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. 2018 Mar 15;25(3):262-267.e5.
doi: 10.1016/j.chembiol.2017.12.013. Epub 2018 Jan 27.

Inhibition of Dpp8/9 Activates the Nlrp1b Inflammasome

Marian C Okondo  1 , Sahana D Rao  2 , Cornelius Y Taabazuing  1 , Ashley J Chui  2 , Sarah E Poplawski  3 , Darren C Johnson  2 , Daniel A Bachovchin  4
Affiliations

Inhibition of Dpp8/9 Activates the Nlrp1b Inflammasome

"V体育官网入口" Marian C Okondo et al. Cell Chem Biol. .

"VSports手机版" Abstract

Val-boroPro (PT-100, Talabostat) induces powerful anti-tumor immune responses in syngeneic cancer models, but its mechanism of action has not yet been established. Val-boroPro is a non-selective inhibitor of post-proline-cleaving serine proteases, and the inhibition of the highly related cytosolic serine proteases Dpp8 and Dpp9 (Dpp8/9) by Val-boroPro was recently demonstrated to trigger an immunostimulatory form of programmed cell death known as pyroptosis selectively in monocytes and macrophages VSports手机版. Here we show that Dpp8/9 inhibition activates the inflammasome sensor protein Nlrp1b, which in turn activates pro-caspase-1 to mediate pyroptosis. This work reveals a previously unrecognized mechanism for activating an innate immune pattern recognition receptor and suggests that Dpp8/9 serve as an intracellular checkpoint to restrain Nlrp1b and the innate immune system. .

Keywords: Dpp8/9; Nlrp1b inflammasome; Val-boroPro; caspase-1; pyroptosis V体育安卓版. .

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Conflict of interest statement (VSports在线直播)

DECLARATION OF INTERESTS

The authors declare no competing financial interests.

"VSports在线直播" Figures

Figure 1
Figure 1. Nlrp1b is required for Dpp8/9 inhibitor-induced pyroptosis. See also Figure S1
(A) Structures of Val-boroPro, 1G244, and compound 8j. (B) Diagram of mouse Nlrp1b allele 1. The LF cleavage and FIIND autoproteolysis sites are indicated. (C,D) Casp1 and Nlrp1b KO RAW 264.7 macrophages are resistant to LT (1 μg/mL, 6 h) (C) and the Dpp8/9 inhibitors Val-boroPro (2 μM, 24 h), 1G244 (5 μM, 24 h), and compound 8j (20 μM, 24 h) (D). Data are means ± SEM of three biological replicates. ***p < 0.001 by two-sided Student’s t-test for vehicle versus treated cells. LT, lethal toxin; VbP, Val-boroPro.
Figure 2
Figure 2. Nlrp1b controls primary mouse macrophages sensitivity to Val-boroPro. See also Figure S2
(A,B) Primary macrophages from the indicated inbred mouse strains were treated with LT (1 μg/mL, 6 h) (A) or Val-boroPro (10 μM, 24 h) (B) before LDH release was assessed. The Nlrp1b allele encoded in each macrophage is shown below the strain in red (see also Fig. S2A). Data are means ± SEM of three biological replicates. ** p < 0.01, ***p < 0.001 by two-sided Student’s t-test for vehicle versus treated cells. (C,D) Val-boroPro (100 μg/mouse) induces high levels of serum G-CSF (C) and CXCL1/KC (D) after 6 h in C57BL/6 and 129S6 ice, but not in Nlrp1b−/−, mice as measured by ELISA. Data are means ± SEM, n = 5 mice/group. **p < 0.01, ***p < 0.001 by two-sided Student’s t-test for vehicle versus Val-boroPro-treated mice. NS, not significant.
Figure 3
Figure 3. Activation of Nlrp1b requires FIIND autoproteolysis. See also Figure S3
(A) Plasmids encoding WT or autoproteolysis-deficient mutant (S984A) Nlrp1b were nucleofected into Nlrp1b−/− RAW 264.7 cells, which were then treated with LT (1 μg/mL, 6 h) or Val-boroPro (10 μM, 24 h). Cytotoxicity was assessed by LDH release. (B,C) HEK 293T cells ectopically expressing mCasp1 and either Nlrp1b WT or S984A were treated with LT (1 μg/mL, 6 h) or Val-boroPro (5 μM, 24 h). Cytotoxicity was assessed by LDH release in (B) and Nlrp1b N-terminal cleavage was assessed by immunoblotting (construct contains an N-terminal GST tag) in (C). Asterisks indicate background bands. FL, full-length. In (A) and (B), data are means ± SEM of three biological replicates. *p<0.05, **p < 0.01, ***p < 0.001 by two-sided Student’s t-test for vehicle versus compound-treated cells. NS, not significant.
Figure 4
Figure 4. Proteasome inhibitors block Nlrp1b activation
RAW 264.7 cells were treated with VbP (2 μM), LT (1 μg/mL), or both for the 6 h (a and c) or indicated time (b). In (a) and (c), cells were pretreated bortezomib (100 °C) or MG132 (100 °C) for 30 min prior to the addition of VbP and/or LT. Cytotoxicity was assessed by LDH release. In (b), LT and VbP induce synergistic cell death. Data are means ± SEM of three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001 by two-sided Students t-test.
Scheme 1
Scheme 1
Synthesis of Val-boroPro Reagents and conditions: i. HATU, DIEA, DMF, 0°C to r.t, 92%; ii. BCl3 in CH2Cl2, −78°C, 78%.
Scheme 2
Scheme 2
Synthesis of 8j Reagents and conditions: i. HATU, DIEA, DMF, 0°C to r.t; ii. 4N HCl in dioxane, 0°C to r.t, 91% for two steps.
Scheme 3
Scheme 3
Synthesis of 1G244 Reagents and conditions: i. Isoindoline, HATU, DIEA, DMF, 0°C to r.t; ii. H 2, 10% Pd-C, MeOH; iii. 1-Bis(4-fluorophenyl)methyl piperazine, HATU, DIEA, DMF, 0°C to r.t, 75% for 3 steps; iv. 4N HCl in dioxane, 0°C to r.t, 90%.
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