Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models

Abstract

Necrostatin-1 (Nec-1) is widely used in disease models to examine the contribution of receptor-interacting protein kinase (RIPK) 1 in cell death and inflammation. We studied three Nec-1 analogs: Nec-1, the active inhibitor of RIPK1, Nec-1 inactive (Nec-1i), its inactive variant, and Nec-1 stable (Nec-1s), its more stable variant. We report that Nec-1 is identical to methyl-thiohydantoin-tryptophan, an inhibitor of the potent immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). Both Nec-1 and Nec-1i inhibited human IDO, but Nec-1s did not, as predicted by molecular modeling. Therefore, Nec-1s is a more specific RIPK1 inhibitor lacking the IDO-targeting effect. Next, although Nec-1i was ∼100 × less effective than Nec-1 in inhibiting human RIPK1 kinase activity in vitro, it was only 10 times less potent than Nec-1 and Nec-1s in a mouse necroptosis assay and became even equipotent at high concentrations. Along the same line, in vivo, high doses of Nec-1, Nec-1i and Nec-1s prevented tumor necrosis factor (TNF)-induced mortality equally well, excluding the use of Nec-1i as an inactive control. Paradoxically, low doses of Nec-1 or Nec-1i, but not Nec -1s, even sensitized mice to TNF-induced mortality. Importantly, Nec-1s did not exhibit this low dose toxicity, stressing again the preferred use of Nec-1s in vivo. Our findings have important implications for the interpretation of Nec-1-based data in experimental disease models.

Figure 1

Activity of Nec-1, Nec-1i and Nec-1s in an in vitro RIPK kinase assay and a cellular assay for necroptosis. (a) Chemical structures of Nec-1/MTH-Trp, Nec-1i and Nec-1s (b) Effect of Nec-1 variants on human RIPK1 kinase activity. Recombinant GST-hRIPK1 was preincubated with the indicated amount of inhibitor, and autophosphorylation was determined by a radioactive ATP assay, followed by SDS-PAGE and transfer to nitrocellulose membrane. All reactions contained the same amount of DMSO. The autophosphorylation observed in the presence of only DMSO was set to 100%. (c). Effect of Nec-1 variants on human RIPK3 kinase activity. The procedure was identical to b, but GST-hRIPK3 was used instead of GST-hRIPK1. (d). Effect of Nec-1 variants on TNF-induced necrosis. L929sA cells were preincubated for 1 h with the indicated amounts of inhibitor or an equal amount of DMSO, and then treated with 1000 IU/ml of mTNF for 6 h. Cell death was measured as percentage of propidium iodide (PI)-positive nuclei on images acquired with BD pathway

Figure 2

Molecular docking of substrates, inhibitors and Nec-1 variants on human IDO. (a) Chemical structures of ℒ-Trp, 1-MT, Nec-1/MTH-Trp, Nec-1i and Nec-1s. (b) Surface view of IDO's pocket-like active site, with the docked pose of ℒ-Trp as seen from the enzyme's heme in the foreground. (c) Same viewing position partly surfaced, now showing Ser167 in relation to the ligand's indole ring. (d) Same docking pose of ℒ-Trp (amino-acid moiety clipped off for clarity) now as seen from the pocket entrance with the heme on the right, with meshed surface showing the narrow pocket. The gap between the heme's iron versus positions 1 and 2 of the indole ring leaves sufficient space for the missing iron-bound dioxygen. (e) The docked pose of Nec-1 shows a symmetrical distance between the heme's iron and position 1 or 2 of the indole ring, also allowing sufficient space for dioxygen. (f) For the analogous pose of Nec-1s, the indole ring is slightly tilted due to a steric clash of Ser167 with the indole's 7-Cl substituent. This brings the indole ring substantially closer to the heme, suggesting that this pose no longer leaves sufficient space for molecular oxygen, or that Nec-1s may be unable to fit into the pocket of dioxygen-bound IDO

Figure 3

Molecular docking of substrates, inhibitors and Nec-1 variants on human IDO representing a similar docking pose for all ligands. (a) Vina dockings on human IDO yielded highly similar poses for all ligands whether as ℒ or 𝒟 enantioner; only ℒ enantiomers are shown. Top left: ℒ-Trp with amino-acid moiety and meshed surface clipped off, showing the narrow pocket; top center: L-Trp complete view; top right: L-1-MT; bottom left: L-Nec-1; bottom center: L-Nec-1i; and bottom right: L-Nec-1s. The latter shows a larger tilt of the indole ring with respect to the pocket, due to a steric clash between Ser167 and the indole's 7-Cl substituent. (b) Table with calculated distances between IDO's heme iron versus positions 1 and 2 of the respective indoles of the docked ligand poses. The red highlight indicates the smaller gap for molecular oxygen with the pose of Nec-1s

Figure 4

Nec-1/1-M-Trp and Nec-1i, but not Nec-1s, inhibit IDO activity. (a) IDO enzymatic assay using recombinant IDO shows IDO inhibitory activities of Nec-1 and Nec-1i, but not of Nec-1s, which is in accordance with the predictions of the docking experiment. (b) Prototype IDO inhibitor D-1-MT has no inhibitory activity on cellular necroptosis assay using L929 cells. Cell survival was measured by MTT colorimetric method. (c) Increasing the concentration of 1-MT up to 100 μM has no effect on necroptosis. Cell survival was measured by MTT colorimetric method

Figure 5

Nec-1 and Nec-1i show a paradoxical dose response in TNF-induced SIRS. (a) Effect of Nec-1 and Nec-1i on TNF shock-associated hypothermia. DMSO, Nec-1 or Nec-1i were injected i.v. at a dose of 6 mg/kg b.w. 17 min before challenge with 10 μg mTNF i.v. (about LD₁₀₀); Nec-1 and Nec-1i were equipotent in protection. (b) Survival curve for the experiment described in a. (c) TNF shock-associated hypothermia for different doses of Nec-1 and Nec-1i. Both compounds were injected at different doses with the same regimen as above, before injection with 7.5 μg mTNF (about LD₅₀). Hi: 6 mg/kg b.w; Mo: 3 mg/kg b.w; and Lo: 0.6 mg/kg b.w. Nec-1 and Nec-1i paradoxically sensitized mice to TNF-induced SIRS. (d) Survival curve for different doses of Nec-1 and Nec-1i. (e) Levels of damage-associated markers detected in the plasma of mice treated as indicated with TNF and different doses of Nec-1 and Nec-1i. A cumulative result of two independent experiments is represented (a–d). Number of mice is indicated between brackets. ^*P<0.05, ^**P<0.001 and ^***P<0.0001

Figure 6

Nec-1s does not show a paradoxical dose response in TNF-induced SIRS. (a) TNF shock-associated hypothermia. Nec-1 and Nec-1s were injected i.v. at a dose of Hi: 6 mg/kg b.w. or Lo: 0.6 mg/kg b.w. 17 min before the challenge with mTNF; i.v. Nec-1s was as protective as Nec-1 but lacked the sensitizing effect of Nec-1 at the lower dose. (b) Survival curve for the experiment described in a. Symbols of Nec-1 Hi and Nec-1s Hi coincide and are both completely protective. A cumulative result of two independent experiments is represented. Number of mice is indicated between brackets. ^*P<0.05, ^**P<0.001 and ^***P<0.0001