Identification of Azalamellarin N as a Pyroptosis Inhibitor

Abstract

Pyroptosis is a form of regulated cell death that promotes inflammation; it attracts much attention because its dysregulation leads to various inflammatory diseases. To help explore the precise mechanisms by which pyroptosis is regulated, in this study, we searched for chemical compounds that inhibit pyroptosis. From our original compound library, we identified azalamellarin N (AZL-N), a hexacyclic pyrrole alkaloid, as an inhibitor of pyroptosis induced by R837 (also called imiquimod), which is an agonist of the intracellular multiprotein complex nucleotide-binding and oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome. However, whereas the effect of AZL-N on R837-induced pyroptosis was relatively weak, AZL-N strongly inhibited pyroptosis induced by extracellular ATP or nigericin, which are different types of NLRP3 inflammasome agonists. This was in contrast with the results that MCC950, a well-established NLRP3 inhibitor, consistently inhibited pyroptosis irrespective of the type of stimulus. We also found that AZL-N inhibited activation of caspase-1 and apoptosis-associated speck-like proteins containing a caspase activation and recruitment domain (ASC), which are components of the NLRP3 inflammasome. Analysis of the structure–activity relationship revealed that a lactam ring of AZL-N, which has been shown to contribute to the strong binding of AZL-N to its known target protein kinases, is required for its inhibitory effects on pyroptosis. These results suggest that AZL-N inhibits pyroptosis by targeting molecule(s), which may be protein kinase(s), that act upstream of NLRP3 inflammasome activation, rather than by directly targeting the components of the NLRP3 inflammasome. Further identification and analysis of target molecule(s) of AZL-N will shed light on the regulatory mechanisms of pyroptosis, particularly those depending on proinflammatory stimuli.

INTRODUCTION

Inflammation is a biological response that eliminates foreign invaders such as bacteria and viruses upon recognition of their pathogen-associated molecular patterns (PAMPs).¹⁾ Recent studies have also revealed that damage-associated molecular patterns (DAMPs), which are host biomolecules released from stressed or damaged cells, cause sterile inflammation, promoting autoimmune reactions and tissue repair.²⁾ However, growing evidence suggests that inflammation that lasts longer than necessary, even if it is mild, is detrimental to health and implicated in various pathologies, such as cancer and autoimmune, neurodegenerative, and metabolic diseases.²⁾

Pyroptosis is a form of regulated cell death characterized by nuclear condensation, cell swelling, and the formation of large bubbles at the plasma membrane that ultimately ruptures.^3–5) Pyroptosis is induced in innate immune system cells (e.g., macrophages and dendritic cells), and promotes inflammation through the release of inflammatory cytokines such as interleukin (IL)-1β and IL-18. Inflammasomes are intracellular multiprotein complexes that are formed in response to various PAMPs and DAMPs, triggering pyroptosis.^6,7) An inflammasome typically consists of a sensor, an adaptor, and pro-caspase-1. The sensor molecules are pattern recognition receptors, including nucleotide-binding and oligomerization domain-like receptors (NLRs), absent in melanoma-2 (AIM2), and pyrin. Upon recognition of inflammasome agonists, the sensor molecules assemble and then recruit and oligomerize apoptosis-associated speck-like proteins containing a caspase activation and recruitment domain (ASCs). ASCs in turn recruit and activate pro-caspase-1 through its self-cleavage.⁸⁾ Activated caspase-1 proteolytically processes pro-IL-1β and pro-IL-18 into their respective mature forms and cleaves gasdermin D (GSDMD), an executor of pyroptosis, to give a 31-kDa N-terminal fragment (GSDMD-N).⁹⁾ GSDMD-N, unleashed from the autoinhibitory C-terminal fragment (GSDMD-C), translocases to the plasma membrane where it oligomerizes to form pores that release mature IL-1β and IL-18. After passive osmotic swelling, the transmembrane protein ninjurin-1 (NINJ1) has been reported to cause plasma membrane rupture, allowing the release of large DAMPs such as high mobility group box 1 (HMGB1).¹⁰⁾

Considering the above-mentioned physiological and pathophysiological significance of pyroptosis, chemical compounds that manipulate pyroptosis would contribute to further elucidation of the regulatory mechanisms of pyroptosis and to the development of therapeutics for inflammatory diseases caused by dysregulation of pyroptosis. Thus, we searched for such compounds in this study.

MATERIALS AND METHODS

Reagents

R837 (also called imiquimod) was purchased from InvivoGen (San Diego, CA, U.S.A.). Lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), ATP, and cycloheximide (CHX) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). ABT-737 was purchased from ChemScene (Monmouth Junction, NJ, U.S.A.). Q-VD-OPH was purchased from Tonbo Biosciences (Cologne, Germany). MCC950 was purchased from Cayman Chemical (Ann Arbor, MI, U.S.A.). Nigericin was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

Cell Culture

Human leukemia monocytic THP-1 cells (American Type Culture Collection, Manassas, VA, U.S.A.) and murine macrophagic J774.1 cells (RIKEN BRC, Tsukuba, Japan; RCB0434) were cultured in RPMI 1640 medium containing 8% fetal bovine serum, 100 U/mL penicillin G, and 0.1 mg/mL streptomycin under a 5% CO₂ atmosphere at 37 °C overnight. THP-1 cells were differentiated into macrophagic cells by overnight treatment with 100 nM PMA. Before stimulation with inflammasome agonists, THP-1 and J774.1 cells were washed twice with phosphate-buffered saline (PBS) and further incubated for 4 and 5 h, respectively, in Opti-MEM I Reduced-Serum Medium (Thermo Fisher Scientific, Waltham, MA, U.S.A.) containing 100 ng/mL LPS, which was the “priming” stimulus to induce transcription of pro-IL-1β and other proinflammatory factors.^6,7) The phase contrast images of cells were acquired by confocal microscopy (LSM710; Zeiss, Jena, Germany).

Establishment of THP-1 Cells Deficient in GSDMD

Recombinant lentiviruses were produced by transfection of HEK293T cells with GSDMD sgRNA CRISPR/Cas9 all-in-one lentivirus plasmid (Applied Biological Materials, Richmond, BC, Canada; K0911206), pMD2G, pMDLg/RRE, and pRSV-Rev (gifted from Dr. Didier Trono, École polytechnique Fédérale de Lausanne)¹¹⁾ using PEI-MAX (transfection grade linear polyethylenimine hydrochloride; Polysciences, Warrington, PA, U.S.A.). After culture for 72 h, the culture medium was centrifuged at 1200 × g for 10 min, and the supernatant was passed through a 0.45-µm filter (Merck Millipore, Burlington, MA, U.S.A.). Lentiviruses in the filtrate were collected by precipitation with polyethylene glycol and suspended in Opti-MEM I. THP-1 cells were infected with the prepared lentivirus for 24 h, and then transiently treated with 2 µg/mL puromycin. The expression level of GSDMD in the resulting cells (mixed population) was examined by immunoblot analysis.

Immunoblot Analysis

For immunoblot analysis of culture supernatants, culture medium was collected and centrifuged at 860 × g for 1 min, and the resulting supernatant was vigorously mixed with the same volume of methanol and a one-fourth volume of chloroform. After incubation on ice for 10 min, the mixture was centrifuged at 21500 × g for 10 min, and the upper phase of the mixture was removed. The remaining mixture was vigorously mixed with 0.5 mL of methanol. After incubation on ice for 10 min, the mixture was centrifuged at 21500 × g for 20 min, and the supernatant was removed. The pellet was resuspended in methanol, and the resulting suspension was centrifuged at 21500 × g for 2 min. The supernatant was removed, and the pellet was air-dried and then dissolved in a buffer containing 125 mM Tris–HCl (pH 6.8), 20% glycerol, 4% sodium dodecyl sulfate (SDS), and 10 mM dithiothreitol (DTT).

For immunoblot analysis of cell lysates, cells were lysed in a buffer containing 1% Triton X-100, 25 mM Tris–HCl (pH 7.5), 150 mM NaCl, 5 mM ethylene glycol tetraacetic acid (EGTA), 5 µg/mL aprotinin, and 1 mM phenylmethylsulfonyl fluoride (PMSF) on ice for 20 min. The lysate was centrifuged at 21500 × g for 20 min. The supernatant was mixed with one-third amounts of a buffer containing 250 mM Tris–HCl (pH 6.8), 40% glycerol, 8% SDS, and 20 mM DTT.

After heating at 99 °C for 3 min, both types of SDS-containing samples were fractionated by SDS-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membranes. The membranes were probed with primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies. Protein bands were visualized using the enhanced chemiluminescence system and analyzed with a ChemiDoc Touch MP Imaging System (Bio-Rad, Hercules, CA, U.S.A.). Some membranes were reblotted with different antibodies without stripping off the antibody between the blotting procedures. Sometimes this caused residual signals, which are indicated by asterisks in the figures. The following primary antibodies were used in this study: anti-ASC (Cell Signaling, Danvers, MA, U.S.A.; D2W8U #67824), anti-β-actin (Santa Cruz, Dallas, TX, U.S.A.; AC-15 sc-69879), anti-caspase-1 (Adipogen, Basel, Switzerland; AG-20B-0042), anti-caspase-1 (Cell Signaling; D7F10 #3866), anti-GSDMD (Proteintech, Rosemont, IL, U.S.A.; 20770-1-AP), and anti-IL-1β (Cell Signaling; D3H1Z #12507 and D3U3E #12703). HRP-conjugated anti-mouse immunoglobulin G (IgG) (Cytiva, Marlborough, MA, U.S.A.; NA931-1ML) and HRP-conjugated anti-rabbit IgG (Cell Signaling; #7074) were used as secondary antibodies.

Analysis of ASC Oligomerization

J774.1 cells were primed with 100 ng/mL LPS for 4 h and pretreated with 10 µM azalamellarin N (AZL-N) or MCC950 for 30 min, followed by treatment with 2 mM ATP for 1 h. The cells were lysed with a buffer containing 1% Triton X-100, 25 mM Tris–HCl (pH 7.5), 150 mM NaCl, 5 mM EGTA, 5 µg/mL aprotinin, and 1 mM PMSF on ice for 20 min, followed by centrifugation at 21500 × g for 20 min. The supernatants (soluble fractions) were set aside as lysates, and the pellets (insoluble fractions) were washed twice with PBS, and then suspended and incubated in 2 mM solution of the crosslinker disuccinimidyl suberate (Thermo Fischer Scientific) in PBS at 37 °C for 45 min. Both fractions were subjected to immunoblot analysis.

SYTOX Green Assay

THP-1 cells were seeded in assay plates and pretreated with 100 nM PMA overnight. The medium was replaced with RPMI 1640 medium, no-phenol red (FUJIFILM Wako Pure Chemical Corporation). For evaluation of pyroptosis, cells were stimulated with 100 ng/mL LPS for 4 h, and then treated with test compounds for 30 min, followed by stimulation with 10 µg/mL R837 for 2 h. For evaluation of apoptosis, cells were treated with compounds for 30 min, followed by treatment with 0.4 µM ABT-737 and 10 µg/mL CHX for 6 h.¹²⁾ The cells were further treated with 1 µM SYTOX Green (Thermo Fischer Scientific), a high-affinity nucleic acid stain that easily penetrates cells with compromised plasma membranes, for 30 min, and the fluorescence was measured using a plate reader (Bio Tek, Winooski, VT, U.S.A.; Cytation3, Ex/Em = 488/523 nm, gain 70).

Propidium Iodide Assay

Propidium iodide (PI; 2 µg/mL) was added to the culture medium 10 min before cell harvest. Cells were dissociated with trypsin and suspended into single cells by pipetting or passing through 23G needles. The suspended cells were centrifuged at 860 × g for 3 min and resuspended in PBS. The fluorescence emitted by dead cells was analyzed using a BD Accuri C6 flow cytometer (BD Bioscience, Franklin Lakes, NJ, U.S.A.).

RESULTS

Identification of AZL-N as a Compound That Inhibits Pyroptosis

We previously screened and found chemical compounds that inhibit the extracellular release of IL-1β from human leukemia monocytic THP-1 cells stimulated with the imidazoquinoline compound R837 (also called imiquimod).¹³⁾ R837 has been shown to act as an agonist of the NLR family pyrin domain containing 3 (NLRP3) inflammasome, which responds to the broadest range of stimuli among inflammasomes and is crucial in triggering pyroptosis.^14–16) To establish a screening system to search for compounds that inhibit pyroptosis, we first examined whether R837 induces pyroptosis in THP-1 cells. THP-1 cells were differentiated into macrophagic cells by overnight treatment with PMA, followed by 4-h treatment with LPS, and then stimulated with R837. Morphological analysis using a phase contrast microscopy showed that flattened phase-dark cells, which appeared to be dead cells, increased upon stimulation with R837 (Fig. 1A). Immunoblot analysis revealed that cleavage of GSDMD in the cell lysates and the extracellular release of the cleaved GSDMD-C, concomitant with the release of IL-1β, were induced time-dependently upon stimulation with R837 (Fig. 1B). Consistent with this, the number of PI-positive (i.e., dead) cells increased in wild-type cells, but not in the cells deficient in GSDMD, in response to R837 (Figs. 1C, D). These results indicate that R837 induces GSDMD-dependent death, i.e., pyroptosis, in THP-1 cells.

Fig. 1. Identification of Azalamellarin N (AZL-N) as a Compound That Inhibits Pyroptosis

(A) Differentiated THP-1 cells primed with lipopolysaccharide (LPS) were treated with 10 µg/mL R837 for 2 h. The phase contrast images of these cells are shown. Arrowheads indicate flattened phase-dark cells, which appear to be dead cells. Scale bar, 20 µm. (B) Differentiated THP-1 cells primed with LPS were treated with 10 µg/mL R837 for 0.5, 1, and 2 h. The culture supernatants (Sup) and cell lysates (Lysate) were subjected to immunoblot analysis. The asterisks indicate the residual signals caused by reblotting of the membrane. FL, full-length. (C) The cell lysates of differentiated THP-1 cells (wild-type, WT) and gasdermin D (GSDMD)-deficient THP-1 cells were subjected to immunoblot analysis. (D) Differentiated WT and GSDMD-deficient THP-1 cells both primed with LPS were treated with 10 µg/mL R837 for 2 h and then stained with propidium iodide. Data are shown as the mean ± standard error of the mean (S.E.M.) (n = 3 samples). *** p < 0.001, Student’s t-test, compared with untreated cells. (E) Differentiated THP-1 cells primed with LPS were pretreated with various compounds at 1 µM for 30 min, and then treated with 10 µg/mL R837 for 2 h, followed by staining with SYTOX Green. (F) Differentiated THP-1 cells primed with LPS were pretreated with 10 µM AZL-N or MCC950 (MCC) for 30 min, and then treated with 10 µg/mL R837 for 2 h, followed by staining with SYTOX Green. Data are shown as the mean ± S.E.M. (n = 3 samples). *** p < 0.001, Tukey–Kramer’s HSD test, compared with cells treated only with R837. (G) Differentiated THP-1 cells were pretreated with 10 µM Q-VD-OPH (QVD) or AZL-N or for 30 min, and then treated with 0.4 µM ABT-737 and 10 µg/mL cycloheximide (CHX) for 6 h, followed by staining with SYTOX Green. Data are shown as the mean ± S.E.M. (n = 3 samples). *** p < 0.001, Tukey–Kramer’s HSD test, compared with cells treated only with ABT-737 and CHX.

Using this pyroptosis model in combination with the SYTOX Green assay for detecting cell death, we screened a Nagasaki University compound library consisting of 1040 natural and synthetic compounds. Twenty-eight compounds (C1–C28) that inhibited pyroptosis were selected from this initial screening, in which the compounds were applied at 10 µM. We further evaluated the effects of the 28 compounds at 1 µM and found that compound C25 most strongly inhibited R837-induced pyroptosis in THP-1 cells (Fig. 1E). Compound C25 is already known as AZL-N, which is a synthetic congener of lamellarins, a group of 3,4-dihydroxyphenylalanine (DOPA)-derived hexacyclic pyrrole alkaloids originally isolated from marine invertebrates.^17–20) We confirmed that 10 µM AZL-N inhibited R837-induced death of THP-1 cells to a similar extent to the same concentration of MCC950, the most potent and selective inhibitor of NLRP3²¹⁾ (Fig. 1F). However, AZL-N did not inhibit apoptosis, which was induced by the BH3-mimetic ABT-737 and the protein synthesis inhibitor CHX and was sensitive to the pan-caspase inhibitor Q-VD-OPH¹²⁾ (Fig. 1G), suggesting that AZL-N is not a general inhibitor of cell death but is a relatively specific inhibitor of pyroptosis.

AZL-N Preferentially Inhibits ATP- and Nigericin-Induced Pyroptosis

To evaluate the potential of AZL-N as a pyroptosis inhibitor, we examined the effects of lower concentrations (<1 µM) of AZL-N on R837-induced extracellular release of IL-1β, which was a more sensitive marker of pyroptosis than cell death itself detected by PI or SYTOX Green assay. AZL-N inhibited R837-induced release of IL-1β in a concentration-dependent manner even in the range of below 1 µM in THP-1 cells; however, the inhibitory effects of AZL-N were much weaker than those of MCC950 (Fig. 2A). Similar results were obtained when different macrophagic cells, murine J774.1 cells, were used (Fig. 2B).

Fig. 2. Inhibitory Effect of AZL-N on R837-Induced Interleukin (IL)-1β Release Is Relatively Weak

(A) Differentiated THP-1 cells primed with LPS were pretreated with the indicated concentrations of AZL-N or MCC950 for 30 min, and then treated with 10 µg/mL R837 for 2 h. The culture supernatants (Sup) and cell lysates (Lysate) were subjected to immunoblot analysis. (B) J774.1 cells primed with LPS were pretreated with the indicated concentrations of AZL-N or MCC950 for 30 min, and then treated with 20 µg/mL R837 for 1 h. The culture supernatants (Sup) and cell lysates (Lysate) were subjected to immunoblot analysis. The asterisk indicates the residual signals caused by reblotting of the membrane.

We next focused on other agonists of the NLRP3 inflammasome because it is activated by a variety of stimuli. Extracellular ATP binds to the purinergic receptor P2X7 and causes K⁺ efflux, which activates the NLRP3 inflammasome.^22,23) Expression of P2X7 has been reported to be very low, if any, in undifferentiated THP-1 cells.²⁴⁾ Although P2X7 is induced in response to PMA in THP-1 cells, the extent of P2X7 induction is lower than that in response to stronger proinflammatory stimuli such as interferon-γ in combination with LPS or tumor necrosis factor-α.^24,25) Thus, we used J774.1 cells to examine the effects of AZL-N on ATP-induced pyroptosis. As shown in Fig. 3A, AZL-N suppressed ATP-induced release of IL-1β to a similar extent to MCC950, suggesting that the effects of AZL-N differ depending on how pyroptosis is induced. To confirm this point, we directly compared the effects of AZL-N on pyroptosis induced by ATP, R837, or another NLRP3 agonist nigericin, a bacterial toxin acting as a K⁺ ionophore that activates NLRP3 inflammasomes.²⁶⁾ All three agonists strongly induced the release of IL-1β (Fig. 3B, top panel; lanes 3, 6, 9). Although 10 µM MCC950 suppressed IL-1β release irrespective of the pyroptosis-inducing stimulus (lanes 5, 8, 11), the inhibitory effects of AZL-N depended on the stimulus; AZL-N strongly inhibited ATP- and nigericin-induced IL-1β release, but only modestly inhibited R837-induced IL-1β release (lanes 4, 7, 10). These results suggest that AZL-N preferentially attenuates ATP- and nigericin-induced pyroptosis.

Fig. 3. AZL-N Suppresses ATP- and Nigericin-Induced Activation of the NLRP3 Inflammasome

(A) J774.1 cells primed with LPS were pretreated with the indicated concentrations of AZL-N or MCC950 for 30 min, and then treated with 2 mM ATP for 1 h. The culture supernatants (Sup) and cell lysates (Lysate) were subjected to immunoblot analysis. The asterisk indicates the residual signals caused by reblotting of the membrane. (B) J774.1 cells primed with LPS were pretreated with 10 µM AZL-N or MCC950 for 30 min, and then treated with 2 mM ATP, 20 µM nigericin, or 20 µg/mL R837 for 1 h. The culture supernatants (Sup) and cell lysates (Lysate) were subjected to immunoblot analysis. The asterisk indicates the residual signals caused by reblotting of the membrane. (C) J774.1 cells primed with LPS were pretreated with 10 µM AZL-N or MCC950 for 30 min, and then treated with 2 mM ATP for 1 h. The cell lysates (Lysate) and 1% Triton X-100-insoluble fractions treated with 2 mM disuccinimidyl suberate (Insoluble fraction + DSS) were subjected to immunoblot analysis.

AZL-N Suppresses ATP- and Nigericin-Induced Activation of the NLRP3 Inflammasome

To gain insight into how AZL-N suppresses pyroptosis, we focused on the cleavage, and thus activation, of caspase-1, a hallmark of inflammasome activation, which was detected in lysates of J774.1 cells (Fig. 3B, third panel). Nigericin strongly induced the cleavage of caspase-1 (lane 6), and, importantly, AZL-N clearly inhibited this cleavage (lane 7), suggesting that AZL-N targets nigericin-induced inflammasome activation. However, ATP-induced cleavage of caspase-1 was not obvious enough to evaluate the effect of AZL-N on ATP-induced caspase-1 activation (Fig. 3B, lanes 3, 4). Thus, we examined the oligomerization of ASC, another hallmark of inflammasome activation. As shown in Fig. 3C, AZL-N suppressed ATP-induced oligomerization of ASC to a similar extent to MCC950, suggesting that AZL-N also targets inflammasome activation in ATP-induced pyroptosis. Taken together, the point of action of AZL-N appears to be inflammasome activation or its upstream events.

The Lactam Structure of AZL-N Is Required for Its Inhibitory Effect on Pyroptosis

To dissect the mechanism of action of AZL-N, we examined its structure–activity relationship. The library used in this study included six lamellarins as the closest analogues of AZL-N (Fig. 4A; compounds L1–L6), but they were not among the compounds C1–C28 that were selected in our initial screening (Fig. 1E). We then confirmed that none of the lamellarins in the library exhibited inhibitory effects on R837-induced pyroptosis in THP-1 cells (Fig. 4B). The primary difference in the molecular structures between AZL-N and lamellarins is that AZL-N has a lactam ring instead of the lactone ring that lamellarins consistently have.^17,18) AZL-N and compound L2, which corresponds to lamellarin N, have the same structure except for their lactam/lactone rings (Fig. 4A). These results strongly suggest that the lactam ring is required for the inhibitory effect of AZL-N on pyroptosis.

Fig. 4. Lamellarins Do Not Exhibit Inhibitory Effects on Pyroptosis

(A) Structures of AZL-N and lamellarins (compounds L1–6) in the library used in this study. (B) Differentiated THP-1 cells primed with LPS were pretreated with 10 µM AZL-N (N) or L1–6 for 30 min, and then treated with 10 µg/mL R837 for 2 h, followed by staining with SYTOX Green. Data are shown as the mean ± S.E.M. (n = 3 samples). *** p < 0.001, Tukey–Kramer’s HSD test, compared with cells treated only with R837.

We finally examined whether analogues of AZL-N that have lactam rings also exhibit inhibitory effects on pyroptosis. Here, we used four A-ring-modified analogues of AZL-N (Fig. 5A; compounds AZL-2–AZL-5), which we previously synthesized to evaluate their effects on the epidermal growth factor receptor (EGFR) T790M/L858R mutant that causes resistance of non-small cell lung cancer to some tyrosine kinase inhibitors.¹⁸⁾ All four analogues inhibited R837-induced pyroptosis as effectively as AZL-N, whereas they did not inhibit ABT-737/CHX-induced apoptosis in THP-1 cells (Figs. 5B, C). These results again suggest the structural importance of the lactam ring of AZL-N in its inhibitory effects on pyroptosis.

Fig. 5. Analogues of AZL-N Possessing the Lactam Structure Exhibit Inhibitory Effects on Pyroptosis

(A) Structures of AZL-N and its A-ring-modified analogues (compounds AZL-2–5). (B) Differentiated THP-1 cells primed with LPS were pretreated with 10 µM AZL-N or AZL-2–5 for 30 min, and then treated with 10 µg/mL R837 for 2 h, followed by staining with SYTOX Green. Data are shown as the mean ± S.E.M. (n = 3 samples). *** p < 0.001, Tukey–Kramer’s HSD test, compared with cells treated only with R837. (C) Differentiated THP-1 cells were pretreated with 10 µM Q-VD-OPH (Q), AZL-N, or AZL-2~AZL-5 for 30 min, and then treated with 0.4 µM ABT-737 and 10 µg/mL CHX for 6 h, followed by staining with SYTOX Green. Data are shown as the mean ± S.E.M. (n = 3 samples). *** p < 0.001, Tukey–Kramer’s HSD test, compared with cells treated only with R837.

DISCUSSION

In this study, we identified AZL-N as an inhibitor of pyroptosis. On the basis of analyses of caspase-1 activation and ASC oligomerization, the point of action of AZL-N appears to be inflammasome activation or its upstream events. AZL-N inhibited pyroptosis induced by ATP, nigericin, or R837, all of which are NLRP3 inflammasome agonists. However, the inhibitory effects of AZL-N differed depending on the type of stimulus; the effects of AZL-N on ATP- and nigericin-induced pyroptosis were stronger than those on R837-induced pyroptosis. This was in contrast with MCC950, which consistently inhibited pyroptosis irrespective of the type of stimulus. Considering that MCC950 directly and specifically binds NLRP3,²¹⁾ AZL-N may not inhibit components of the NLRP3 inflammasome but rather target molecules that act in the upstream events of inflammasome activation.

The NLRP3 inflammasome has been regarded as being activated by most of its agonists, including ATP and nigericin in a K⁺ efflux-dependent manner.^22,26) However, R837 was previously reported to activate the NLRP3 inflammasome in a manner independent of K⁺ efflux, being dependent on the generation of large amounts of reactive oxygen species.¹⁴⁾ More recently, it has been shown that the trans-Golgi network (TGN) is dispersed in response to K⁺ efflux-dependent stimuli such as ATP or nigericin and that a phospholipid on the dispersed TGN (dTGN), phosphatidylinositol-4-phosphate, recruits the NLRP3 inflammasome and induces its activation.²⁷⁾ Although K⁺ efflux-independent stimuli such as R837 also disperse the TGN, activation of the NLRP3 inflammasome by such stimuli takes place not only on the dTGN but also on the plasma membrane. This finding suggests that the dependence of R837-induced NLRP3 inflammasome activation on the dTGN is relatively low. Thus, AZL-N may target regulatory molecules that are involved in the recruitment and activation of the NLRP3 inflammasome on the dTGN.

Analysis of the structure–activity relationship revealed that the lactam ring of AZL-N is required for its inhibitory effect on pyroptosis. Lamellarin N (Fig. 4A, compound L2) is the prototypic compound of AZL-N that possesses a lactone ring instead of a lactam ring and has been found to inhibit the catalytic activity of several protein kinases related to cancer and neurodegenerative diseases, including cyclin-dependent kinases, glycogen synthase kinase-3 (GSK-3), and EGFR T790M/L858R.^28–30) Importantly, the inhibitory effects of AZL-N on GSK-3 and EGFR T790M/L858R are stronger than those of lamellarin N, which is supported by molecular-modeling studies showing that the lactam NH group in AZL-N forms an additional hydrogen bond with the target kinases.^17,18) These findings strongly suggest that AZL-N also targets protein kinases in the context of pyroptosis induction. GSK-3 may be at least one target of AZL-N because it has recently been shown to facilitate activation of the NLRP3 inflammasome on the TGN.³¹⁾

Many studies have focused on the general regulatory mechanisms of pyroptosis, particularly those by the NLRP3 inflammasome, irrespective of the type of stimulus. In this regard, AZL-N is a unique tool that inhibits pyroptosis differentially depending on the type of stimulus. Thus, further identification and analysis of target molecule(s) of AZL-N will shed light on the precise mechanisms by which pyroptosis is induced in response to a variety of proinflammatory stimuli.

Acknowledgments

This work was supported in part by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from the Japan Agency for Medical Research and Development (AMED) (JP23ama121032), JSPS KAKENHI (Grant Numbers: JP21K06069 to S. T. and JP23K06117 to K. T.), the Takeda Science Foundation, the Mitsubishi Foundation, the NOVARTIS Foundation (Japan) for the Promotion of Science, and the Naito Foundation.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Li D, Wu M. Pattern recognition receptors in health and diseases. Signal Transduct. Target. Ther., 6, 291 (2021).
• 2) Gong T, Liu L, Jiang W, Zhou R. DAMP-sensing receptors in sterile inflammation and inflammatory diseases. Nat. Rev. Immunol., 20, 95–112 (2020).
• 3) Shi J, Gao W, Shao F. Pyroptosis: gasdermin-mediated programmed necrotic cell death. Trends Biochem. Sci., 42, 245–254 (2017).
• 4) Li T, Zheng G, Li B, Tang L. Pyroptosis: a promising therapeutic target for noninfectious diseases. Cell Prolif., 54, e13137 (2021).
• 5) Kist M, Vucic D. Cell death pathways: intricate connections and disease implications. EMBO J., 40, e106700 (2021).
• 6) Broz P, Dixit VM. Inflammasomes: mechanism of assembly, regulation and signalling. Nat. Rev. Immunol., 16, 407–420 (2016).
• 7) Barnett KC, Li S, Liang K, Ting JP. A 360° view of the inflammasome: mechanisms of activation, cell death, and diseases. Cell, 186, 2288–2312 (2023).
• 8) Van Opdenbosch N, Lamkanfi M. Caspases in cell death, inflammation, and disease. Immunity, 50, 1352–1364 (2019).
• 9) Broz P, Pelegrin P, Shao F. The gasdermins, a protein family executing cell death and inflammation. Nat. Rev. Immunol., 20, 143–157 (2020).
• 10) Kayagaki N, Kornfeld OS, Lee BL, et al. NINJ1 mediates plasma membrane rupture during lytic cell death. Nature, 591, 131–136 (2021).
• 11) Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat. Biotechnol., 15, 871–875 (1997).
• 12) Vince JE, De Nardo D, Gao W, Vince AJ, Hall C, McArthur K, Simpson D, Vijayaraj S, Lindqvist LM, Bouillet P, Rizzacasa MA, Man SM, Silke J, Masters SL, Lessene G, Huang DCS, Gray DHD, Kile BT, Shao F, Lawlor KE. The mitochondrial apoptotic effectors BAX/BAK activate caspase-3 and -7 to trigger NLRP3 inflammasome and caspase-8 driven IL-1beta activation. Cell Rep., 25, 2339–2353.e4 (2018).
• 13) Honda S, Sadatomi D, Yamamura Y, Nakashioya K, Tanimura S, Takeda K. WP1066 suppresses macrophage cell death induced by inflammasome agonists independently of its inhibitory effect on STAT3. Cancer Sci., 108, 520–527 (2017).
• 14) Groß CJ, Mishra R, Schneider KS, et al. K⁺ efflux-independent NLRP3 inflammasome activation by small molecules targeting mitochondria. Immunity, 45, 761–773 (2016).
• 15) Xu J, Nunez G. The NLRP3 inflammasome: activation and regulation. Trends Biochem. Sci., 48, 331–344 (2023).
• 16) Swanson KV, Deng M, Ting JP. The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat. Rev. Immunol., 19, 477–489 (2019).
• 17) Theppawong A, Ploypradith P, Chuawong P, Ruchirawat S, Chittchang M. Facile and divergent synthesis of lamellarins and lactam-containing derivatives with improved drug likeness and biological activities. Chem. Asian J., 10, 2631–2650 (2015).
• 18) Fukuda T, Anzai M, Nakahara A, Yamashita K, Matsukura K, Ishibashi F, Oku Y, Nishiya N, Uehara Y, Iwao M. Synthesis and evaluation of azalamellarin N and its A-ring-modified analogues as non-covalent inhibitors of the EGFR T790M/L858R mutant. Bioorg. Med. Chem., 34, 116039 (2021).
• 19) Fan H, Peng J, Hamann MT, Hu JF. Lamellarins and related pyrrole-derived alkaloids from marine organisms. Chem. Rev., 108, 264–287 (2008).
• 20) Boonya-udtayan S, Yotapan N, Woo C, Bruns CJ, Ruchirawat S, Thasana N. Synthesis and biological activities of azalamellarins. Chem. Asian J., 5, 2113–2123 (2010).
• 21) Coll RC, Hill JR, Day CJ, Zamoshnikova A, Boucher D, Massey NL, Chitty JL, Fraser JA, Jennings MP, Robertson AAB, Schroder K. MCC950 directly targets the NLRP3 ATP-hydrolysis motif for inflammasome inhibition. Nat. Chem. Biol., 15, 556–559 (2019).
• 22) Di A, Xiong S, Ye Z, Malireddi RKS, Kometani S, Zhong M, Mittal M, Hong Z, Kanneganti TD, Rehman J, Malik AB. The TWIK2 potassium efflux channel in macrophages mediates NLRP3 inflammasome-induced inflammation. Immunity, 49, 56–65.e4 (2018).
• 23) Wang J, Takemura N, Saitoh T. Macrophage response driven by extracellular ATP. Biol. Pharm. Bull., 44, 599–604 (2021).
• 24) Humphreys BD, Dubyak GR. Modulation of P2X7 nucleotide receptor expression by pro- and anti-inflammatory stimuli in THP-1 monocytes. J. Leukoc. Biol., 64, 265–273 (1998).
• 25) Gadeock S, Tran JN, Georgiou JG, Jalilian I, Taylor RM, Wiley JS, Sluyter R. TGF-beta1 prevents up-regulation of the P2X7 receptor by IFN-gamma and LPS in leukemic THP-1 monocytes. Biochim. Biophys. Acta, 1798, 2058–2066 (2010).
• 26) Muñoz-Planillo R, Kuffa P, Martínez-Colón G, Smith BL, Rajendiran TM, Núñez GK. K⁺ Efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter. Immunity, 38, 1142–1153 (2013).
• 27) Chen J, Chen ZJ. PtdIns4P on dispersed trans-Golgi network mediates NLRP3 inflammasome activation. Nature, 564, 71–76 (2018).
• 28) Baunbæk D, Trinkler N, Ferandin Y, Lozach O, Ploypradith P, Rucirawat S, Ishibashi F, Iwao M, Meijer L. Anticancer alkaloid lamellarins inhibit protein kinases. Mar. Drugs, 6, 514–527 (2008).
• 29) Yoshida K, Itoyama R, Yamahira M, Tanaka J, Loaec N, Lozach O, Durieu E, Fukuda T, Ishibashi F, Meijer L, Iwao M. Synthesis, resolution, and biological evaluation of atropisomeric (aR)- and (aS)-16-methyllamellarins N: unique effects of the axial chirality on the selectivity of protein kinases inhibition. J. Med. Chem., 56, 7289–7301 (2013).
• 30) Fukuda T, Umeki T, Tokushima K, Xiang G, Yoshida Y, Ishibashi F, Oku Y, Nishiya N, Uehara Y, Iwao M. Design, synthesis, and evaluation of A-ring-modified lamellarin N analogues as noncovalent inhibitors of the EGFR T790M/L858R mutant. Bioorg. Med. Chem., 25, 6563–6580 (2017).
• 31) Arumugam S, Qin Y, Liang Z, Han SN, Boodapati SLT, Li J, Lu Q, Flavell RA, Mehal WZ, Ouyang X. GSK3beta mediates the spatiotemporal dynamics of NLRP3 inflammasome activation. Cell Death Differ., 29, 2060–2069 (2022).