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.

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.

Discovery of NLRP3 inhibitors using machine learning: Identification of a hit compound to treat NLRP3 activation-driven diseases.

NLRP3 is vital in developing many human diseases as one of the most critical inflammasomes. Developing related inhibitors has been instrumental in advancing the development of therapies for associated diseases. To date, there are no NLRP3 inhibitors on the market. This study identified a series of NLRP3 inhibitors using the self-developed machine learning model. Among them, CSC-6 was validated as the hit molecule with optimal activity and significantly inhibited IL-1ß secreted by PMA-THP-1 cells (IC50 = 2.3 ± 0.38 µM). The results show that CSC-6 specifically binds NLRP3 and inhibits NLRP3 activation by blocking ASC oligomerization during NLRP3 assembly. In vivo experiments have demonstrated that CSC-6 effectively reduces the symptoms of NLRP3 overactivation-mediated sepsis and Gout in mouse models. Importantly, CSC-6 has lower cytotoxicity and exhibits better stability in human-derived liver microsomes, which is more favorable for the drug to maintain its efficacy in vivo for longer. The discovery of CSC-6 may contribute to the design and discovery of related NLRP3 inhibitors.

A review of studies on the implication of NLRP3 inflammasome for Parkinson's disease and related candidate treatment targets.

Parkinson's disease (PD) is a neurodegenerative disease for which the prevalence is second only to Alzheimer's disease (AD). This disease primarily affects people of middle and old age, significantly impacting their health and quality of life. The main pathological features include the degenerative nigrostriatal dopaminergic (DA) neuron loss and Lewy body (LB) formation. Currently, available PD medications primarily aim to alleviate clinical symptoms, however, there is no universally recognized therapy worldwide that effectively prevents, clinically treats, stops, or reverses the disease. Consequently, the evaluation and exploration of potential therapeutic targets for PD are of utmost importance. Nevertheless, the pathophysiology of PD remains unknown, and neuroinflammation mediated by inflammatory cytokines that prompts neuron death is fundamental for the progression of PD. The nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome is a key complex of proteins linking the neuroinflammatory cascade in PD. Moreover, mounting evidence suggests that traditional Chinese medicine (TCM) alleviates PD by suppressing the NLRP3 inflammasome. This article aims to comprehensively review the available studies on the composition and activating mechanism of the NLRP3 inflammasome, along with its significance in PD pathogenesis and potential treatment targets. We also review natural products or synthetic compounds which reduce neuroinflammation via modulating NLRP3 inflammasome activity, aiming to identify new targets for future PD diagnosis and treatment through the exploration of NLRP3 inhibitors. Additionally, this review offers valuable references for developing new PD treatment methods.

Medicinal chemistry strategies targeting NLRP3 inflammasome pathway: A recent update from 2019 to mid-2023.

Nod-like receptor protein 3 (NLRP3), a therapeutic target that has a close relationship with inflammatory diseases, has drawn significant attention from researchers in the field. An increasing number of NLRP3 inhibitors have been reported since NLRP3 was identified as a biomarker and inflammatory therapeutic target. Inhibiting NLRP3 has been widely studied as therapeutics for the treatment of cryopyrin associated periodic syndrome (CAPS), inflammatory bowel disease (IBD), nonalcoholic steatohepatitis (NASH), arthrolithiasis, Alzheimer's disease (AD) and Parkinson's disease (PD). This review updates the recently reported (2019 to mid-2023) molecule inhibitors targeting the NLRP3 inflammasome pathway, summarizes their structure-activity relationships (SARs), and discusses the therapeutic effects on inflammatory diseases. I hope this review will contribute to the development of novel inhibitors targeting NLRP3 inflammasome pathway as potential drugs.

Research progress of NLRP3 inflammasome and its inhibitors with aging diseases.

In recent years, a new target closely linked to a variety of diseases has appeared in the researchers' vision, which is the NLRP3 inflammasome. With the deepening of the study of NLRP3 inflammasome, it was found that it plays an extremely important role in a variety of physiological pathological processes, and NLRP3 inflammasome was also found to be associated with some age-related diseases. It is associated with the development of insulin resistance, Alzheimer's disease, Parkinson's, cardiovascular aging, hearing and vision loss. At present, the only clinical approach to the treatment of NLRP3 inflammasome-related diseases is to use anti-IL-1ß antibodies, but NLRP3-specific inhibitors may be better than the IL-1ß antibodies. This article reviews the relationship between NLRP3 inflammasome and aging diseases: summarizes some of the relevant experimental results reported in recent years, and introduces the biological signals or pathways closely related to the NLRP3 inflammasome in a variety of aging diseases, and also introduces some promising small molecule inhibitors of NLRP3 inflammasome for clinical treatment, such as: ZYIL1, DFV890 and OLT1177, they have excellent pharmacological effects and good pharmacokinetics.

Multinomial classification of NLRP3 inhibitory compounds based on large scale machine learning approaches.

The role of NLRP3 inflammasome in innate immunity is newly recognized. The NLRP3 protein is a family of nucleotide-binding and oligomerization domain-like receptors as well as a pyrin domain-containing protein. It has been shown that NLRP3 may contribute to the development and progression of a variety of diseases, such as multiple sclerosis, metabolic disorders, inflammatory bowel disease, and other auto-immune and auto-inflammatory conditions. The use of machine learning methods in pharmaceutical research has been widespread for several decades. An important objective of this study is to apply machine learning approaches for the multinomial classification of NLRP3 inhibitors. However, data imbalances can affect machine learning. Therefore, a synthetic minority oversampling technique (SMOTE) has been developed to increase the sensitivity of classifiers to minority groups. The QSAR modelling was performed using 154 molecules retrieved from the ChEMBL database (version 29). The accuracy of the multiclass classification top six models was found to fall within ranges of 0.99 to 0.86, and log loss ranges of 0.2 to 2.3, respectively. The results showed that the receiver operating characteristic curve (ROC) plot values significantly improved when tuning parameters were adjusted and imbalanced data was handled. Moreover, the results demonstrated that SMOTE offers a significant advantage in handling imbalanced datasets as well as substantial improvements in overall accuracy of machine learning models. The top models were then used to predict data from unseen datasets. In summary, these QSAR classification models exhibited robust statistical results and were interpretable, which strongly supported their use for rapid screening of NLRP3 inhibitors.

Discovery of a novel 1,3,4-oxadiazol-2-one-based NLRP3 inhibitor as a pharmacological agent to mitigate cardiac and metabolic complications in an experimental model of diet-induced metaflammation.

Inspired by the recent advancements in understanding the binding mode of sulfonylurea-based NLRP3 inhibitors to the NLRP3 sensor protein, we developed new NLRP3 inhibitors by replacing the central sulfonylurea moiety with different heterocycles. Computational studies evidenced that some of the designed compounds were able to maintain important interaction within the NACHT domain of the target protein similarly to the most active sulfonylurea-based NLRP3 inhibitors. Among the studied compounds, the 1,3,4-oxadiazol-2-one derivative 5 (INF200) showed the most promising results being able to prevent NLRP3-dependent pyroptosis triggered by LPS/ATP and LPS/MSU by 66.3 ± 6.6% and 61.6 ± 11.5% and to reduce IL-1ß release (35.5 ± 8.8% µM) at 10 µM in human macrophages. The selected compound INF200 (20 mg/kg/day) was then tested in an in vivo rat model of high-fat diet (HFD)-induced metaflammation to evaluate its beneficial cardiometabolic effects. INF200 significantly counteracted HFD-dependent "anthropometric" changes, improved glucose and lipid profiles, and attenuated systemic inflammation and biomarkers of cardiac dysfunction (particularly BNP). Hemodynamic evaluation on Langendorff model indicate that INF200 limited myocardial damage-dependent ischemia/reperfusion injury (IRI) by improving post-ischemic systolic recovery and attenuating cardiac contracture, infarct size, and LDH release, thus reversing the exacerbation of obesity-associated damage. Mechanistically, in post-ischemic hearts, IFN200 reduced IRI-dependent NLRP3 activation, inflammation, and oxidative stress. These results highlight the potential of the novel NLRP3 inhibitor, INF200, and its ability to reverse the unfavorable cardio-metabolic dysfunction associated with obesity.

Portrayal of NLRP3 Inflammasome in Atherosclerosis: Current Knowledge and Therapeutic Targets.

We are witnessing the globalization of a specific type of arteriosclerosis with rising prevalence, incidence and an overall cardiovascular disease burden. Currently, atherosclerosis increasingly affects the younger generation as compared to previous decades. While early preventive medicine has seen improvements, research advances in laboratory and clinical investigation promise to provide us with novel diagnosis tools. Given the physio-pathological complexity and epigenetic patterns of atherosclerosis and the discovery of new molecules involved, the therapeutic field of atherosclerosis has room for substantial growth. Thus, the scientific community is currently investigating the role of nucleotide-binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, a crucial component of the innate immune system in different inflammatory disorders. NLRP3 is activated by distinct factors and numerous cellular and molecular events which trigger NLRP3 inflammasome assembly with subsequent cleavage of pro-interleukin (IL)-1ß and pro-IL-18 pathways via caspase-1 activation, eliciting endothelial dysfunction, promotion of oxidative stress and the inflammation process of atherosclerosis. In this review, we introduce the basic cellular and molecular mechanisms of NLRP3 inflammasome activation and its role in atherosclerosis. We also emphasize its promising therapeutic pharmaceutical potential.

Mechanisms of NLRP3 inflammasome-mediated hepatic stellate cell activation: Therapeutic potential for liver fibrosis.

The liver injury leads to an inflammatory response, which causes the activation of hepatic stellate cells (HSCs) that further secrete ECM proteins and play an important role in liver fibrosis. Moreover, the inflammatory response is a driving force for fibrogenesis, which is triggered by many types of injuries. Exaggerated inflammatory immune responses are mediated by cytoplasmic protein complexes known as inflammasomes, which are involved in many chronic liver diseases. Inflammasomes are pattern recognition receptors (PRRs) that can sense any microbial motifs known as pathogen-associated molecular patterns (PAMPs), and host- or environmental-derived stress signals known as damage-associated molecular patterns (DAMPs). The inflammasomes cause caspase-mediated proteolytic cleavage of pro-IL-1ß and pro-IL-18 into active IL-1ß and IL-18. In this review, we provide a comprehensive summary of the important roles of NLRP3 inflammasome in the pathogenesis of liver fibrosis with an emphasis on several direct and indirect pathways responsible for the NLRP3 inflammasome-mediated HSCs activation and fibrogenesis. In addition, we discuss the general pharmacological and genetics strategies for the inhibition of NLRP3 inflammasome activation and its downstream signaling with examples of emerging pharmacotherapeutics, targeting the NLRP3 inflammasome signaling as well as a possible way to develop effective and safer NLRP3 inflammasome inhibitors.

Chalcone: A potential scaffold for NLRP3 inflammasome inhibitors.

Overactivated NLRP3 inflammasome has been shown to associate with an increasing number of disease conditions. Activation of the NLRP3 inflammasome results in caspase-1-catalyzed formation of active pro-inflammatory cytokines (IL-1ß and IL-18) resulting in pyroptosis. The multi-protein composition of the NLRP3 inflammasome and its sensitivity to several damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) make this extensively studied inflammasome an attractive target to treat chronic conditions. However, none of the known NLRP3 inhibitors has been approved for clinical use. Sulfonylurea and covalent inhibitors with electrophilic warhead (Michael acceptor) are among the prominent classes of compounds explored for their NLRP3 inhibitory effects. Chalcone, a small molecule with α, ß unsaturated carbonyl group (Michael acceptor), has also been studied as a promising scaffold for the development of NLRP3 inhibitors. Low molecular weight, easy to manipulate lipophilicity and cost-effectiveness have attracted many to use chalcone scaffold for drug development. In this review, we highlight chalcone derivatives with NLRP3 inflammasome inhibitory activities. Recent developments and potential new directions summarized here will, hopefully, serve as valuable perspectives for investigators including medicinal chemists and drug discovery researchers to utilize chalcone as a scaffold for developing novel NLRP3 inflammasome inhibitors.

DFV890: a new oral NLRP3 inhibitor-tested in an early phase 2a randomised clinical trial in patients with COVID-19 pneumonia and impaired respiratory function.

BACKGROUND: Coronavirus-associated acute respiratory distress syndrome (CARDS) has limited effective therapy to date. NLRP3 inflammasome activation induced by SARS-CoV-2 in COVID-19 contributes to cytokine storm. METHODS: This randomised, multinational study enrolled hospitalised patients (18-80 years) with COVID-19-associated pneumonia and impaired respiratory function. Eligible patients were randomised (1:1) via Interactive Response Technology to DFV890 + standard-of-care (SoC) or SoC alone for 14 days. Primary endpoint was APACHE II score at Day 14 or on day-of-discharge (whichever-came-first) with worst-case imputation for death. Other key assessments included clinical status, CRP levels, SARS-CoV-2 detection, other inflammatory markers, in-hospital outcomes, and safety. FINDINGS: Between May 27, 2020 and December 24, 2020, 143 patients (31 clinical sites, 12 countries) were randomly assigned to DFV890 + SoC (n = 71) or SoC alone (n = 72). Primary endpoint to establish clinical efficacy of DFV890 vs. SoC, based on combined APACHE II score, was not met; LSM (SE), 8·7 (1.06) vs. 8·6 (1.05); p = 0.467. More patients treated with DFV890 vs. SoC showed ≥ 1-level improvement in clinical status (84.3% vs. 73.6% at Day 14), earlier clearance of SARS-CoV-2 (76.4% vs. 57.4% at Day 7), and mechanical ventilation-free survival (85.7% vs. 80.6% through Day 28), and there were fewer fatal events in DFV890 group (8.6% vs. 11.1% through Day 28). DFV890 was well tolerated with no unexpected safety signals. INTERPRETATION: DFV890 did not meet statistical significance for superiority vs. SoC in primary endpoint of combined APACHE II score at Day 14. However, early SARS-CoV-2 clearance, improved clinical status and in-hospital outcomes, and fewer fatal events occurred with DFV890 vs. SoC, and it may be considered as a protective therapy for CARDS. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04382053.

PM / 中国生物化学与分子生物学报

Particulate matter (PM) can damage respiratory system, cardiovascular system, nervous system and immune system, but there are few researches on reproductive damage of particulate matter. The objectives of this study were to investigate the effect of short-term particulate matter 2.5 (PM

Chemical Modulation of the 1-(Piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazole-2-one Scaffold as a Novel NLRP3 Inhibitor.

In the search for new chemical scaffolds able to afford NLRP3 inflammasome inhibitors, we used a pharmacophore-hybridization strategy by combining the structure of the acrylic acid derivative INF39 with the 1-(piperidin-4-yl)1,3-dihydro-2H-benzo[d]imidazole-2-one substructure present in HS203873, a recently identified NLRP3 binder. A series of differently modulated benzo[d]imidazole-2-one derivatives were designed and synthesised. The obtained compounds were screened in vitro to test their ability to inhibit NLRP3-dependent pyroptosis and IL-1ß release in PMA-differentiated THP-1 cells stimulated with LPS/ATP. The selected compounds were evaluated for their ability to reduce the ATPase activity of human recombinant NLRP3 using a newly developed assay. From this screening, compounds 9, 13 and 18, able to concentration-dependently inhibit IL-1ß release in LPS/ATP-stimulated human macrophages, emerged as the most promising NLRP3 inhibitors of the series. Computational simulations were applied for building the first complete model of the NLRP3 inactive state and for identifying possible binding sites available to the tested compounds. The analyses led us to suggest a mechanism of protein-ligand binding that might explain the activity of the compounds.

Techniques to Study Inflammasome Activation and Inhibition by Small Molecules.

Inflammasomes are immune cytosolic oligomers involved in the initiation and progression of multiple pathologies and diseases. The tight regulation of these immune sensors is necessary to control an optimal inflammatory response and recover organism homeostasis. Prolonged activation of inflammasomes result in the development of chronic inflammatory diseases, and the use of small drug-like inhibitory molecules are emerging as promising anti-inflammatory therapies. Different aspects have to be taken in consideration when designing inflammasome inhibitors. This review summarizes the different techniques that can be used to study the mechanism of action of potential inflammasome inhibitory molecules.

Cellular Models and Assays to Study NLRP3 Inflammasome Biology.

The NLRP3 inflammasome is a multi-protein complex that initiates innate immunity responses when exposed to a wide range of stimuli, including pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). Inflammasome activation leads to the release of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-18 and to pyroptotic cell death. Over-activation of NLRP3 inflammasome has been associated with several chronic inflammatory diseases. A deep knowledge of NLRP3 inflammasome biology is required to better exploit its potential as therapeutic target and for the development of new selective drugs. To this purpose, in the past few years, several tools have been developed for the biological characterization of the multimeric inflammasome complex, the identification of the upstream signaling cascade leading to inflammasome activation, and the downstream effects triggered by NLRP3 activation. In this review, we will report cellular models and cellular, biochemical, and biophysical assays that are currently available for studying inflammasome biology. A special focus will be on those models/assays that have been used to identify NLRP3 inhibitors and their mechanism of action.

Development of covalent NLRP3 inflammasome inhibitors: Chemistry and biological activity.

The NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome is the best recognized and most widely implicated regulator of caspase-1 activation. It is a key regulator of innate immune response and is involved in many pathophysiological processes. Recent evidences for its inappropriate activation in autoinflammatory, autoimmune, as well as in neurodegenerative diseases attract a growing interest toward the development of small molecules NLRP3 inhibitors. Based on the knowledge of biochemical and structural aspects of NLRP3 activation, one successful strategy in the identification of NLRP3 inhibitors relies on the development of covalent irreversible inhibitors. Covalent inhibitors are reactive electrophilic molecules able to alkylate nucleophiles in the target protein. These inhibitors could ensure good efficacy and prolonged duration of action both in vitro and in vivo. In spite of these advantages, effects on other signalling pathways, prone to alkylation, may occur. In this review, we will illustrate the chemistry and the biological action of the most studied covalent NLRP3 inhibitors developed so far. A description of what we know about their mechanism of action will address the reader toward a critical understanding of NLRP3 inhibition by electrophilic compounds.

NLRP3 Inhibitors as Potential Therapeutic Agents for Treatment of Inflammatory Bowel Disease.

BACKGROUND: Inflammatory bowel disease (IBD) is a group of intestinal disorders characterised by chronic relapsing inflammation of the small intestine and colon. IBD manifests as either ulcerative colitis or Crohn's disease and increases the risk of developing colorectal cancer. METHODS: Here, we have reviewed current treatment regimen for IBD utilising anti-inflammatory drugs, immune system suppressors and antibiotics or a combination of these. However, these therapeutics lead to a number of adverse effects, remission or significant non-responsiveness creating an urgent need to develop potent drugs with novel mechanisms of action for IBD. RESULTS: The inflammasome is a multiprotein complex that assembles to a key innate immune system signalling platform and is involved in the pathogenesis of inflammatory and autoimmune diseases. A number of investigations show that the NLRP3 inflammasome recognizes microbial and cell stress components and serve as a platform for caspase-1 activation and pro-inflammatory cytokine IL-1ß, IL18 maturation. Although the exact aetiology of IBD is unknown, uncontrolled NLRP3 Inflammasome activation has shown to play a major role in the chronic intestinal inflammation and mature IL-1ß and IL18 are consistently associated with increased colitis and colitis associated colorectal cancer development. CONCLUSION: In this review, we discuss the experimental NLRP3 inhibitors that have been investigated in IBD experimental models. The potential mechanism of action of these inhibitors such as inhibiting NF-κB activation and decreasing mitochondrial reactive oxygen species are discussed in detail. We further expand the controversial role of NLRP3 in IBD and future issues that might arise from the long term use of NLRP3 inhibitors in IBD therapy.