Mafenide derivatives inhibit neuroinflammation in Alzheimer's disease by regulating pyroptosis

Abstract The main mechanism of pyroptosis is Caspase‐1–mediated GSDMD cleavage, and GSDMD is also the executive protein of pyroptosis. Our previous study has shown that mafenide can inhibit pyroptosis by inhibiting the GSDMD‐Asp275 site to suppress cleavage. In this study, sulfonamide was used as the parent nucleus structure to synthesize sulfa‐4 and sulfa‐20. Screening of drug activity in the pyroptosis model of BV2 and iBMDM cell lines revealed the efficacy of five compounds were superior to mafenide, which exerted a better inhibitory effect on the occurrence of pyroptosis. For in vivo assay, Sulfa‐4 and Sulfa‐22 were intervened in the neuroinflammation APP/PS1 mice. As a result, the administration of Sulfa‐4 and Sulfa‐22 could significantly inhibit the activation of microglia, decrease the expression of inflammatory factors in the central nervous system and simultaneously suppress the production of p30‐GSDMD as well as the expression of upstream NLRP3 inflammasome and Caspase‐1 protein. Immunoprecipitation and Biotin‐labelled assay confirmed the targeted binding relationship of Sulfa‐4 and Sulfa‐22 with GSDMD protein in the iBMDM model in vitro. In this study, we investigated a new type inhibitor of GSDMD cleavage, which exerted a good inhibitory effect on pyroptosis and provided new references for the development of inflammatory drugs in the future.

press cleavage. In this study, sulfonamide was used as the parent nucleus structure to synthesize sulfa-4 and sulfa-20. Screening of drug activity in the pyroptosis model of BV2 and iBMDM cell lines revealed the efficacy of five compounds were superior to mafenide, which exerted a better inhibitory effect on the occurrence of pyroptosis.
iBMDM and BV2 cells of the logarithmic phase were divided into two groups: one group was intervened with small molecule compounds for 2 h, and the control group was added with DMSO. After changing into the serum-free medium, cells were induced by LPS (1 μg/ml) for 4 h, followed by pyroptosis induction by Nigericin (10 μM) for 2 h. After cell intervention, cells and culture medium were collected, followed by detection of the expression of IL-1β and TNFα by ELISA to calculate IC50.
Detection of the pyroptosis-inhibiting role and mechanism of Sulfa-4 and Sulfa-22 small molecules in BV2 cells:

| The targeted binding relationship of Sulfa-4 and Sulfa-22 small molecules with GSDMD
Co-immunoprecipitation (Co-IP) assay: BV2 cells were inoculated into a 6-well plate and separately treated with DMSO, LPS+Nigericin and Sulfa-4 or Sulfa-22 for 4 h. After washing with PBS, cells were added with 120 μl lysis buffer on ice, followed by collection of cells into EP tube. After centrifugation, the supernatant was transferred to a new EP tube and subjected to BCA assay to detect protein concentration. The sample was added with anti-GSDMD monoclonal antibody (Abcam) (200 μg antibody/2μl sample) at 4°C overnight and reacted with protein G agarose affinity matrix in a shaker at 4°C for 4 h. The supernatant was collected and washed with pre-cooled PBS for four times, followed by detection of GSDMD by WB.

| IBA-1 and CD11c by IF assay
The brain tissue of mice was fixed with 4% FPA. The brain tissue was dehydrated with 15%-30% sucrose solutions. The slices were washed with PBS, incubated with monoclonal antibodies against

| Statistical analysis
Measurement data were shown as x±s. SPSS 22.0 was used for data analysis and processing. Two independent sample t test analysis was used for comparison between two groups; LSD method was used for the subsequent comparisons between the two groups. One-way ANOVA was used for comparison among three groups and above, and all the above-mentioned tests were two-sided, and p < 0.05 indicated statistical significance.

| The effect and mechanism of Sulfa-4 and Sulfa-22 on pyroptosis in BV2 cells
In this study, Sulfa-4 (IC50 of 3μM) and Sulfa-22 (IC50 of 5μM) were used for pretreatment in BV2 cells ( Figure 1A), and 15μM mafenide (MAF) was used as the positive control. LDH release rate assay showed obvious LDH release in cells of the DMSO group, which was significantly down-regulated in MAF group (positive control). In addition, the LDH release rate was down-regulated in BV2 cells after Sulfa-4/22 small molecule treatment ( Figure 1B). PI uptake rate assay revealed that the PI uptake rate was significantly up-regulated in the DMSO group, while the uptake rate in the MAF group was significantly  (Figure 1D-G). IF staining showed that the fluorescence intensity of GSDMD in the DMSO group was relatively low, which was significantly lower than that of the MAF and Sulfa-4/22 groups, indicating that GSDMD was cleaved in the DMSO group. In addition, p30-GSDMD had stronger fluorescence intensity in the DMSO group, which was significantly higher than that of MAF and Sulfa-4/22groups, and p30-GSDMD was mainly expressed on the cell membrane (Figures 2A, 3A). Inflammatory factor release assay showed that the expression levels of inflammatory factors IL-18, IL-1β and TNFα in the DMSO group were increased over time, indicating that the opening of cell membrane pores promoted the release of inflammatory factors. However, the expression level of inflammatory factors was significantly lower in the MAF and Sulfa-4/22 groups than that of the DMSO group ( Figures 2B,C, 3B).
Taken together, Sulfa-4 and Sulfa-22 could inhibit the cleavage of GSDMD to play an anti-pyroptosis effect.

| Sulfa-4/22 on inhibiting pyroptosis in BV2 cell and the validation of targeting GSDMD
The expression of NLRP3 and Caspase-1 was up-regulated in the DMSO group, while the expression of Pro-Caspase-1 was not significantly changed. The expression of NLRP3 and Caspase-1 was downregulated in the MAF and Sulfa4/22 groups, which was consistent  Figures 4F, 5E).

| DISCUSS ION
Alzheimer's disease is a degenerative disease and the main cause of cognitive impairment. 12 The pathological features of AD are mainly β-amyloid protein deposition and Tau protein phosphorylation. 13,14 However, the exact pathological mechanism of AD has not yet been revealed. In recent years, many studies have found that neuroinflammation plays an important role in the occurrence and development of AD. In neuroinflammation, microglia play an important role and are also one of the main cells that release inflammatory factors. 15,16 Pyroptosis is a type of cell death, which involves the activation of Caspase-1-GSDMD. Caspase-1 is the key protein for pro-IL-1β cleavage and maturation; therefore, pyrolysis is mostly mediated by Caspase-1. However, it is GSDMD protein that definitely mediates the occurrence of pyroptosis. 7 The level of GSDMD in normal cells is low, and GSDMD can be induced by LPS. The massive expression of Caspase-1 can cleave GSDMD to form p30-GSDMD protein, and the oligomerized p30-GSDMD is located on the cell membrane to open the cell channel to promote the release of inflammation, which is considered as the main cause of pyroptosis. 17 Previous studies have shown that inhibition of pyroptosis can inhibit the release of inflammatory factors. The key to inhibition of pyroptosis is in the inhibition of GSDMD protein, which theoretically can inhibit the cleavage of GSDMD. Our team has previously shown that MAF can target the GSDMD and inhibit the cleavage of GSDMD.
In this study, MAF was used as the lead compound, and we found that the sulfonamide structure was the pharmacodynamic structure of Asp275. Therefore, we tried to perform small molecule modification at the binding sites by skeleton migration, aiming to supplement the group to fill the binding cavity. In consideration of binding stability, the amino structure was converted to the cyano group, which could, on the one hand, increase the binding of π-π bonds, and on the other hand, improve the fat solubility. In vitro assays revealed

| CON CLUS ION
In this study, we designed Sulfa-4 and Sulfa-22 by using MAF as the lead compound. In vitro activity screening revealed that the pharmaceutical efficacy of two small molecules was superior to MAF. In vivo and in vitro assays also fully demonstrated that Sulfa4/22 could target the cleavage of GSDMD, inhibit the occurrence of pyroptosis F I G U R E 6 Effects of Sulfa4/22 on microglia activation and the release of inflammatory factors in APP/PS1 mice. (A) Detection of IBA-1 and CD11c expression by IF staining: There was obvious activation of microglia, obvious expression of IBA-1 and CD11c in the Con group. However, the expression of IBA-1 and CD11c was significantly down-regulated, and the activation of microglia was inhibited in Sulfa-4 and Sulfa-22 group. (B-C) Expression of inflammatory factors in peripheral blood and CSF: The expression levels of inflammatory factors in peripheral blood and CSF were relatively high in Con group, which were significantly down-regulated in Sulfa-4 and Sulfa-22 group. Comparison with Con group, *p < 0.05. (D-I) Expression level of GSDMD and p30-GSDMD in mouse cortex, hippocampus, and striatum: The expression GSDMD was relatively low, while p30-GSDMD expression was relatively high in cortex, hippocampus and striatum. The cleavage of GSDMD was inhibited in Sulfa-4 and Sulfa-22 groups, GSDMD expression level was significantly up-regulated, while the level of p30-GSDMD was down-regulated. Comparison with the Con group, *p < 0.05 and the release of inflammatory factors, exerting a certain effect on the neuroinflammation of AD.

ACK N OWLED G EM ENTS
First of all, I am very grateful to my mentor, Dr. Qingcai Jiao, for his careful guidance of my graduation thesis in the past six months, which greatly improved my understanding of academic writing and taught me a lot of specific research skills; knowledge is a vast ocean, Thanks to the classmates who have given me care and support in life, because of you, the university life is colourful.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.