Comprehensive insights into potential roles of purinergic P2 receptors on diseases: Signaling pathways involved and potential therapeutics.

BACKGROUND: Purinergic P2 receptors, which can be divided into ionotropic P2X receptors and metabotropic P2Y receptors, mediate cellular signal transduction of purine or pyrimidine nucleoside triphosphates and diphosphate. Based on the wide expression of purinergic P2 receptors in tissues and organs, their significance in homeostatic maintenance, metabolism, nociceptive transmission, and other physiological processes is becoming increasingly evident, suggesting that targeting purinergic P2 receptors to regulate biological functions and signal transmission holds significant promise for disease treatment. AIM OF REVIEW: This review highlights the detailed mechanisms by which purinergic P2 receptors engage in physiological and pathological progress, as well as providing prospective strategies for discovering clinical drug candidates. KEY SCIENTIFIC CONCEPTS OF REVIEW: The purinergic P2 receptors regulate complex signaling and molecular mechanisms in nervous system, digestive system, immune system and as a result, controlling physical health states and disease progression. There has been a significant rise in research and development focused on purinergic P2 receptors, contributing to an increased number of drug candidates in clinical trials. A few influential pioneers have laid the foundation for advancements in the evaluation, development, and of novel purinergic P2 receptors modulators, including agonists, antagonists, pharmaceutical compositions and combination strategies, despite the different scaffolds of these drug candidates. These advancements hold great potential for improving therapeutic outcomes by specifically targeting purinergic P2 receptors.

Macrophage P2Y6 receptor deletion attenuates atherosclerosis by limiting foam cell formation through phospholipase Cß/store-operated calcium entry/calreticulin/scavenger receptor A pathways.

BACKGROUND AND AIMS: Macrophage-derived foam cells play a causal role during the pathogenesis of atherosclerosis. P2Y6 receptor (P2Y6R) highly expressed has been considered as a disease-causing factor in atherogenesis, but the detailed mechanism remains unknown. This study aims to explore P2Y6R in regulation of macrophage foaming, atherogenesis, and its downstream pathways. Furthermore, the present study sought to find a potent P2Y6R antagonist and investigate the feasibility of P2Y6R-targeting therapy for atherosclerosis. METHODS: The P2Y6R expression was examined in human atherosclerotic plaques and mouse artery. Atherosclerosis animal models were established in whole-body P2Y6R or macrophage-specific P2Y6R knockout mice to evaluate the role of P2Y6R. RNA sequencing, DNA pull-down experiments, and proteomic approaches were performed to investigate the downstream mechanisms. High-throughput Glide docking pipeline from repurposing drug library was performed to find potent P2Y6R antagonists. RESULTS: The P2Y6R deficiency alleviated atherogenesis characterized by decreasing plaque formation and lipid deposition of the aorta. Mechanically, deletion of macrophage P2Y6R significantly inhibited uptake of oxidized low-density lipoprotein through decreasing scavenger receptor A expression mediated by phospholipase Cß/store-operated calcium entry pathways. More importantly, P2Y6R deficiency reduced the binding of scavenger receptor A to CALR, accompanied by dissociation of calreticulin and STIM1. Interestingly, thiamine pyrophosphate was found as a potent P2Y6R antagonist with excellent P2Y6R antagonistic activity and binding affinity, of which the pharmacodynamic effect and mechanism on atherosclerosis were verified. CONCLUSIONS: Macrophage P2Y6R regulates phospholipase Cß/store-operated calcium entry/calreticulin signalling pathway to increase scavenger receptor A protein level, thereby improving foam cell formation and atherosclerosis, indicating that the P2Y6R may be a potential therapeutic target for intervention of atherosclerotic diseases using P2Y6R antagonists including thiamine pyrophosphate.

Network pharmacology-based analysis of Jin-Si-Wei on the treatment of Alzheimer's disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Jin-Si-Wei (JSW), a traditional Chinese medicine (TCM) formula, have cognitive enhancing effect and delay the memory decline in an animal model of AD， which has been reported. However, the therapeutic mechanism of JSW in the treatment of AD remains unclear. AIM OF THE STUDY: This study aimed to verify the pharmacodynamics of JSW in the treatment of AD, and to explore its potential mechanism based on network pharmacology, molecular docking and experimental validation both in vitro and in vivo. MATERIALS AND METHODS: In this study, the underlying mechanism of JSW against AD was investigated by the integration of network pharmacology. Then, the core pathways and biological process of JSW were verified by experiment, including behavioral test and pathological and biochemical assays with 6-month-old APPswe/PS1ΔE9 transgenic (APP/PS1) mice in vivo and verified with Aß1-42-stimulated SH-SY5Y cells in vitro. At last, molecular docking was used to show the binding activity of each active ingredient to the core genes of JSW treatment in AD. RESULTS: A Drug-Ingredient-Target network was established, which included 363 ingredients and 116 targets related to the JSW treatment of AD. The main metabolic pathway of JSW treatment for AD is neuroactive ligand-receptor interaction pathway, and biological processes are mainly involved in Aß metabolic process. In vivo experiments, compared with APP/PS1 mice, the cognitive and memory ability of mice was significantly improved after JSW administration. In brain tissue of APP/PS1 mice, JSW could increase the contents of low-density lipoprotein receptor-related protein 1 (LRP-1), enkephalinase (NEP) and Acetyl choline (ACh), and decrease the contents of Aß1-42, amyloid precursor protein (APP) and receptor for advanced glycation end products (RAGE), decrease the vitality of cholinesterase (AChE) and choline acetyltransferase (ChAT). Besides, JSW could increase α-secretase expression and decrease ß/γ-secretase expression, and improve the number and morphology of synapses in CA1 region of the hippocampus of APP/PS1 mice. In vitro experiments, Drug-Containing Serum (JSW-serum) has a neuroprotective effect by reducing the apoptosis on Aß1-42-stimulated SH-SY5Y cells. Molecular docking results showed that 2-Isopropyl-8-methylphenanthrene-3,4-dione had strong binding activity with PTGS2, which maybe a potential ingredient for the treatment of AD. CONCLUSIONS: JSW improves AD in APP/PS1 mice, and this therapeutic effect may be achieved in part by altering the neuroactive ligand-receptor interaction pathway.

Polysaccharide from Strongylocentrotus nudus eggs regulates intestinal epithelial autophagy through CD36/PI3K-Akt pathway to ameliorate inflammatory bowel disease.

Sea urchin is a popular food all over the world, of which eggs are main edible part. Previous studies suggested that polysaccharides from eggs of Strongylocentrotus nudus (SEP) exhibited immunomodulatory activities during anti-tumor therapy, nevertheless, effects of SEP on inflammatory bowel disease and its underlying mechanisms have never been reported. In the present study, we showed that the SEP inhibited dextran sodium sulfate-induced ulcerative colitis characterized by decreased disease activity index, restored colon length and body weight, improved histopathological changes, down-regulation of inflammatory cytokines levels and Th17/Treg ratios in C57BL/6 J mice. Moreover, immunofluorescence analysis suggested that SEP repaired gut barrier in UC mice, while 16S rDNA sequencing exhibited improved intestinal flora. Mechanistically, we found SEP significantly modulated autophagy-related factors in intestinal epithelial cells (IECs), while might contributed to pathogenesis of UC. Furthermore, we demonstrated PI3K/Akt pathway was involved in regulatory effect of SEP on lipopolysaccharide-induced autophagy of HT-29 cells. Besides, among possible polysaccharide binding receptors, change of the CD36 expression was most significant, which was associated with PI3K/Akt signals. Collectively, our study showed for the first time that the SEP might be used a prebiotic agent to improve IBD through regulating CD36-PI3K/Akt mediated autophagy of IECs.

Structure-based virtual screening for discovery of paederosidic acid from Paederia scandens as novel P2Y14R antagonist.

BACKGROUND: The activation of P2Y14 receptor (P2Y14R) promotes osteoclast formation and causes neuropathic pain, exhibiting possible link to osteoarthritis (OA). Given lack of P2Y14R antagonist, the present study aims to search a novel P2Y14R antagonist with low toxicity and high activity from natural products as a possible drug candidate in treatment of OA. METHODS: The role of P2Y14R on OA was verified using P2Y14R knockout (KO) rats. Molecular docking virtual screening strategy and activity test in P2Y14R stably-expressed HEK293 cells were used to screen target compound from natural product library. The MM/GBSA free energy calculation/decomposition technique was used to determine the principal interaction mechanism. Next, the binding of target compound to P2Y14R was examined using cellular thermal shift assay and drug affinity responsive target stability test. Finally, the therapeutic effect of target compound was performed in monosodium iodoacetate (MIA)-induced OA mouse model. To verify whether the effect of target compound was attributed to P2Y14R, we establish the osteoarthritis model in P2Y14R KO mice to perform pharmacodynamic evaluation. Importantly, to investigate the potential mechanism by which target compound attenuate OA, expressions of the major transcription factors involved in osteoclast differentiation were detected by western blot, while markers of nerve damage in dorsal root ganglion (DRG) were evaluated by RT-qPCR and immunofluorescence techniques. RESULTS: Deficiency of P2Y14R alleviated pain behavior and cartilage destruction in MIA-induced OA rats. 14 natural compounds were screened by Glide docking-based virtual screening, among which paederosidic acid exhibited the highest antagonistic activity to P2Y14R with IC50 of 8.287 µM. As a bioactive component extracted from Paederia scandens, paederosidic acid directly interacted with P2Y14R to enhance the thermostability and decrease the protease sensitivity of target protein, which significantly inhibited receptor activator for nuclear factor-κB ligand (RANKL)-mediated osteoclastogenesis. More importantly, paederosidic acid suppressed osteoclast formation by downregulating expressions of NFAT2 and ATP6V0D2, as well as relieved neuropathic pain by decreasing expressions of CGRP, CSF1 and galanin in DRG. CONCLUSIONS: Paederosidic acid targeted P2Y14R to improve OA through alleviating osteoclast formation and neuropathic pain, which provided an available strategy for developing novel drug leads for treatment of OA.

Discovery of Selective P2Y6R Antagonists with High Affinity and In Vivo Efficacy for Inflammatory Disease Therapy.

As a member of purinoceptors, the P2Y6 receptor (P2Y6R) plays a crucial role in modulating immune signals and has been considered as a potential therapeutic target for inflammatory diseases. On the basis of the speculated probable conformation and binding determinants of P2Y6R, a hierarchical strategy that combines virtual screening, bioassays, and chemical optimization was presented. A potent P2Y6R antagonist (compound 50) was identified to possess excellent antagonistic activity (IC50 = 5.914 nM) and high selectivity. In addition, binding assays and chemical pull-down experiments confirmed that compound 50 was nicely bound to P2Y6R. Notably, compound 50 could effectively ameliorate DSS-induced ulcerative colitis in mice through inhibiting the activation of NLRP3 inflammasome in colon tissues. Moreover, treatment with compound 50 reduced LPS-induced pulmonary edema and infiltration of inflammatory cells in mice. These findings suggest that compound 50 could serve as a specific P2Y6R antagonist for treating inflammatory diseases and deserve further optimization studies.

HQL6 serves as a novel P2Y14 receptor antagonist to ameliorate acute gouty arthritis through inhibiting macrophage pyroptosis.

Acute gouty arthritis (AGA) has been classified as an autoinflammatory disease caused by deposition of monosodium urate crystals (MSU), accompanied by swellingofjoint and severe pain. Limited clinical therapy and highincidence indicate that the development of effective drugs for AGA is an urgent need. Our previous study found that P2Y14 receptor (P2Y14R) was a potential target in anti-gout treatment through regulating pyroptosis of macrophages under exposure of MSU. Based on previous work, we carried out further structure modifications and led to a more effective antagonist HQL6 with IC50 of 3.007 nM. Extensive profiling of HQL6 has demonstrated that its high selectivity, good pharmacokinetic properties, and reliable in vivo anti-gout efficacy. Moreover, P2Y14R has been demonstrated to be the key target of HQL6 since the diminished effects on adenylate cyclase inhibitor-induced acute gouty arthritis in P2Y14R knockout rats. More importantly, results of single point mutant experiments exhibited that HQL6 might interact with Lys277 as favorable residue in the binding pocket of P2Y14R. Therefore, we confirmed that P2Y14R was a promising drug target for AGA, and HQL6 would be an available candidate for further drug development.

Discovery of a Series of 5-Amide-1H-pyrazole-3-carboxyl Derivatives as Potent P2Y14R Antagonists with Anti-Inflammatory Characters.

UDPG/P2Y14R signaling pathway has been considered as a potential therapeutic target for innate immune system diseases. Based on the scaffold hopping strategy, a series of pyrazole analogues were designed and synthesized as novel P2Y14R antagonists with improved physicochemical properties, together with potential anti-inflammatory activities. Additionally, we designed and synthesized a fluorescent probe based on highly selective and potent PPTN to study the affinity of synthesized compounds. The optimized compound 16 (1-(4-fluorobenzyl)-5-(4-methylbenzamido)-1H-pyrazole-3-carboxylic acid, P2Y14R IC50 = 1.93 nM) showed strong binding ability to P2Y14R, high selectivity, notably improved solubility, and more favorable pharmacokinetic profiles. Moreover, compound 16 possessed extremely low cytotoxicity and anti-inflammatory effect in vitro. In an acute peritonitis model, compound 16 could effectively reduce the levels of inflammatory factor IL-6, IL-1ß, and TNF-α of mice induced by LPS. Compound 16, with potent in vitro and in vivo efficacy and favorable druggability, can be a promising candidate for further research.

GPR105-Targeted Therapy Promotes Gout Resolution as a Switch Between NETosis and Apoptosis of Neutrophils.

The fate of infiltrating neutrophils in inflamed joints determines the development of acute gouty arthritis (AGA). GPR105 highly expressed in human neutrophils is sensitive to monosodium urate crystals (MSU); nevertheless, the roles of GPR105 in AGA remain unclear. Here, we show that GPR105 is significantly upregulated in peripheral polymorphonuclear neutrophils of AGA patients. GPR105 knockout (GPR105-/-) prevented NETosis and induced apoptosis of neutrophils under MSU exposure, as well as attenuating inflammatory cascades in AGA. Mechanistically, GPR105 deletion activated cAMP-PKA signals, thereby disrupting Raf-Mek1/2-Erk1/2 pathway-mediated NADPH oxidase activation, contributing to inhibition of NETosis. Whereas, cAMP-PKA activation resulting in GPR105 deficiency modulated PI3K-Akt pathway to regulate apoptosis. More importantly, suppression of cAMP-PKA pathway by SQ22536 and H-89 restored NETosis instead of apoptosis in GPR105-/- neutrophils, promoting MSU-induced gout flares. Interestingly, lobetyolin was screened out as a potent GPR105 antagonist using molecular docking-based virtual screening and in vitro activity test, which efficiently attenuated MSU-induced inflammatory response interacting with GPR105. Taken together, our study implicated that modulating cell death patterns between NETosis and apoptosis through targeting GPR105 could be a potential therapeutic strategy for the treatment of AGA.

Discovery and computational studies of 2-phenyl-benzoxazole acetamide derivatives as promising P2Y14R antagonists with anti-gout potential.

The P2Y14 nucleotide receptor, a subtype of P2Y receptors, is implicated in many human inflammatory diseases. Based on the identification of favorable residues of two screening hits in the almost symmetrical P2Y14 binding domain, we describe the structural optimization of previously identified virtual screening hits 6 and 7 that result in the development of P2Y14R antagonists with a novel 2-phenyl-benzoxazole acetamide chemical scaffold. Notably, compound 52 showed potent P2Y14R antagonistic activity (IC50 = 2 nM), and a stronger inhibitory effect on MSU-induced inflammatory in vitro, better than a previously described P2Y14R antagonist PPTN. In vivo evaluation demonstrated that compound 52 also had satisfactory inhibitory activity on the inflammatory response of gout flares in mice. Moreover, P2Y14R antagonist 52 decreased paw swelling and inflammatory cell infiltration through cAMP/NLRP3/GSDMD signaling pathways in MSU-induced acute gouty arthritis mice. The discussions on the binding mechanism that employ MM/GBSA free energy calculations/decompositions also provide some useful clues for further structural designing of compound 52. Taken together, 2-phenyl-benzoxazole acetamide derivative 52 with potent P2Y14R antagonistic activity and in vivo potency could be a promising strategy for gout therapy and deserves further optimization.

Cardiovascular Protective Effects of Plant Polysaccharides: A Review.

Cardiovascular disease is a kind of heart, brain, and blood vessel injury disease by the interaction of various pathological factors. The pathogenesis of cardiovascular disease is complex with various risk factors, including abnormally elevated blood pressure, glucose, and lipid metabolism disorders, atherosclerosis, thrombosis, etc. Plant polysaccharides are a special class of natural products derived from plant resources, which have the characteristics of wide sources, diverse biological activities, and low toxicity or side effects. Many studies have shown that plant polysaccharides improve cardiovascular diseases through various mechanisms such as anti-oxidative stress, restoring the metabolism of biological macromolecules, regulating the apoptosis cascade to reduce cell apoptosis, and inhibiting inflammatory signal pathways to alleviate inflammation. This article reviews the pharmacological effects and protective mechanisms of some plant polysaccharides in modulating the cardiovascular system, which is beneficial for developing more effective drugs with low side effects for management of cardiovascular diseases.

Silencing of Gasdermin D by siRNA-Loaded PEI-Chol Lipopolymers Potently Relieves Acute Gouty Arthritis through Inhibiting Pyroptosis.

Gasdermin D (GSDMD) plays a causal role in NOD-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis eruption, which has been regarded as a potential therapeutic target for pyroptosis-related diseases including acute gouty arthritis. In the present study, the synthesized PEI-Chol (cholesterol grafted polyethylenimine) was assembled with GSDMD small interfering RNA (siRNA) to form PEI-Chol/siGSDMD polyplexes, which provided high transfection efficiency for siRNA-mediated GSDMD knockdown. Then we evaluated the effect of GSDMD siRNA-loaded PEI-Chol on inflammatory cascades in bone-marrow-derived macrophages (BMDMs) and acute gouty arthritis animal models under MSU exposure. When accompanied by pyroptosis blockade and decreased release of interleukin-1 beta (IL-1ß), NLRP3 inflammasome activation was also suppressed by GSDMD knockdown in vivo and in vitro. Moreover, in MSU-induced acute gouty arthritis mice, blocking GSDMD with siRNA significantly improved ankle swelling and inflammatory infiltration observed in histopathological analysis. Furthermore, investigation using a mouse air pouch model verified the effect of siGSDMD-loaded PEI-Chol on pyroptosis of recruited macrophages and related signaling pathways in response to MSU. These novel findings exhibited that GSDMD knockdown relieved acute gouty arthritis through inhibiting pyroptosis, providing a possible therapeutic approach for MSU-induced acute gouty arthritis molecular therapy using PEI-Chol as a nucleic acid delivery carrier.

Targeting BRD4 prevents acute gouty arthritis by regulating pyroptosis.

Background: Acute gouty arthritis is a common inflammatory arthropathy resulting from urate deposition in joints during persistent hyperuricemia. Nevertheless, effective therapeutic strategies are still unavailable. Here, we propose the crucial role of bromodomain-containing protein 4 (BRD4) in acute gouty arthritis. Methods: Therapeutic effect of BRD4 specific inhibitor JQ-1 on acute gouty arthritis was evaluated in vivo and in vitro. Pyroptosis was analyzed by Caspase-1/PI double staining and cleavage of gasdermin D (GSDMD). Expression of key factors involved in BRD4/NF-κB/NLRP3/GSDMD signaling pathway were measured by western blot, and colocalization of NLRP3 and ASC was detected using immunofluorescence. In addition, the role of BRD4 on monosodium uric acid crystals (MSU)-induced pyroptosis was verified in BRD4 siRNA-transfected THP-1 cells. Results: Pretreatment of JQ1 and BRD4 siRNA significantly suppressed pyroptosis and inhibited activation of p65 NF-κB signaling as well as NLRP3 inflammasome in THP-1 cells exposed to MSU. In vivo, JQ-1 administration could effectively attenuate joint swelling and synovial inflammation in rats treated by intra-articular injection of MSU. More importantly, MSU led to macrophage pyroptosis and Brd4/NF-κB/NLRP3/GSDMD signaling induction in rat synoviums, which was improved by JQ-1. Conclusions: Our study identifies the role of BRD4 in MSU-induced pyroptosis through regulating NF-κB/NLRP3/GSDMD signaling pathways, which provides a potential target for treatment of acute gouty arthritis.

P2Y14 receptor has a critical role in acute gouty arthritis by regulating pyroptosis of macrophages.

Nod-like receptor protein 3 (NLRP3)-mediated pyroptosis has a causal role in the pathogenesis of gout. P2Y14 receptor (P2Y14R) distributed in immune cells including macrophages is a Gi-coupled receptor that inhibits the synthesis of cAMP, which has been regarded as a potential regulator of inflammatory response. Nevertheless, the role of P2Y14R in MSU-induced pyroptosis of macrophages involved in acute gouty arthritis is still unclear. In our present study, P2Y14R knockout (P2Y14R-KO) disrupted MSU-induced histopathologic changes in rat synoviums, accompanied with a significant inhibition of pyroptotic cell death characterized by Caspase-1/PI double-positive and blockade of NLRP3 inflammasome activation in synovial tissues, which was consistent with that observed in in vitro studies. Owing to the interaction of NLRP3 inflammasome and cAMP, we then investigated the effect of adenylate cyclase activator (Forskolin) on macrophage pyroptosis and gout flare caused by MSU stimulation. The reversal effect of Forskolin verified the negative regulatory role of cAMP in MSU-induced pyroptosis. More importantly, adenylate cyclase inhibitor (SQ22536) intervention led to a reversal of protection attributed to P2Y14R deficiency. Findings in air pouch animal models also verified aforementioned experimental results. Our study first identified the role of P2Y14R/cAMP/NLRP3 signaling pathway in acute gouty arthritis, which provides a novel insight into the pathological mechanisms of pyroptosis-related diseases.

HJ22, a Novel derivative of piperine, Attenuates ibotenic acid-induced cognitive impairment, oxidativestress, apoptosis and inflammation via inhibiting the protein-protein interaction of Keap1-Nrf2.

Kelch-like ECH-associated protein (Keap1)-nuclear factor erythroid-2-related factor 2 (Nrf2) protein-protein interaction has become an important drug target for the treatment of Alzheimer's disease. In this study, we found a novel piperine derivative (HJ22) synthesized by our group with great ability to bind to Keap-1 and activate Keap1-Nrf2-ARE signaling pathway in vitro, driving us to investigate the beneficial effects of HJ22 on ibotenic acid (IBO)-induced neurological disorders in rats and underlying mechanisms. Interestingly, HJ22 significantly ameliorated IBO-induced cognitive impairment in Morris water maze, Y-maze and passive avoidance tests. Moreover, HJ22 significantly attenuated cholinergic dysfunction and neuronal morphological changes via inhibiting apoptotic cell death induced by IBO. Notably, HJ22 inhibited the interaction between Keap1 and Nrf2, and subsequently up-regulated nuclear Nrf2 expression, thereby inhibiting oxidative stress and Thioredoxin-interacting protein (TXNIP)-mediated Nod-like receptor protein 3 (NLRP3) inflammasome activation. These findings demonstrated that HJ22 exhibited potent therapeutic effects against IBO-induced cognitive impairment by alleviating cholinergic damage, oxidative stress, apoptosis and neuroinflammation, which might be partly attributed to its inhibitory activity on Keap1-Nrf2 protein-protein interaction.

The role of P2Y6R in cardiovascular diseases and recent development of P2Y6R antagonists.

As a member of the P2Y receptor family with a typical 7-transmembrane structure, P2Y6 purinergic receptor (P2Y6R) belongs to the G-protein-coupled nucleotide receptor activating the phospholipase-C signaling pathway. P2Y6R is widely involved in a range of human diseases, including atherosclerosis and other cardiovascular diseases, gradually attracting attention owing to its inappropriate or excessive activation. In addition, it was reported that P2Y6R might regulate inflammatory responses by governing the maturation and secretion of proinflammatory cytokines. Hence, several P2Y6R antagonists have been subjected to evaluation as new therapeutic strategies in recent years. This review was aimed at summarizing the role of P2Y6R in the pathogenesis of cardiovascular diseases, with an insight into the recent progress on discovery of P2Y6R antagonists.

Development of novel NLRP3-XOD dual inhibitors for the treatment of gout.

Gout is a crystalline-related arthropathy caused by the deposition of monosodium urate (MSU). Acute gouty arthritis is the most common first symptom of gout. Studies have shown that NOD-like receptor protein 3 (NLRP3) inflammasome as pattern recognition receptors can be activated by uric acid crystallization, triggering immune inflammation and causing acute gouty arthritis symptoms. Currently, the treatment of gout mainly includes two basic methods: reducing uric acid and alleviating inflammation. In this paper, 22 novel benzoxazole and benzimidazole derivatives were synthesized from deoxybenzoin oxime derivatives. These compounds have good inhibitory effects on NLRP3 and XOD screened by our research group in the early stage. The inhibitory activities of XOD and NLRP3 and their derivatives were also screened. Notably, compound 9b is a multi-targeting inhibitor of NLRP3 and XOD with excellent potency in treating hyperuricemia and acute gouty arthritis.

Design, synthesis and anti-inflammatory evaluation of 3-amide benzoic acid derivatives as novel P2Y14 receptor antagonists.

The P2Y14 receptor (P2Y14R) plays a key role in the modulation of inflammatory process, but very few classes of antagonists have been reported. A series of 3-amide benzoic acid derivatives were identified as novel and potent P2Y14R antagonists. The most potent antagonist, 16c, showed comparable activity (IC50 = 1.77 nM) to PPTN, the most potent P2Y14R antagonist reported. Compound 16c demonstrated dramatically improved aqueous solubility and excellent metabolic stability in rat and human microsomes. Investigation of the anti-inflammatory effect of 16c was performed in MSU treated THP-1 cells by flow cytometry, Western Blot and immunofluorescence labeling technology, which exhibited that 16c might be a promising candidate for further research.

4-(2-(4-chlorophenyl)-1-((4-chlorophenyl)amino)ethyl)benzene-1, 3-diol is a potential agent for gout therapy as a dual inhibitor of XOD and NLRP3.

BACKGOUND: Gout is an inflammatory arthritis characterized by abrupt self-limiting attacks of inflammation caused by precipitation of monosodium urate crystals (MSU) in the joint. Both anti-hyperuricemia and anti-inflammation could be gout therapeutic strategies, whereas ideal drugs for gout treatment are deficient. PURPOSE: 4-(2-(4-chlorophenyl)-1-((4-chlorophenyl)amino)ethyl)benzene-1, 3-diol (CBED) was obtained from a cluster of deoxybenzoins derivatives synthesized by our research group with potent anti-hyperuricemic and anti-inflammatory activities, which was expected to be a dual inhibitor of xanthine oxidase (XOD) and NOD-like receptor protein 3 (NLRP3). This study aimed to investigate effects of CBED on XOD and NLRP3 in vitro, as well as the possible mechanisms by which CBED improved gout in vivo. METHODS: After molecular docking detection, inhibitory effects of CBED on XOD and NLRP3 were evaluated in vitro. Subsequently, hyperuricemia and acute gouty arthritis animal models were established by potassium oxonate or MSU, respectively. After CBED treatment, serum uric acid levels, synovial interleukin (IL)-1ß concentrations, hepatic XOD activities, as well as synovial morphological changes were examined. More importantly, synovial expressions of NLRP3 inflammasome components including NLRP3, apoptosis-associated speck-like protein (ASC) and caspase-1 in rats were analyzed by immunofluorescence and western blot. RESULTS: In vitro, CBED obviously inhibited XOD activity with an IC50 value of 3.87 µM, moreover, it effectively inhibited MSU-induced NLRP3 inflammasome activation and IL-1ß over-production in THP-1 cells. In addition, CBED dose-dependently decreased serum uric acid levels suppressed hepatic XOD activities in oxonate-induced hyperuricemic mice. On the other hand, CBED significantly improved MSU-induced ankle swelling and histopathological damage with elevated IL-1ß. In addition, NLRP3 inflammasome activation could be blocked by CBED treatment in rats with acute gouty arthritis. Notbly, CBED exhibited no effects on all these indicators in normal animals, predicting its safety. CONCLUSIONS: CBED might serve as a dual XOD and NLRP3 inhibitor for treatment of gout.

Development of benzoxazole deoxybenzoin oxime and acyloxylamine derivatives targeting innate immune sensors and xanthine oxidase for treatment of gout.

Both the inhibition of inflammatory flares and the treatment of hyperuricemia itself are included in the management of gout. Extending our efforts to development of gout therapy, two series of benzoxazole deoxybenzoin oxime derivatives as inhibitors of innate immune sensors and xanthine oxidase (XOD) were discovered in improving hyperuricemia and acute gouty arthritis. In vitro studies revealed that most compounds not only suppressed XOD activity, but blocked activations of NOD-like receptor (NLRP3) inflammasome and Toll-like receptor 4 (TLR4) signaling pathway. More importantly, (E)-1-(6-methoxybenzo[d]oxazol-2-yl)-2-(4-methoxyphenyl)ethanone oxime (5d) exhibited anti-hyperuricemic and anti-acute gouty arthritis activities through regulating XOD, NLRP3 and TLR4. Compound 5d may serve as a tool compound for further design of anti-gout drugs targeting both innate immune sensors and XOD.