1C). These results clearly distinguish
the SAR of Necs against the two different enzyme targets.
available Necrostain-1 (MTH-Trp),a molecule originally identified
assay) because of the presence of sulfur of thiohydantoin.5
Chemical optimization of Nec-1 (4200 derivatives analyzed
to date) led to the development of a much improved analog,
termed 7-Cl-O-Nec-1 (or 7-Cl-O-MH-Trp) (Supplemantary
Figure S1A),5which showed a robust improvement in metabolic
stability to T1/2of B1h in liver microsomal assay and in in vivo
pharmacokinetics (PK) study.5
Target specificity is a critical consideration for any small-
molecule tool. Notably, optimized 7-Cl-O-Nec-1 displayed
exclusive selectivity towards RIP1 in a screen of a 4400
human kinases, including several other RIP family members.6
Furthermore, we observed perfect correlation between
inhibition of RIP1 kinase and cellular necroptosis by Nec-1
analogs.3We also found, using wild-type and RIP1-deficient
Jurkat and mouse fibroblast cells, that the ability of Nec-1 to
inhibit cell death is entirely dependent on the expression of
RIP1 kinase.3,6Thus, Nec-1 and, especially, 7-Cl-O-Nec-1
represent useful chemical probes for defining the role of RIP1
kinase in cellular regulation. It is also worth noting that very
closely related inactive analogs of both Nec-1 and 7-Cl-O-
Nec-1 have also been described to facilitate confirmation of
on-target activity of the inhibitors in vitro and in vivo
(Supplemantary Figures S1A and B).3
Overall, these data lead us to suggest that in interpreting the
results obtained using original Nec-1, a limited bioavailability
and inhibitory activity against IDO should always be consid-
ered. It is also useful to bear in mind that Nec-1i, commercially
available inactive derivative of Nec-1, cannot be used alone to
differentiate between targeting IDO and RIP1. Optimized 7-Cl-
O-Nec-1 represents a superior tool, especially, in vivo. Addi-
tional tools, such as described in the literature RIP3 and IDO-
deficient animals and/or short hairpin RNA knockdowns, can
help further distinguish between these two pathways.
Conflict of Interest
The authors declare no conflict of interest.
A Degterev1, JL Maki1and J Yuan*,2
1Department of Biochemistry, Tufts University, Boston, MA, USA and
2Department of Cell Biology, Harvard Medical School, Boston, MA, USA
* Corresponding authors: J Yuan, Department of Cell Biology, Harvard Medical
School, 240 Longwood Avenue, Boston, MA 02115, USA.
Tel: 617 432 4187; Fax: 617 432 4177;
1. Galluzzi L et al. Int Rev Cell Mol Biol 2011; 289: 1–35.
2. Degterev A et al. Nat Chem Biol 2005; 1: 112–119.
3. Degterev A et al. Nat Chem Biol 2008; 4: 313–321.
4. Muller AJ et al. Nat Med 2005; 11: 312–319.
5. Teng X et al. Bioorg Med Chem Lett 2005; 15: 5039–5044.
6. Christofferson DE et al. Cell Death Dis 2012; 3: e320.
Supplementary Information accompanies the paper on Cell Death and Differentiation website (http://www.nature.com/cdd)
Cell Death and Differentiation (2013) 20, 366
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