SGLT2 inhibitors and NLRP3 inflammasome: potential target in diabetic kidney disease.

Diabetic kidney disease (DKD) remains the leading cause of chronic kidney disease (CKD) worldwide. The pathogenesis of DKD is influenced by functional, histopathological, and immune mechanisms, including NLRP3 inflammasome activity and oxidative stress. The sodium-glucose cotransporter 2 inhibitors (SGLT2i) have shown metabolic benefits and the ability to slow the progression of DKD in several clinical studies over the years. Recent studies suggest that the antidiabetic activity also extends to inhibition of the inflammatory response, including modulation of the NLRP3 inflammasome, reduction of pro-inflammatory markers and reduction of oxidative stress. Here we review the efficacy of SGLT2i in the treatment of CKD and discuss the role of the inflammatory response in the development of DKD, including its relationship to the NLRP3 inflammasome and oxidative stress.

Deep-Learning-Driven Discovery of SN3-1, a Potent NLRP3 Inhibitor with Therapeutic Potential for Inflammatory Diseases.

The NLRP3 inflammasome plays a central role in the pathogenesis of various intractable human diseases, making it an urgent target for therapeutic intervention. Here, we report the development of SN3-1, a novel orally potent NLRP3 inhibitor, designed through a lead compound strategy centered on deep-learning-based molecular generative models. Our strategy enables rapid fragment enumeration and takes into account the synthetic accessibility of the compounds, thereby significantly enhancing the optimization of lead compounds and facilitating the discovery of potent inhibitors. X-ray crystallography provided insights into the SN3-1 inhibitory mechanism. SN3-1 has shown a favorable safety profile in both acute and chronic toxicity assessments and exhibits robust pharmacokinetic properties. Furthermore, SN3-1 demonstrated significant therapeutic efficacy in various disease models characterized by NLRP3 activation. This study introduces a potent candidate for developing NLRP3 inhibitors and significantly expands the repertoire of tools available for the discovery of novel inhibitors.

Design, Synthesis, and Bioevaluation of Novel NLRP3 Inhibitor with IBD Immunotherapy from the Virtual Screen.

NLRP3, a crucial member of the NLRP family, plays a pivotal role in immune regulation and inflammatory modulation. Here, we report a potent and specific NLRP3 inhibitor Z48 obtained though docking-based virtual screening and structure-activity relationship studies with an IC50 of 0.26 µM in THP-1 cells and 0.21 µM in mouse bone marrow-derived macrophages. Mechanistic studies indicated that Z48 could bind directly to the NLRP3 protein (KD = 1.05 µM), effectively blocking the assembly and activation of the NLRP3 inflammasome, consequently manifesting anti-inflammatory properties. Crucially, with acceptable mouse pharmacokinetic profiles, Z48 demonstrated notable therapeutic efficacy in a mouse model of DSS-induced ulcerative colitis, while displaying no significant therapeutic impact on NLRP3KO mice. In conclusion, this study provided a promising NLRP3 inflammasome inhibitor with novel molecular scaffold, poised for further development as a therapeutic candidate in the treatment of inflammatory bowel disease.

Design, synthesis and biological evaluation of novel diphenylamine analogues as NLRP3 inflammasome inhibitors.

The aberrant activation of the NLRP3 inflammasome has been implicated in the pathogenesis of numerous inflammation-related diseases. Development of NLRP3 inflammasome inhibitors is expected to provide a new strategy for the treatment of these diseases. Herein, a novel series of diphenylamine derivatives were designed based on the lead compounds H20 and H28, and the preliminary structure-activity relationship was studied. The representative compound 19 displayed significantly higher inhibitory activity against NLRP3 inflammasome compared to lead compounds H20 and H28, with an IC50 of 0.34 µM. Mechanistic studies indicated that compound 19 directly targets the NLRP3 protein (KD: 0.45 µM), blocking the assembly and activation of the NLRP3 inflammasome, leading to anti-inflammatory effects and inhibition of cellular pyroptosis. Our findings indicated that compound 19 is a promising NLRP3 inhibitor and could potentially serve as a lead compound for further optimization.

Discovery of Potent, Specific, and Orally Available NLRP3 Inflammasome Inhibitors Based on Pyridazine Scaffolds for the Treatment of Septic Shock and Peritonitis.

The NLRP3 inflammasome is a multiprotein complex that is a component of the innate immune system, involved in the production of pro-inflammatory cytokines. Its abnormal activation is associated with many inflammatory diseases. In this study, we designed and synthesized a series of NLRP3 inflammasome inhibitors based on pyridazine scaffolds. Among them, P33 exhibited significant inhibitory effects against nigericin-induced IL-1ß release in THP-1 cells, BMDMs, and PBMCs, with IC50 values of 2.7, 15.3, and 2.9 nM, respectively. Mechanism studies indicated that P33 directly binds to NLRP3 protein (KD = 17.5 nM), inhibiting NLRP3 inflammasome activation and pyroptosis by suppressing ASC oligomerization during NLRP3 assembly. Additionally, P33 displayed excellent pharmacokinetic properties, with an oral bioavailability of 62%. In vivo efficacy studies revealed that P33 significantly ameliorated LPS-induced septic shock and MSU crystal-induced peritonitis in mice. These results indicate that P33 has great potential for further development as a candidate for treating NLRP3 inflammasome-mediated diseases.

Design, synthesis, and biological evaluation of oridonin derivatives as novel NLRP3 inflammasome inhibitors for the treatment of acute lung injury.

Acute lung injury (ALI) is a severe respiratory disorder closely associated with the excessive activation of the NLRP3 inflammasome. Oridonin (Ori), a natural diterpenoid compound, had been confirmed as a specific covalent NLRP3 inflammasome inhibitor, which was completely different from that of MCC950. However, the further clinical application of Ori was limited by its weak inhibitory activity against NLRP3 inflammasome (IC50 = 1240.67 nM). Fortunately, through systematic structure-optimization of Ori, D6 demonstrated the enhancement of IL-1ß inhibitory activity (IC50 = 41.79 nM), which was better than the parent compound Ori. Then, by using SPR, molecular docking and MD simulation, D6 was verified to directly interact with NLRP3 via covalent and non-covalent interaction. The further anti-inflammatory mechanism studies were revealed that D6 could inhibit the activation of NLRP3 inflammasome without affecting the initiation phase of NLRP3 inflammasome activation, and D6 was a broad-spectrum and selective NLRP3 inflammasome inhibitor. Finally, D6 demonstrated a favorable therapeutic effect on LPS-induced ALI in mice model, and the potent pharmacodynamic effect of D6 was correlated with the specific inhibition of NLRP3 inflammasome activation in vivo. Thus, D6 is proved as a potent NLRP3 inhibitor, and has the potential to develop as a novel anti-ALI agent.

Recent advances in the treatment of gout with NLRP3 inflammasome inhibitors.

Gout is an autoinflammatory disorder characterized by the accumulation of monosodium urate crystals in joints and other tissues, representing the predominant type of inflammatory arthritis with a notable prevalence and propensity for severe outcomes. The NLRP3 inflammasome, a member of the pyrin domain-containing NOD-like receptor family, exerts a substantial impact on both innate and adaptive immune responses and serves as a pivotal factor in the pathogenesis of gout. In recent years, there has been significant academic and industrial interest in the development of NLRP3-targeted small molecule inhibitors as a promising therapeutic approach for gout. To assess the advancements in NLRP3 inflammasome inhibitors for gout treatment, this review offers a comprehensive analysis and evaluation of current clinical candidates and other inhibitors targeting NLRP3 inflammasome from a chemical structure standpoint, with the goal of identifying more efficacious options for clinical management of gout.

High-Throughput Molecular Modeling and Evaluation of the Anti-Inflammatory Potential of Açaí Constituents against NLRP3 Inflammasome.

The search for bioactive compounds in natural products holds promise for discovering new pharmacologically active molecules. This study explores the anti-inflammatory potential of açaí (Euterpe oleracea Mart.) constituents against the NLRP3 inflammasome using high-throughput molecular modeling techniques. Utilizing methods such as molecular docking, molecular dynamics simulation, binding free energy calculations (MM/GBSA), and in silico toxicology, we compared açaí compounds with known NLRP3 inhibitors, MCC950 and NP3-146 (RM5). The docking studies revealed significant interactions between açaí constituents and the NLRP3 protein, while molecular dynamics simulations indicated structural stabilization. MM/GBSA calculations demonstrated favorable binding energies for catechin, apigenin, and epicatechin, although slightly lower than those of MCC950 and RM5. Importantly, in silico toxicology predicted lower toxicity for açaí compounds compared to synthetic inhibitors. These findings suggest that açaí-derived compounds are promising candidates for developing new anti-inflammatory therapies targeting the NLRP3 inflammasome, combining efficacy with a superior safety profile. Future research should include in vitro and in vivo validation to confirm the therapeutic potential and safety of these natural products. This study underscores the value of computational approaches in accelerating natural product-based drug discovery and highlights the pharmacological promise of Amazonian biodiversity.

Constituents from Dolichos lablab L. Flowers and Their Anti-Inflammatory Effects via Inhibition of IL-1ß Release.

The occurrence of inflammation is closely related to the activation of the NLRP3 inflammasome. IL-1ß produced during the activation of the NLRP3 inflammasome has strong pro-inflammatory activity and can also promote the release of inflammatory factors by other immune cells, exacerbating inflammatory damage to tissues. Utilizing IL-1ß as the detection index to find small-molecule inhibitors targeting NLRP3 from natural products will benefit the search for drugs for inflammation-related diseases. During the exploration of anti-inflammatory active components derived from the flowers of Dolichos lablab L., an ingredient in traditional Chinese medicine with dual applications in both medicinal treatment and dietary consumption, fourteen compounds (1-14), including seven previously unreported ones, named flosdolilabnitrogenousols A-D (1-4) and flosdolilabsaponins A-C (5-7), were found. Their structures were established through extensive NMR spectra determination, HR-ESI-MS analysis, ECD calculations, and chemical reactions. Flosdolilabsaponin A (5) stands out as an exceptionally rare tetracyclic lactone oleane-type saponin. Additionally, the inhibitory activity on IL-1ß release of all compounds, without cytotoxicity, was evaluated using BMDMs stimulated with LPS/Nigericin. An Elisa assay revealed that compounds 1, 8, 9, and 11-14 exhibited significant inhibition of IL-1ß release at a concentration of 30 µM. Structure-activity relationships were also discussed. This study indicates that D. lablab flowers possess anti-inflammatory activity, which might exert its effect by suppressing the activation of the NLRP3 inflammasome.

Targeting COVID-19 and varicocele by blocking inflammasome: Ligand-based virtual screening.

COVID-19 is a new generation of outbreaks that invade not only local emerging region, continental but also the whole globe. Varicocele on the other hand, is a testicular vascular disease that underlies 40 % of male infertility cases. Fortunately, the two diseases can be blocked through targeting one common target, NLRP3 inflammasome. Upon searching for similar drugs that gained FDA-approval in ChEMBL library along with examining their potential blockade of the receptor through docking using CB-DOCK-2, three potential approved drugs can be repurposed, ChEMBL 4297185, ChEMBL 1201749, ChEMBL 1200545 which had binding energy of -9.8 and -9.7 kcal/mol (stronger than the reference inhibitor, -9.3 kcal/mol). Also, ADME profile of the top 3 drugs showed better attributes. Also, the simulated proteins exhibited stable pattern with strong free binding energies. Among the potential inhibitor drugs ChEMBL 4297185 was found to remain inside the binding site of the protein during the 200 ns simulation time. Hence, it is anticipated to have the highest binding and thus inhibition potential against the protein. The suggested drugs, especially ChEMBL 4297185, are potentially repurposable toward treating COVID-19 and varicocele which deserve further experimental validation.

13,14-seco withaphysalins from Physalis minima and their inhibitory effects on NLRP3 inflammasome activation.

Seven new 13,14-seco withaphysalins including two new skeletons (1 and 9) were isolated from the whole plants of Physalis minima, together with three known analogues (6-8). Among them, compound 1 was an extremely rare steroid with a 6, 8-cyclo ring. Their structures were established by extensive analysis of spectroscopic data, experimental electronic circular dichroism measurements, and single-crystal X-ray crystallographic analysis. In Raw264.7 cells, compounds 1-3, 5, 6, and 8 demonstrated potent ability to reduce the NLRP3-dependent caspase-1 activation. Among these compounds, 1 and 2 showed a superior potential, consistently concentration-dependent downregulating NLRP3-dependent proinflammatory cytokine IL-1ß production in macrophage. Mechanistically, compounds 1 and 2 reduced the cleavage of caspase-1 and GSDMD, and exhibited no obvious impact both on the NF-κB activation and the expression of NLRP3 and IL-1ß, suggesting that the compounds target the activation of the NLRP3 pathway mainly by inhibiting the NLRP3 inflammasome activation step rather than the priming step.

A novel selective NLRP3 inhibitor shows disease-modifying potential in animal models of Parkinson's disease.

Pathological activation of the Nod-like receptor family pyrin domain containing protein 3 (NLRP3) inflammasome signaling underlies many autoimmune and neuroinflammatory conditions. Here we report that, a rationally designed, novel, orally active, selective NLRP3 inflammasome inhibitor, Usnoflast (ZYIL1), showed potent inhibition of ATP, Nigericin and monosodium urate-mediated interleukin (IL)-1ß release in THP-1 cells and human PBMC. In isolated microglia cells, the IC50 of ZYIL1 mediated inhibition of IL-1ß was 43 nM. ZYIL1 displayed good pharmacokinetic profile in mice, rats and primates after oral administration and the concentrations found in the brain and cerebrospinal fluid (CSF) were markedly higher than the IC50 values. In an in vivo model of neuroinflammation, ZYIL1 demonstrated robust suppression of NLRP3 inflammasome activation and IL-1ß upon oral administration. This translated into efficacy in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-Hydroxydopamine (6-OHDA)-induced Parkinson's disease (PD) models in mice. In MPTP and/or 6-OHDA models, treatment with ZYIL1 ameliorated motor deficits, degeneration of nigrostriatal dopaminergic neurons and abnormal accumulation of α-synuclein. There were positive changes in the genes related to walking, locomotor activity, neurogenesis, neuroblast proliferation and neuronal differentiation in the PD brain indicating improvement in neural health which translated into improved mobility. These findings clearly indicate that selective NLRP3 inhibitor ZYIL1, ameliorates neuroinflammation and appears to have the potential for disease modification and progression associated with PD.

Lindenane sesquiterpenoid dimers with NLRP3 inflammasome inhibitory activities from Chloranthus holostegius var. shimianensis.

Thirteen previously undescribed lindenane sesquiterpenoid dimers (LSDs), named chlorahololides G-S (1-13), were isolated from the whole plants of Chloranthus holostegius var. shimianensis, along with ten known analogues (14-23). The structures and absolute configurations of compounds 1-13 were elucidated through comprehensive spectroscopic analysis, NMR and electronic circular dichroism (ECD) calculations, and X-ray single-crystal diffraction. Chlorahololide G (1) represents the first instance of LSDs formed via a C-15-C-9' carbon-carbon single bond, whose plausible biosynthetic pathway was also proposed. Chlorahololides I and J (3 and 4) were deduced to be rare 8,9-seco and 9-deoxy LSDs with C-11-C-7' carbon-carbon bond, respectively. The inhibitory activity against NLRP3 inflammasome activation was evaluated for all isolates, with six compounds (5, 7, 8, 17, 22, and 23) exhibiting significant effects, and IC50 values ranging from 2.99 to 8.73 µM. Additionally, a preliminary structure-activity relationship analysis regarding their inhibition of NLRP3 inflammasome activation was summarized. Compound 17 exhibited dose-dependent inhibition of nigericin-induced pyroptosis in J774A.1 cells. Molecular docking studies suggested a strong interaction between compound 17 and NLRP3.

Novel NLRP3 inhibitor INF195: Low doses provide effective protection against myocardial ischemia/reperfusion injury.

BACKGROUND: Several factors contribute to ischemia/reperfusion injury (IRI), including activation of the NLRP3 inflammasome and its byproducts, such as interleukin-1ß (IL-1ß) and caspase-1. However, NLRP3 may paradoxically exhibit cardioprotective properties. This study aimed to assess the protective effects of the novel NLRP3 inhibitor, INF195, both in vitro and ex vivo. METHODS: To investigate the relationship between NLRP3 and myocardial IRI, we synthetized a series of novel NLRP3 inhibitors, and investigated their putative binding mode via docking studies. Through in vitro studies we identified INF195 as optimal for NLRP3 inhibition. We measured infarct-size in isolated mouse hearts subjected to 30-min global ischemia/one-hour reperfusion in the presence of three different doses of INF195 (5, 10, or 20-µM). We analyzed caspase-1 and IL-1ß concentration in cardiac tissue homogenates by ELISA. Statistical significance was determined using one-way ANOVA followed by Tukey's test. RESULTS AND CONCLUSION: INF195 reduces NLRP3-induced pyroptosis in human macrophages. Heart pre-treatment with 5 and 10-µM INF195 significantly reduces both infarct size and IL-1ß levels. Data suggest that intracardiac NLRP3 activation contributes to IRI and that low doses of INF195 exert cardioprotective effects by reducing infarct size. However, at 20-µM, INF195 efficacy declines, leading to a lack of cardioprotection. Research is required to determine if high doses of INF195 have off-target effects or dual roles, potentially eliminating both harmful and cardioprotective functions of NLRP3. Our findings highlight the potential of a new chemical scaffold, amenable to further optimization, to provide NLRP3 inhibition and cardioprotection in the ischemia/reperfusion setting.

Discovery, Total Synthesis, and Anti-Inflammatory Evaluation of Naturally Occurring Naphthopyrone-Macrolide Hybrids as Potent NLRP3 Inflammasome Inhibitors.

Numerous clinical disorders have been linked to the etiology of dysregulated NLRP3 (NACHT, LRR, and PYD domain-containing protein 3) inflammasome activation. Despite its potential as a pharmacological target, modulation of NLRP3 activity remains challenging. Only a sparse number of compounds have been reported that can modulate NLRP3 and none of them have been developed into a commercially available drug. In this research, we identified three potent NLRP3 inflammasome inhibitors, gymnoasins A-C (1-3), with unprecedented pentacyclic scaffolds, from an Antarctic fungus Pseudogymnoascus sp. HDN17-895, which represent the first naturally occurring naphthopyrone-macrolide hybrids. Additionally, biomimetic synthesis of gymnoasin A (1) was also achieved validating the chemical structure and affording ample amounts of material for exhaustive bioactivity assessments. Biological assays indicated that 1 could significantly inhibited in vitro NLRP3 inflammasome activation and in vivo pro-inflammatory cytokine IL-1ß release, representing a valuable new lead compound for the development of novel therapeutics with the potential to inhibit the NLRP3 inflammasome.

Characterization of a small molecule inhibitor of the NLRP3 inflammasome and its potential use for acute lung injury.

Accumulating data support the key roles of the NLRP3 inflammasome, an essential component of the innate immune system, in human pathophysiology. As an emerging drug target and a potential biomarker for human diseases, small molecule inhibitors of the NLRP3 inflammasome have been actively pursued. Our recent studies identified a small molecule, MS-II-124, as a potent NLRP3 inhibitor and potential imaging probe. In this report, MS-II-124 was further characterized by an unbiased and comprehensive analysis through Eurofins BioMAP Diversity PLUS panel that contains 12 human primary cell-based systems. The analysis revealed promising activities of MS-II-124 on inflammation and immune functions, further supporting the roles of the NLRP3 inflammasome in these model systems. Further studies of MS-II-124 in mouse model of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and NLRP3 knockout mice demonstrated its target engagement, efficacy to suppress inflammatory cytokines and infiltration of immune cells in the lung tissues. In summary, the results support the therapeutic potential of MS-II-124 as a NLRP3 inhibitor and warrant future studies of this compound and its analogs to develop therapeutics for ALI/ARDS.

NLRP3 Inflammasome Inhibitors for Antiepileptogenic Drug Discovery and Development.

Epilepsy is one of the most prevalent and serious brain disorders and affects over 70 million people globally. Antiseizure medications (ASMs) relieve symptoms and prevent the occurrence of future seizures in epileptic patients but have a limited effect on epileptogenesis. Addressing the multifaceted nature of epileptogenesis and its association with the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated neuroinflammation requires a comprehensive understanding of the underlying mechanisms of these medications for the development of targeted therapeutic strategies beyond conventional antiseizure treatments. Several types of NLRP3 inhibitors have been developed and their effect has been validated both in in vitro and in vivo models of epileptogenesis. In this review, we discuss the advances in understanding the regulatory mechanisms of NLRP3 activation as well as progress made, and challenges faced in the development of NLRP3 inhibitors for the treatment of epilepsy.

Sorbremnoids A and B: NLRP3 Inflammasome Inhibitors Discovered from Spatially Restricted Crosstalk of Biosynthetic Pathways.

Crosstalk-oriented chemical evolution of natural products (NPs) is an efficacious strategy for generating novel skeletons through coupling reactions between NP fragments. In this study, two NOD-like receptor protein 3 (NLRP3) inflammasome inhibitors, sorbremnoids A and B (1 and 2), with unprecedented chemical architectures were identified from a fungus Penicillium citrinum. Compounds 1 and 2 exemplify rare instances of hybrid NPs formed via a major facilitator superfamily (MFS)-like enzyme by coupling reactive intermediates from two separate biosynthetic gene clusters (BGCs), pcisor and pci56. Both sorbremnoids A and B are NLRP3 inflammasome inhibitors. Sorbremnoid A demonstrated strong inhibition of IL-1ß by directly binding to the NLRP3 protein, inhibiting the assembly and activation of the NLRP3 inflammasome in vitro, with potential application in diabetic refractory wound healing through the suppression of excessive inflammatory responses. This research will inspire the development of anti-NLRP3 inflammasome agents as lead treatments and enhance knowledge pertaining to NPs derived from biosynthetic crosstalk.

Astaxanthin alleviates fibromyalgia pain and depression via NLRP3 inflammasome inhibition.

Fibromyalgia is characterised by widespread chronic pain and is often accompanied by comorbidities such as sleep disorders, anxiety, and depression. Because it is often accompanied by many adverse symptoms and lack of effective treatment, it is important to search for the pathogenesis and treatment of fibromyalgia. Astaxanthin, a carotenoid pigment known for its anti-inflammatory and antioxidant properties, has demonstrated effective analgesic effects in neuropathic pain. However, its impact on fibromyalgia remains unclear. Therefore, in this study, we constructed a mouse model of fibromyalgia and investigated the effect of astaxanthin on chronic pain and associated symptoms through multiple intragastrical injections. We conducted behavioural assessments to detect pain and depression-like states in mice, recorded electroencephalograms to monitor sleep stages, examined c-Fos activation in the anterior cingulate cortex, measured activation of spinal glial cells, and assessed levels of inflammatory factors in the brain and spinal cord, including interleukin (IL)-1ß, IL-6, and tumour necrosis factor- α(TNF-α).Additionally, we analysed the expression levels of IL-6, IL-10, NOD-like receptor thermal protein domain associated protein 3 (NLRP3), Apoptosis-associated speck-like protein containing CARD, and Caspase-1 proteins. The findings revealed that astaxanthin significantly ameliorated mechanical and thermal pain in mice with fibromyalgia and mitigated sleep disorders and depressive-like symptoms induced by pain. A potential mechanism underlying these effects is the anti-inflammatory action of astaxanthin, likely mediated through the inhibition of the NLRP3 inflammasome, which could be one of the pathways through which astaxanthin alleviates fibromyalgia. In conclusion, our study suggests that astaxanthin holds promise as a potential analgesic medication for managing fibromyalgia and its associated symptoms.