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The NACHT-, leucine-rich-repeat-, and pyrin domain-containing protein 3 (NLRP3) is a critical intracellular inflammasome sensor and an important clinical target against inflammation-driven human diseases. Recent studies have elucidated its transition from a closed cage to an activated disk-like inflammasome, but the intermediate activation mechanism remains elusive. Here we report the cryo-electron microscopy structure of NLRP3, which forms an open octamer and undergoes a ~ 90° hinge rotation at the NACHT domain. Mutations on open octamer’s interfaces reduce IL-1β signaling, highlighting its essential role in NLRP3 activation/inflammasome assembly. The centrosomal NIMA-related kinase 7 (NEK7) disrupts large NLRP3 oligomers and forms NEK7/NLRP3 monomers/dimers which is a critical step preceding the assembly of the disk-like inflammasome. These data demonstrate an oligomeric cooperative activation of NLRP3 and provide insight into its inflammasome assembly mechanism. NLRP3 is a critical intracellular inflammasome sensor and an important clinical target against inflammation-driven human diseases. Here, the authors determined Cryo-EM structures of human NLRP3 in its closed and open states, elucidating the mechanism of NLRP3 inflammasome activation.

The NLRP3 inflammasome plays a pivotal role in host defense and drives inflammation against microbial threats, crystals, and danger-associated molecular patterns (DAMPs). Dysregulation of NLRP3 activity is associated with various human diseases, making it an attractive therapeutic target. Patients with NLRP3 mutations suffer from Cryopyrin-Associated Periodic Syndrome (CAPS) emphasizing the clinical significance of modulating NLRP3. In this study, we present the identification of a novel chemical class exhibiting selective and potent inhibition of the NLRP3 inflammasome. Through a comprehensive structure–activity relationship (SAR) campaign, we optimized the lead molecule, compound A, for in vivo applications. Extensive in vitro and in vivo characterization of compound A confirmed the high selectivity and potency positioning compound A as a promising clinical candidate for diseases associated with aberrant NLRP3 activity. This research contributes to the ongoing efforts in developing targeted therapies for conditions involving NLRP3-mediated inflammation, opening avenues for further preclinical and clinical investigations. Synopsis In this study a novel clinical candidate is introduced as a potent inhibitor of the NLRP3 inflammasome. The chemical diversification of this molecule from existing clinical candidates opens newer avenues for clinical development. Human and mouse NLRP3 inflammasomes are potently and selectively inhibited by compound A. ATPase activity of NLRP3 is targeted by compound A through binding to Walker A motif. Tested NLRP3 auto-activating mutants are more potently inhibited by compound A in comparison to reference compound. In this study a novel clinical candidate is introduced as a potent inhibitor of the NLRP3 inflammasome. The chemical diversification of this molecule from existing clinical candidates opens newer avenues for clinical development.

Neuroinflammation is widely recognized as a key factor in the pathogenesis of Alzheimer's disease (AD), alongside ß-amyloid deposition and the formation of neurofibrillary tangles. The NLR family pyrin domain containing 3 (NLRP3) inflammasome, part of the innate immune system, has been implicated in the neuropathology of both preclinical amyloid and tau transgenic models. Activation of the NLRP3 pathway involves an initial priming step, which increases the expression of Nlrp3 and interleukin (IL)-1β, followed by the assembly of the NLRP3 inflammasome complex, comprising NLRP3, ASC, and caspase-1. This assembly leads to the proteolytic maturation of the pro-inflammatory cytokines IL-1β and IL-18. Additionally, the NLRP3 inflammasome induces Gasdermin D (GSDMD) cleavage, forming membrane pores through which IL-1β and IL-18 are secreted. Inhibition of NLRP3 has been shown to enhance plaque clearance by modulating microglial activation. Furthermore, blocking NLRP3 in tau transgenic mice has been found to reduce tau phosphorylation by affecting the activity of certain tau kinases and phosphatases. In this study, organotypic brain slice cultures from P301S transgenic mice were treated with lipopolysaccharide (LPS) plus nigericin as a positive control or exposed to tau seeds (K18) to evaluate NLRP3 inflammasome activation. The effect of tau seeding on NLRP3 activity was further examined using Meso Scale Discovery (MSD) assays to measure IL1β secretion levels in the presence and absence of NLRP3 inhibitors. The role of NLRP3 activity was investigated in full-body knockout mice crossbred with the tau transgenic P301S model. Additionally, full-body and microglia-selective knockout mice were crossbred with P301S mice, and tau pathology and neurodegeneration were evaluated at early and late stages of the disease using immunohistochemistry and biochemical assays. Activation of the NLRP3 pathway was observed in the mouse organotypic slice culture (OSC) model following stimulation with LPS and nigericin or exposure to tau seeds. However, deficiency did not mitigate tauopathy or neurodegeneration in P301S mice , showing only a minor effect on plasma neurofilament (NF-L) levels. Consistently, deficiency did not alter tau pathology in P301S mice. Furthermore, neither full-body nor microglia-selective deletion had an impact on neuronal pathology or the release of pro-inflammatory cytokines. The absence of key components of the NLRP3 inflammasome pathway did not yield a beneficial effect on tau pathology or neurodegeneration in the preclinical Tau-P301S mouse model of AD. Nonetheless, organotypic slice cultures could serve as a valuable mechanistic model for evaluating NLRP3 pathway activation and pharmacological inhibitors.

Posttranslational modification of chemokines is one of the mechanisms that regulate leukocyte migration during inflammation. Multiple natural NH(2)-terminally truncated forms of the major human neutrophil attractant interleukin-8 or CXCL8 have been identified. Although differential activity was reported for some CXCL8 forms, no biological data are available for others. Aminopeptidase-cleaved CXCL8(2-77) and CXCL8(3-77), the product of alternative cleavage of the signal peptide CXCL8(-2-77) and the previously studied forms containing 77 and 72 amino acids, CXCL8(1-77) and CXCL8(6-77), were prepared by solid-phase peptide synthesis, purified and folded into active proteins. No differences in binding and calcium signaling potency were detected between CXCL8(1-77), CXCL8(-2-77), CXCL8(2-77) and CXCL8(3-77) on cells transfected with one of the human CXCL8 receptors, i.e. CXCR1 and CXCR2. However, CXCL8(-2-77) was more potent compared to CXCL8(1-77), CXCL8(2-77) and CXCL8(3-77) in signaling and in vitro chemotaxis of peripheral blood-derived human neutrophils. Moreover, CXCL8(-2-77) was less efficiently processed by plasmin into the more potent CXCL8(6-77). The truncated forms CXCL8(2-77) and CXCL8(3-77) had higher affinity for heparin than CXCL8(1-77), a property important for the presentation of CXCL8 on endothelial layers. Upon intraperitoneal injection in mice, elongated, truncated and intact CXCL8 were equally potent to recruit neutrophils to the peritoneal cavity. In terms of their ability to induce neutrophil recruitment in vivo, the multiple CXCL8 forms may be divided in three groups. The first group includes CXCL8 proteins consisting of 75 to 79 amino acids, cleaved by aminopeptidases, with intermediate activity on neutrophils. The second group, generated through proteolytic cleavage (e.g. by Ser proteases), contains 69 to 72 amino acid forms which are highly potent neutrophil attractants in vivo. A third category is generated through the modification of the arginine in the NH(2)-terminal region into citrulline by peptidylarginine deiminases and has weak potency to induce neutrophil extravasation.

Posttranslational modification of chemokines is one of the mechanisms that regulate leukocyte migration during inflammation. Multiple natural NH.sub.2 -terminally truncated forms of the major human neutrophil attractant interleukin-8 or CXCL8 have been identified. Although differential activity was reported for some CXCL8 forms, no biological data are available for others. Aminopeptidase-cleaved CXCL8(2-77) and CXCL8(3-77), the product of alternative cleavage of the signal peptide CXCL8(-2-77) and the previously studied forms containing 77 and 72 amino acids, CXCL8(1-77) and CXCL8(6-77), were prepared by solid-phase peptide synthesis, purified and folded into active proteins. No differences in binding and calcium signaling potency were detected between CXCL8(1-77), CXCL8(-2-77), CXCL8(2-77) and CXCL8(3-77) on cells transfected with one of the human CXCL8 receptors, i.e. CXCR1 and CXCR2. However, CXCL8(-2-77) was more potent compared to CXCL8(1-77), CXCL8(2-77) and CXCL8(3-77) in signaling and in vitro chemotaxis of peripheral blood-derived human neutrophils. Moreover, CXCL8(-2-77) was less efficiently processed by plasmin into the more potent CXCL8(6-77). The truncated forms CXCL8(2-77) and CXCL8(3-77) had higher affinity for heparin than CXCL8(1-77), a property important for the presentation of CXCL8 on endothelial layers. Upon intraperitoneal injection in mice, elongated, truncated and intact CXCL8 were equally potent to recruit neutrophils to the peritoneal cavity. In terms of their ability to induce neutrophil recruitment in vivo, the multiple CXCL8 forms may be divided in three groups. The first group includes CXCL8 proteins consisting of 75 to 79 amino acids, cleaved by aminopeptidases, with intermediate activity on neutrophils. The second group, generated through proteolytic cleavage (e.g. by Ser proteases), contains 69 to 72 amino acid forms which are highly potent neutrophil attractants in vivo. A third category is generated through the modification of the arginine in the NH.sub.2 -terminal region into citrulline by peptidylarginine deiminases and has weak potency to induce neutrophil extravasation.

Posttranslational modification of chemokines is one of the mechanisms that regulate leukocyte migration during inflammation. Multiple natural NH.sub.2 -terminally truncated forms of the major human neutrophil attractant interleukin-8 or CXCL8 have been identified. Although differential activity was reported for some CXCL8 forms, no biological data are available for others. Aminopeptidase-cleaved CXCL8(2-77) and CXCL8(3-77), the product of alternative cleavage of the signal peptide CXCL8(-2-77) and the previously studied forms containing 77 and 72 amino acids, CXCL8(1-77) and CXCL8(6-77), were prepared by solid-phase peptide synthesis, purified and folded into active proteins. No differences in binding and calcium signaling potency were detected between CXCL8(1-77), CXCL8(-2-77), CXCL8(2-77) and CXCL8(3-77) on cells transfected with one of the human CXCL8 receptors, i.e. CXCR1 and CXCR2. However, CXCL8(-2-77) was more potent compared to CXCL8(1-77), CXCL8(2-77) and CXCL8(3-77) in signaling and in vitro chemotaxis of peripheral blood-derived human neutrophils. Moreover, CXCL8(-2-77) was less efficiently processed by plasmin into the more potent CXCL8(6-77). The truncated forms CXCL8(2-77) and CXCL8(3-77) had higher affinity for heparin than CXCL8(1-77), a property important for the presentation of CXCL8 on endothelial layers. Upon intraperitoneal injection in mice, elongated, truncated and intact CXCL8 were equally potent to recruit neutrophils to the peritoneal cavity. In terms of their ability to induce neutrophil recruitment in vivo, the multiple CXCL8 forms may be divided in three groups. The first group includes CXCL8 proteins consisting of 75 to 79 amino acids, cleaved by aminopeptidases, with intermediate activity on neutrophils. The second group, generated through proteolytic cleavage (e.g. by Ser proteases), contains 69 to 72 amino acid forms which are highly potent neutrophil attractants in vivo. A third category is generated through the modification of the arginine in the NH.sub.2 -terminal region into citrulline by peptidylarginine deiminases and has weak potency to induce neutrophil extravasation.