Caspase-1

The expression of IL-1β and IL-18 in activated astrocytes is decreased in vitro by Ac-YVAD-CMK (40 μM or 80 μM) [2].

---

The caspase-1 inhibitor suppresses alcohol-induced pyroptosis and inflammation [1]
Because alcohol treatment could induce pyroptosis, next we detected the effect of the caspase-1 inhibitor N-Ac-Tyr-Val-Ala-Asp-chloromethyl ketone (Ac-YVAD-CMK) on pyroptosis and inflammatory cytokines in esophagitis. We first needed to clarify the inhibitory effect of Ac-YVAD-CMK on caspase-1. Thus, we carried out immunofluorescence assays [Figure 4A]. The results showed that both Ac-YVAD-CMK and caspase-1 siRNA could remarkably inhibit the caspase-1 expression level. Cell death induced by pyroptosis was also suppressed by Ac-YVAD-CMK [Figure 4B]. Furthermore, qRT-PCR and western blotting showed that this agent could effectively inhibit caspase-1 expression similar to siRNA [Figure 4C & 4D].

---

Ac-YVAD-CMK reduced the expression of IL-1β and IL-18 in activated microglia in vitro [2]
The microglia cells were purified by shaking of the cell culture flask leading to differential cell adherence, after which thrombin could effectively activate the resting microglia cells by treatment for 24 h [17]. The mRNA expression of NLRP3 and IL-1β/IL-18 increased in thrombin-activated microglia (Fig. 1a). High level Ac-YVAD-CMK treatment (40 μmol/L or 80 μmol/L) significantly decreased the mRNA levels of IL-1β/IL-18. Furthermore, the protein levels of NLRP3, caspase-1 (p20), mature IL-1β/IL-18 were higher in thrombin-activated microglia group than in control group as seen by western blot. Meantime, the 40 μmol/L dose of Ac-YVAD-CMK significantly reduced the protein levels of caspase-1 (p20) and mature IL-1β/IL-18 compared with the thrombin-activated microglia group (Fig. 1b–f).

Mature IL-1β/IL-18 Log-rank analysis revealed that Ac-YVAD-CMK (ac-YVAD-cmk receiving a dose of 12.5 μMol/kg) significantly reduced the test from 83% to 33% when compared to the ICH group. Ac-YVAD-cmk treatment (1 μg/staple; SD stack; injected into the left ventricle) significantly reduced the protein levels of caspase-1 (p20).

---

Alcohol accumulation initiates pyroptosis in vivo [1]
To further confirm that alcohol drinking could act on esophagitis through pyroptosis, we completed animal experiments. We chose nine Balb/c mice, six to eight weeks old, and divided them into three groups. The first group was given normal saline, the second group was given 1% alcohol, and the third group was given the same alcohol concentration and the inhibitor Ac-YVAD-CMK. All three groups were administered the agents by gavage. First, we isolated the esophageal epithelial tissues of these three groups to form hematoxylin and eosin (HE) staining [Figure 5A]. Inflammatory cell evasion was detected in these three groups, and the results showed a significant increase in mouse esophageal mucosal inflammation in the ethanol group and then decreased in caspase-1 inhibitor-treated group. Next, we carried out immunohistochemical assays to detect caspase-1, IL-1β and IL-18 expression, and the results showed that alcohol treatment activated the expression of all three; however, the inhibitor significantly reversed the increased expression [Figure 5B-D]. Next, we extracted the esophageal epithelial cells from all three groups, collected the whole nucleic acids and proteins, detected the mRNA [Figure 5E] and protein expression levels [Figure 5F] of caspase-1, IL-1β and IL-18. The results suggested that alcohol drinking could induce caspase-1 activation in esophagitis; however, Ac-YVAD-CMK could inhibit the activation of IL-1β and IL-18 remarkably, reducing the inflammatory response and greatly relieving the progression of esophagitis.

---

Ac-YVAD-CMK improved neurological function and reduced brain edema after ICH [2]
In order to assess the effects of Ac-YVAD-CMK after ICH, the mNSS was performed at 24 h post-ICH (Fig. 2a). The results of the mNSS test showed that the degree of neurological injury was significantly reduced in the inhibitor treatment group (Ac-YVAD-cmk) compared with the ICH group at 24 h after ICH (9.33 ± 1.21 vs ICH group, 11.00 ± 1.79; P < 0.05), similar results were obtained at 72 h after ICH (7.17 ± 1.17 vs ICH group, 8.50 ± 1.52; P < 0.05). Meanwhile, to investigate whether Ac-YVAD-cmk could attenuate brain edema, we monitored brain water content in the experimental groups 24 h post-ICH. We found that the brain edema alleviated in inhibitor treatment group compared with the ICH group at 24 h following ICH in different positions (81.20 ± 0.61% vs ICH group, 82.53 ± 0.75%; P < 0.01) (Fig. 2b).

---

Ac-YVAD-CMK mitigated the caspase-1 mediated inflammatory response after ICH [2]
To investigate the anti-inflammatory effects of Ac-YVAD-CMK, we measured the protein levels of NLRP3, caspase-1(p20), pro-inflammatory factors (IL-1β and IL-18) in the injured hemisphere at 24 h post-ICH by western blot. The results showed that the protein levels of NLRP3, caspase-1 (p20), mature IL-1β/IL-18 were significantly increased in perihematoma compared with the normal group or the sham group, whereas Ac-YVAD-cmk treatment (Ac-YVAD-cmk dose: 1 μg/rats) significantly decreased the protein levels of caspase-1 (p20), mature IL-1β/IL-18 compared with the ICH group (Fig. 3). Then we detected the protein levels of caspase-1 (p20) and mature IL-1β/IL-18 by immunohistochemistry in perihematoma at 24 h and 72 h after ICH to confirm the above results and make long-term observation (Fig. 4a–c). Furthermore, we found that Ac-YVAD-cmk could reduce the infiltration of inflammatory cells in the perihematoma by H&E staining at 24 h and 72 h after ICH as well (Fig. 4d–e).

---

Ac-YVAD-CMK alleviated microglia infiltration after ICH [2]
Microglia activation is an important feature of the pathological changes following ICH. As shown in Fig. 5, we found that Ac-YVAD-cmk could significantly alleviate microglia infiltration in perihematoma by immunohistochemistry compared with ICH group at 24 h (28.17 ± 3.82% vs ICH group, 45.50 ± 4.39%; P < 0.05) or 72 h after ICH. (38.83 ± 5.42% vs ICH group, 75.00 ± 8.27%; P < 0.01) And both groups were compared with normal group at each time point (P < 0.01).

---

The effect of acetyl - tyrosyl-valyl-alanyl-aspartyl - chloromethylketone (Ac-YVAD-CMK), an irreversible caspase-1 (IL-1beta converting enzyme, ICE) inhibitor on mortality, leukocyte and platelet counts and cytokine levels was investigated in a double-blind rat model of endotoxaemia. Intravenous (i.v.) bolus administration of lipopolysaccharide (LPS) (25-75 mg kg(-1), n=12 per group) to anaesthetized rats induced a dose dependent increase in mortality over 8 h (LD(50)=48 mg kg(-1)). During this period, animals became leukopenic and thrombocytopenic. Serum levels of IL-beta, IL-6, and TNF-alpha were highly elevated. Pretreatment of rats with ac-YVAD-cmk at a dose of 12.5 micromol kg(-1) significantly reduced mortality from 83 to 33% using Log Rank analysis. However, Ac-YVAD-CMK did not modify blood cell counts or cytokine profiles as compared with the LPS-drug vehicle group. These data lay credence to the potential importance of caspase-1-inhibition in modifying the inflammatory response to endotoxin. Further investigations are warranted in understanding the relationship between caspase-1 inhibition, cytokine production and animal survival in different experimental paradigms of sepsis [3].

Cell treatment and experimental groups in vitro [2]
Microglia cells (1 × 105) were stimulated with the caspase-1 inhibitor Ac-YVAD-CMK (dissolved by DMSO) for 1 h. Thrombin was added at a final concentration of 10 U/mL to activate the microglia cells for 24 h. The experiments were divided into three groups: (i) Control group, microglia cells were cultured alone. (ii) Thrombin-activated group, microglia cells cultured with thrombin for 24 h. (iii) Inhibitor treatment group, microglia cells were stimulated with Ac-YVAD-CMK dissolved by DMSO for 1 h, then activated with thrombin for 24 h.

---

MTT assay [1]
Cells were seeded into 96-well plates and were treated as previously described. After treatment, 10 μl of MTT reagent (0.5 mg/ml) was added to each well and was incubated for 4 h at 37°C. The formazine granules in the wells were dissolved with 150 μl of dimethyl sulfoxide (DMSO), and the absorbance at 570 nm was measured using a microplate reader.

---

HE staining [1]
Hematoxylin and eosin (HE) staining was used to observe the histological changes. Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The samples were cut into 5-μm-thick sections and stained with HE.

---

Immunofluorescence [1]
The cells were washed three times with PBS and then were fixed with 4% paraformaldehyde at room temperature for 15 min. The cells were washed three times with PBS again and blocked with PBS containing 1% bovine serum albumin (BSA) for 30 min at room temperature. Cells were then incubated overnight at 4°C with primary antibody diluted in blocking buffer and with secondary antibody for 1 h at room temperature. Caspase-1 was detected via a monoclonal anti-caspase1 antibody at a dilution of 1:200 and a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody at a dilution of 1:1000. The cells were then washed three times with PBS. Images were taken using an Axiovert 200 fluorescence microscope.

Drug administration and intracerebral ventricular injection [2]
The caspase-1 inhibitor Ac-YVAD-CMK was dissolved in DMSO and further diluted in sterile normal saline in order to achieve the final concentration of DMSO <0.2%. The drug Ac-YVAD-CMK was then injected into the left lateral ventricle (coordinates: 1.0 mm posterior, 1.5 mm lateral, and 4.5 mm ventral to the bregma) using a 5-μL needle 30 min before induction of ICH. Vehicle group was treated with the same volume of DMSO diluted in sterile normal saline. The experiments were divided into four groups: (i) Normal group. (ii) Sham group, the sham-operated rats were administered of 2-μL sterile normal saline in left striatum. (iii) ICH group. (iiii) Inhibitor treatment group, the ICH rats were administered of Ac-YVAD-CMK in left lateral ventricle at 1 μg/rats and the concentration of Ac-YVAD-CMK is 1 μg/μL.

---

Animals were randomized into four groups, each animal receiving either PBS containing 2.8% DMSO (group 1, control group, n=12), or the caspase-1 inhibitor Ac-YVAD-CMK at a dosage of 1.25 μmol kg−1 (group 2, n=12), 6.25 μmol kg−1 (group 3, n=12) or 12.5 μmol kg−1 (group 4, n=12), respectively. Thirty minutes after administration of Ac-YVAD-CMK, endotoxaemia was induced by bolus i.v. of 65 mg kg−1 Lipopolysaccharide (LPS) (=LD80). Surviving animals were sacrificed after the last blood sample had been collected.[3]

[1]. Alcohol accumulation promotes esophagitis via pyroptosis activation. Int J Biol Sci. 2018;14(10):1245-1255. Published 2018 Jul 13.

[2]. Ac-YVAD-cmk improves neurological function by inhibiting caspase-1-mediated inflammatory response in the intracerebral hemorrhage of rats. Int Immunopharmacol. 2019;75:105771.

[3]. Caspase-1-inhibitor ac-YVAD-cmk reduces LPS-lethality in rats without affecting haematology or cytokine responses. Br J Pharmacol. 2000;131(3):383-386.

Ac-Tyr-Val-Ala-Asp-chloromethyl ketone is a tetrapeptide composed of L-tyrosine, L-valine, L-alanine, and L-aspartic acid linked by peptide bonds, with its amino terminus replaced by an acetyl group and its carboxyl terminus replaced by a chloromethyl group. It has multiple functions, including acting as an EC 3.4.22.36 (caspase-1) inhibitor, a neuroprotective agent, and a nephroprotective agent. It is a tetrapeptide and also an organochlorine compound. Its functions are associated with N-acetyl-L-tyrosine, L-valine, L-alanine, and L-aspartic acid. Gastroesophageal reflux damages the mucosal barrier of the distal esophagus, exposing the squamous epithelium to various stimuli for extended periods, thus inducing chronic inflammation. Esophagitis is a reaction to inflammation of the esophageal squamous mucosa. Our study showed that alcohol accumulation can exacerbate the progression of esophagitis by inducing pyroptosis; however, the caspase-1 inhibitor Ac-YVAD-CMK can effectively inhibit the expression of IL-1β and IL-18 in vitro and in vivo, thereby alleviating the inflammatory response and is expected to become a drug to inhibit the progression of esophagitis. In addition, caspase-1-mediated pyroptosis is also associated with the occurrence and development of esophageal cancer. [1]

---

Objective: Intracerebral hemorrhage (ICH) is a recognized serious clinical problem, and there is currently no effective treatment. In the progression of ICH, a caspase-1-mediated inflammatory response occurs. Therefore, we aimed to investigate the effect of the caspase-1 inhibitor Ac-YVAD-cmk on intracerebral hemorrhage (ICH).
Materials and Methods: Microglia were isolated and activated with thrombin for 24 hours. Then, the transcription and protein expression of NLRP3 and inflammatory factors were detected by RT-PCR and Western blotting. In addition, Ac-YVAD-cmk was injected into the ICH model. mNSS score and brain water content were detected 24 hours after ICH. Finally, pathological changes of microglial activation after ICH were observed by immunohistochemistry and HE staining. Results: Ac-YVAD-cmk inhibited the activation of pro-caspase-1 and reduced cerebral edema. At the same time, a decrease in activated microglia and a decrease in the expression of inflammatory factors were observed 24 hours after ICH. Therefore, Ac-YVAD-cmk can reduce the release of mature IL-1β/IL-18 around the hematoma in rats with cerebral hemorrhage, improve their behavioral performance, and reduce microglial infiltration in the hematoma area. Conclusion: These results indicate that caspase-1 can release multiple inflammatory responses in cerebral hemorrhage. Administration of Ac-YVAD-cmk may become a new treatment strategy for cerebral hemorrhage. [2] This study aims to investigate whether administration of the specific caspase-1 inhibitor ac-YVAD-cmk can reduce endotoxin-induced mortality and whether this effect may be attributed to the synchronous decrease in IL-1β levels. At the highest dose tested, ac-YVAD-cmk significantly reduced mortality in an acute endotoxemia model without altering circulating IL-1β levels. Furthermore, the improved survival with ac-YVAD-cmk was independent of changes in hematological parameters or the production of TNFα or IL-6. Previous studies have shown that caspase-1 inhibitors can partially inhibit the release of IL-1β in vitro (Schumann et al., 1998) and in vivo (Fletcher et al., 1995; Mignon et al., 1999). However, transient decreases in IL-1β production do not affect mortality (Fletcher et al., 1995). The effect of ac-YVAD-cmk on circulating IL-1β deficiency in this study may be attributed to: (1) insufficient dose of ac-YVAD-cmk; (2) inability of the drug to reach target sites, such as monocytes and macrophages; (3) short half-life of ac-YVAD-cmk; and (4) the existence of an ICE-independent IL-1β synthesis pathway. Evidence suggests that high concentrations of various proteases (e.g., cathepsin G, elastase, metalloproteinases) in inflammatory fluids can cleave proIL-1-β into biologically active forms (Irmler et al., 1995; Hazuda et al., 1988), which are present at both cellular infiltration sites and extracellular sites (Hazuda et al., 1988). Furthermore, in ICE-deficient mice stimulated with turpentine, inflammation was still driven by high levels of biologically active IL-1β (Fantuzzi et al., 1997), while this effect was not observed in IL-1β-deficient mice (Zheng et al., 1995).
In this study, the increased survival rate of 12.5 μmol kg−1 ac-YVAD-cmk was similar to that of ICE-deficient mice in the face of lethal endotoxemia (Li et al., 1995; Kuida et al., 1995), while this effect was not observed in IL-1β (Fantuzzi et al., 1996) or IL-18 knockout mice (Sakao et al., 1998). IL-18 plays a crucial role in inflammatory responses, stimulating the production of chemokines and cytokines in vitro (Puren et al., 1998). However, compared to wild-type mice, IL-18 knockout mice showed significantly elevated levels of cytokines such as IL-1β, IL-6, and TNF-α, indicating a negative feedback effect of IL-18 on cytokine production (Sakao et al., 1998). The inhibition of caspase-1 activity and IL-18 production in this study may be an autocrine mechanism, producing biologically active IL-1β through an IL-18-independent pathway. This hypothesis should be validated once specific reagents (e.g., rat IL-18 antibodies) are developed. Caspase is known to be involved in apoptosis in vitro (Fahy et al., 1999) and in vivo (Mignon et al., 1999). In the latter study, Mignon and colleagues found that ac-YVAD-cmk prevented death in a mouse model of TNFα-dependent endotoxemia sensitized with D-galactosamine. Based on normal liver histology and selective inhibition of caspase-3 activity, the authors concluded that survival was due to suppressed hepatocyte apoptosis. Conversely, ac-YVAD-cmk did not prevent LPS-induced endotoxin shock in unsensitized mice. The protective effect of ac-YVAD-cmk in this study is likely due to its prevention of inflammation-induced apoptosis rather than multi-organ failure, as the timing of deaths was similar in this study and in the Mignon et al. study. This effect may be due to FAS-mediated apoptosis via a caspase-1-dependent pathway, as ICE knockout mice are resistant to FAS-mediated apoptosis but remain sensitive to other apoptotic stimuli (Kuida et al., 1995). Alternatively, ac-YVAD-cmk may be a broad-spectrum caspase inhibitor, including caspase-3, which has been observed in both FAS (Enari et al., 1995) and TGF-β-mediated hepatocyte apoptosis (Inayat-Hassain et al., 1997).
Current data suggest that caspase-1 inhibition may have significant implications for altering the pathophysiological outcomes of experimental sepsis. Further research should be conducted using more selective caspase inhibitors in conjunction with improved non-hypodynamic animal models of human sepsis (Mathiak et al., 2000; Wichterman et al., 1980) [3].

C24H33CLN4O8

540.99400

540.198

C, 53.28; H, 6.15; Cl, 6.55; N, 10.36; O, 23.66

178603-78-6

9915279

Ac-Tyr-Val-Ala-Asp-CH2Cl

YVAD

White to off-white solid powder

1.3±0.1 g/cm3

969.1±65.0 °C at 760 mmHg

539.9±34.3 °C

0.0±0.3 mmHg at 25°C

1.554

0.9

6

8

14

37

845

4

CC([C@H](NC([C@@H](NC(C)=O)CC1=CC=C(O)C=C1)=O)C(N[C@H](C(N[C@H](C(CCl)=O)CC(O)=O)=O)C)=O)C

UOUBHJRCKHLGFB-DGJUNBOTSA-N

InChI=1S/C24H33ClN4O8/c1-12(2)21(24(37)26-13(3)22(35)28-17(10-20(33)34)19(32)11-25)29-23(36)18(27-14(4)30)9-15-5-7-16(31)8-6-15/h5-8,12-13,17-18,21,31H,9-11H2,1-4H3,(H,26,37)(H,27,30)(H,28,35)(H,29,36)(H,33,34)/t13-,17-,18-,21-/m0/s1

(3S)-3-[[(2S)-2-[[(2S)-2-[[(2S)-2-acetamido-3-(4-hydroxyphenyl)propanoyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]-5-chloro-4-oxopentanoic acid

Ac-YVAD-CMK; 178603-78-6; Caspase-1 Inhibitor II; Ac-Tyr-Val-Ala-Asp-chloromethylketone; acetyl-Tyr-Val-Ala-Asp-chloromethylketone; L-Alaninamide, N-acetyl-L-tyrosyl-L-valyl-N-[(1S)-1-(carboxymethyl)-3-chloro-2-oxopropyl]-; (4S,7S,10S,13S)-13-(2-chloroacetyl)-4-(4-hydroxybenzyl)-7-isopropyl-10-methyl-2,5,8,11-tetraoxo-3,6,9,12-tetraazapentadecan-15-oic acid; N-acetyl-tyr-val-ala-asp-chloromethyl ketone;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~184.85 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.84 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (3.84 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)