Kinase Domain-like Pseudokinase Research Articles

Inhibition of RIPK1-driven necroptosis ameliorates inflammatory hyperalgesia caused by lipopolysaccharide: involvement of TLR-, NLRP3-, and caspase-11-mediated signaling pathways.

Increasing evidence suggests that inhibition of receptor-interacting serine/threonine-protein kinase (RIPK) 1/RIPK3/mixed lineage kinase domain-like pseudokinase (MLKL) necrosome has protective effects in vivo models of painful conditions seen in humans associated with inflammation and demyelination in the central nervous system. However, the contribution of RIPK1-driven necroptosis to inflammatory pain remains unknown. Therefore, this study aims to determine the effect of necrostatin (Nec) -1s, a selective RIPK1 inhibitor, on lipopolysaccharide (LPS)-induced inflammatory pain and related underlying mechanisms. In the saline-, LPS-, and/or Nec-1s-injected male mice, thermal hyperalgesia was evaluated by hot plate test. Alterations in the expression of proteins involved in the RIPK1, toll-like receptor (TLR) 4, myeloid differentiation factor (MyD) 88/toll-interleukin (IL)-1 receptor domain-containing adapter-inducing interferon-β (TRIF)/nuclear factor (NF)-kB, nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing (NLRP) 3/apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/pro-caspase-1, and caspase-11/gasdermin D (GSDMD) signaling pathways, as well as proteins related to demyelination and remyelination in the brain and spinal cord were determined by the immunoblotting method. The LPS-induced alleviation of thermal hyperalgesia was prevented by necrostatin-1s. Necrostatin-1s reversed (1) increased activity of RIPK1, RIPK3, MLKL, and NF-kB p65, (2) enhanced expression of TLR4, MyD88, TRIF, NF-kB p65, HMGB1, NLRP3, ASC, caspase-1 p20, IL-1β, caspase-11 p20, p30-GSDMD, and semaphorin 3A, and (3) diminished myelin PLP expression induced by LPS. These findings suggest that the use of RIPK1 inhibitors could be a therapeutic approach in the management of inflammatory pain associated with necroptosis, pyroptosis, and demyelination.

In vitro studies of the combination between berberine chloride and a Copper(II) complex against Triple negative breast cancer

In this study, the copper(II) complex [Cu(chromoneTSC)Cl2]•0·.5H2O•0.0625C2H5OH (where chromoneTSC = (E)-N-Ethyl-2-((4-oxo-4H-chromen-3-yl)methylene)-hydrazinecarbothioamide) was synthesized and characterized; then used to carry out in vitro studies in combination with berberine chloride (BBC). The ligand and complex were characterized by elemental analysis, FTIR and NMR (1H and 13C) spectroscopy, and conductivity measurements. The cytotoxic effect was analyzed by using the CCK-8 viability assay in cancer MDA-MB-231 VIM RFP and non-cancer MCF-10A cell lines. The IC50 values for the complex and BBC were 21.2 ± 1.6 and 48.3 ± 2.4 μM, respectively at 24 h incubation, while the IC50 value of the combination treatment was 9.3 ± 1.5 in cancer cells. The co-treatment group significantly increased the number of cells in G2 phase, indicating the growth arrest of cancer cells. Moreover, the combination group showed induction of both intrinsic and extrinsic apoptotic pathways. There was also a study on the effect of the combination treatment on receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and mixed lineage kinase domain-like pseudokinase (MLKL) as biomarkers of necroptosis. The results showed activation of necroptosis after treatment with the combination of the copper complex and BBC via the activation of RIPK3–MLKL pathway.

Mechanisms Underlying Sensory Nerve-Predominant Damage by Methylmercury in the Peripheral Nervous System.

Sensory disturbances and central nervous system symptoms are important in patients with Minamata disease. In the peripheral nervous system of these patients, motor nerves are not strongly injured, whereas sensory nerves are predominantly affected. In this study, we investigated the mechanisms underlying the sensory-predominant impairment of the peripheral nervous system caused by methylmercury. We found that the types of cell death in rat dorsal root ganglion (DRG) neurons caused by methylmercury included apoptosis, necrosis, and necroptosis. Methylmercury induced apoptosis in cultured rat DRG neurons but not in anterior horn neurons or Schwann cells. Additionally, methylmercury activated both caspase 8 and caspase 3 in DRG neurons. It increased the expression of tumor necrosis factor (TNF) receptor-1 and the phosphorylation of receptor-interacting protein kinase 3 (RIP3) and mixed-lineage kinase domain-like pseudokinase (MLKL). The expression of TNF-α was increased in macrophage-like RAW264.7 cells by methylmercury. The increase was suggested to be mediated by the NF-κB pathway. Moreover, methylmercury induced neurological symptoms, evaluated by a hindlimb extension response, were significantly less severe in TNF-α knockout mice. Based on these results and our previous studies, we propose the following hypothesis regarding the pathogenesis of sensory nerve-predominant damage by methylmercury: First, methylmercury accumulates within sensory nerve neurons and initiates cell death mechanisms, such as apoptosis, on a small scale. Second, cell death triggers the infiltration of macrophages into the sensory fibers. Third, the macrophages are stimulated by methylmercury and secrete TNF-α through the NF-κB pathway. Fourth, TNF-α induces cell death mechanisms, including necrosis, apoptosis through the caspase 8/3 pathway, and necroptosis through the TNFR1-RIP1-RIP3-MLKL pathway, activated by methylmercury in sensory neurons. Consequently, methylmercury exhibits potent cytotoxicity specific to the DRG/sensory nerve cells in the peripheral nervous system. This chain of events caused by methylmercury may contribute to sensory disturbances in patients with Minamata disease.

Discovery of Covalent MLKL PROTAC Degraders via Optimization of a Theophylline Derivative Ligand for Treating Necroptosis.

Mixed lineage kinase domain-like pseudokinase (MLKL) initiates necroptosis and could serve as a therapeutic target related to a series of human diseases. Proteolysis-targeting chimeras (PROTACs) are useful tools for degrading pathological proteins and blocking disease processes. Using computer-aided modeling and molecular dynamics simulations, we developed a series of covalent MLKL PROTACs by linking and optimizing a theophylline derivative that covalently targets MLKL. Via structure-activity relationship studies, MP-11 was identified as a potent MLKL PROTAC degrader. Furthermore, MP-11 showed lower toxicity than the original MLKL ligand, exhibiting nanomolar-scale antinecroptotic activity on human cell lines. Xenograft model studies showed that MP-11 effectively degraded MLKL in vivo. Importantly, our study demonstrates that the covalent binding strategy is an effective approach for designing MLKL-targeting PROTACs, serving as a model for developing PROTACs to treat future necroptosis-related human diseases.

PANoptosis is a prominent cell death feature in thoracic aortic aneurysm or dissection

Thoracic aortic aneurysm and dissection (TAAD) is a devastating macrovascular disease, and its pathogenic mechanisms have not been well clarified. This study aimed to investigate the role of PANoptosis, which is newly defined programmed cell death (PCD) and characterized by pyroptosis, apoptosis, and necroptosis, in the pathogenesis of TAAD. We found that the expression of initiator factor Z-DNA binding protein 1 (ZBP1) and PANoptosis-related genes were upregulated in the β-aminopropionitrile (BAPN) + Angiotensin II (Ang II)-induced TAAD mice. Ang II stimuli enhanced the expression of ZBP1, promoted the generation of bioactive GSDMD (Gasdermin D) fragments, the cleavage of Caspase 3, and increased the phosphorylation of mixed lineage kinase domain-like pseudokinase (MLKL) in human aortic vascular smooth muscle cells (HASMCs), indicating the activation of hallmarks for PANoptosis. Moreover, ZBP1-mediated PANoptosis occurs in the aortic tissues of TAAD patients. These results highlight the significant role of PANoptosis in TAAD pathogenesis, suggesting ZBP1 and other PANoptosis-related genes as potential therapeutic targets for this condition.

Licochalcone A attenuates NMDA-induced neurotoxicity

ABSTRACT This study investigates the effect of Licochalcone A (Lico-A), a flavonoid from licorice roots known for its anti-inflammatory, anti-cancer, and antioxidant properties, on NMDA-induced neurotoxicity in primary cultured rat hippocampal neurons. The study measured cell survival following NMDA and Lico-A exposure, revealing that Lico-A at a 2.5 μg/ml significantly improved cell viability, countering the detrimental effects of NMDA. The study also analyzed synaptic changes by examining both postsynaptic density 95 (PSD95) and synaptophysin-targeted imaging, showing that Lico-A treatment resulted in a significant increase in synaptic puncta, contrasting with the reduction observed under NMDA exposure. Furthermore, levels of phosphorylated mixed lineage kinase domain-like pseudokinase (P-MLKL) and phosphorylated receptor-interacting serine/threonine-protein kinase 3 (P-RIP3), key necroptosis regulators, were measured using Western blotting. The results showed an increase in P-MLKL and P-RIP3 in neurons exposed to NMDA, which was reduced following Lico-A treatment. The response of astrocyte and microglia was also evaluated by immunostaining for glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor molecule 1 (IBA-1) and tumor necrosis factor alpha (TNF-α). These markers exhibited heightened expression in the NMDA group, which was substantially reduced by Lico-A treatment. These findings suggest that Lico-A has neuroprotective effects against NMDA-induced neurotoxicity, potentially contributing to synaptic preservation, inhibition of neuronal necroptosis, and modulation of glial activation. Therefore, Lico-A shows promise as a neuroprotective agent for conditions associated with NMDA-related neurotoxicity.

Necroptosis enhances ‘don’t eat me’ signal and induces macrophage extracellular traps to promote pancreatic cancer liver metastasis

Pancreatic ductal adenocarcinoma (PDAC) is a devastating cancer with dismal prognosis due to distant metastasis, even in the early stage. Using RNA sequencing and multiplex immunofluorescence, here we find elevated expression of mixed lineage kinase domain-like pseudo-kinase (MLKL) and enhanced necroptosis pathway in PDAC from early liver metastasis T-stage (T1M1) patients comparing with non-metastatic (T1M0) patients. Mechanistically, MLKL-driven necroptosis recruits macrophages, enhances the tumor CD47 ‘don’t eat me’ signal, and induces macrophage extracellular traps (MET) formation for CXCL8 activation. CXCL8 further initiates epithelial–mesenchymal transition (EMT) and upregulates ICAM-1 expression to promote endothelial adhesion. METs also degrades extracellular matrix, that eventually supports PDAC liver metastasis. Meanwhile, targeting necroptosis and CD47 reduces liver metastasis in vivo. Our study thus reveals that necroptosis facilitates PDAC metastasis by evading immune surveillance, and also suggest that CD47 blockade, combined with MLKL inhibitor GW806742X, may be a promising neoadjuvant immunotherapy for overcoming the T1M1 dilemma and reviving the opportunity for radical surgery.

Necrosulfonamide promotes hair growth and ameliorates DHT-induced hair growth inhibition

BackgroundAlopecia affects patients' appearance and psychology. Mixed-lineage kinase domain-like pseudokinase (MLKL)-mediated necroptosis plays a role in various skin diseases, but its effect on hair growth is unclear. ObjectiveTo investigate the effects of MLKL on hair growth and its regulatory mechanisms and to determine the potential clinical value of Necrosulfonamide (NSA, a MLKL-targeting inhibitor) in promoting hair growth and counteracting dihydrotestosterone (DHT) inhibition of hair growth. MethodsThe expression level of MLKL was detected in the scalp of androgenetic alopecia (AGA) patients and the skin tissues of mice. Knock down MLKL expression or use NSA to observe hair growth in vivo and in vitro. ResultsIn AGA patients, MLKL expression is elevated in the alopecia areas. In mice, MLKL is significantly expressed in the outer root sheath (ORS) cells of hair follicles, peaking during the catagen phase. Knockdown expression of MLKL in mice skin promoted hair growth. NSA enhanced hair growth and prevented hair follicle regression via the Wnt signaling. Reduced MLKL boosts ORS cell proliferation without directly impacting DPCs' growth. Interestingly, NSA boosts DPCs' proliferation and induction when co-cultured with ORS cells. Besides, NSA alleviated the inhibition of DHT on hair growth in vivo and vitro. ConclusionNSA inhibited the activation of MLKL in ORS cells, promoted the activation of Wnt signal in DPC cells, and improved the inhibition of hair growth by DHT, illuminating a new alopecia mechanism and aiding anti-alopecia drug development.

NLRX1 Inhibits LPS-Induced Microglial Death via Inducing p62-Dependent HO-1 Expression, Inhibiting MLKL and Activating PARP-1.

The activation of microglia and the production of cytokines are key factors contributing to progressive neurodegeneration. Despite the well-recognized neuronal programmed cell death regulated by microglial activation, the death of microglia themselves is less investigated. Nucleotide-binding oligomerization domain, leucine-rich repeat-containing X1 (NLRX1) functions as a scaffolding protein and is involved in various central nervous system diseases. In this study, we used the SM826 microglial cells to understand the role of NLRX1 in lipopolysaccharide (LPS)-induced cell death. We found LPS-induced cell death is blocked by necrostatin-1 and zVAD. Meanwhile, LPS can activate poly (ADP-ribose) polymerase-1 (PARP-1) to reduce DNA damage and induce heme oxygenase (HO)-1 expression to counteract cell death. NLRX1 silencing and PARP-1 inhibition by olaparib enhance LPS-induced SM826 microglial cell death in an additive manner. Less PARylation and higher DNA damage are observed in NLRX1-silencing cells. Moreover, LPS-induced HO-1 gene and protein expression through the p62-Keap1-Nrf2 axis are attenuated by NLRX1 silencing. In addition, the Nrf2-mediated positive feedback regulation of p62 is accordingly reduced by NLRX1 silencing. Of note, NLRX1 silencing does not affect LPS-induced cellular reactive oxygen species (ROS) production but increases mixed lineage kinase domain-like pseudokinase (MLKL) activation and cell necroptosis. In addition, NLRX1 silencing blocks bafilomycin A1-induced PARP-1 activation. Taken together, for the first time, we demonstrate the role of NLRX1 in protecting microglia from LPS-induced cell death. The underlying protective mechanisms of NLRX1 include upregulating LPS-induced HO-1 expression via Nrf2-dependent p62 expression and downstream Keap1-Nrf2 axis, mediating PARP-1 activation for DNA repair via ROS- and autophagy-independent pathway, and reducing MLKL activation.

Non-Necroptotic Roles of MLKL in Diet-Induced Obesity, Liver Pathology, and Insulin Sensitivity: Insights from a High-Fat, High-Fructose, High-Cholesterol Diet Mouse Model.

Chronic inflammation is a key player in metabolic dysfunction-associated fatty liver disease (MAFLD) progression. Necroptosis, an inflammatory cell death pathway, is elevated in MAFLD patients and mouse models, yet its role is unclear due to the diverse mouse models and inhibition strategies. In our study, we inhibited necroptosis by targeting mixed lineage kinase domain-like pseudokinase (MLKL), the terminal effector of necroptosis, in a high-fat, high-fructose, high-cholesterol (HFHFrHC) mouse model of diet-induced MAFLD. Despite the HFHFrHC diet upregulating MLKL (2.5-fold), WT mice livers showed no increase in necroptosis markers or associated proinflammatory cytokines. Surprisingly, Mlkl-/- mice experienced exacerbated liver inflammation without protection from diet-induced liver damage, steatosis, or fibrosis. In contrast, Mlkl+/- mice showed a significant reduction in these parameters that was associated with elevated Pparα and Pparγ levels. Both Mlkl-/- and Mlkl+/- mice on the HFHFrHC diet resisted diet-induced obesity, attributed to the increased beiging, enhanced oxygen consumption, and energy expenditure due to adipose tissue, and exhibited improved insulin sensitivity. These findings highlight the tissue-specific effects of MLKL on the liver and adipose tissue, and they suggest a dose-dependent effect of MLKL on liver pathology.

Stimulation of Hemolysis and Eryptosis by β-Caryophyllene Oxide.

Eryptosis stimulated by anticancer drugs can lead to anemia in patients. β-caryophyllene oxide (CPO) is an anticancer sesquiterpene present in various plants; however, its effect on the structure and function of human red blood cells (RBCs) remains unexplored. The aim of this study was to investigate the hemolytic and eryptotic activities and underlying molecular mechanisms of CPO in human RBCs. Cells were treated with 10-100 μM of CPO for 24 h at 37 °C, and hemolysis, LDH, AST, and AChE activities were photometrically assayed. Flow cytometry was employed to determine changes in cell volume from FSC, phosphatidylserine (PS) externalization by annexin-V-FITC, intracellular calcium by Fluo4/AM, and oxidative stress by 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). Cells were also cotreated with CPO and specific signaling inhibitors and antihemolytic agents. Furthermore, whole blood was exposed to CPO to assess its toxicity to other peripheral blood cells. CPO induced concentration-responsive hemolysis with LDH and AST leakage, in addition to PS exposure, cell shrinkage, Ca2+ accumulation, oxidative stress, and reduced AChE activity. The toxicity of CPO was ameliorated by D4476, staurosporin, and necrosulfonamide. ATP and PEG 8000 protected the cells from hemolysis, while urea and isotonic sucrose had opposite effects. CPO stimulates hemolysis and eryptosis through energy depletion, Ca2+ buildup, oxidative stress, and the signaling mediators casein kinase 1α, protein kinase C, and mixed lineage kinase domain-like pseudokinase. Development of CPO as an anticancer therapeutic must be approached with prudence to mitigate adverse effects on RBCs using eryptosis inhibitors, Ca2+ channel blockers, and antioxidants.

PUMA/RIP3 Mediates Chemotherapy Response via Necroptosis and Local Immune Activation in Colorectal Cancer.

Induction of programmed cell death (PCD) is a key cytotoxic effect of anticancer therapies. PCD is not confined to caspase-dependent apoptosis, but includes necroptosis, a regulated form of necrotic cell death controlled by receptor-interacting protein (RIP) kinases 1 and 3, and mixed lineage kinase domain-like (MLKL) pseudokinase. Necroptosis functions as a defense mechanism against oncogenic mutations and pathogens and can be induced by a variety of anticancer agents. However, the functional role and regulatory mechanisms of necroptosis in anticancer therapy are poorly understood. In this study, we found that RIP3-dependent but RIP1-independent necroptosis is engaged by 5-fluorouracil (5-FU) and other widely used antimetabolite drugs, and functions as a major mode of cell death in a subset of colorectal cancer cells that express RIP3. We identified a novel 5-FU-induced necroptosis pathway involving p53-mediated induction of the BH3-only Bcl-2 family protein, p53 upregulated modulator of apoptosis (PUMA), which promotes cytosolic release of mitochondrial DNA and stimulates its sensor z-DNA-binding protein 1 (ZBP1) to activate RIP3. PUMA/RIP3-dependent necroptosis mediates the in vitro and in vivo antitumor effects of 5-FU and promotes a robust antitumor immune response. Our findings provide a rationale for stimulating necroptosis to enhance tumor cell killing and antitumor immune response leading to improved colorectal cancer treatments.

Pyroptosis and necroptosis inhibitor necrosulfonamide ameliorates lipopolysaccharide-induced inflammatory hyperalgesia in mice

Objectives: This study aimed to investigate the effect of the gasdermin D (GSDMD) and mixed lineage kinase domain-like pseudokinase (MLKL) inhibitor, necrosulfonamide (NSA), on lipopolysaccharide (LPS)-induced hyperalgesia in mice. Methods: Reaction time to a thermal stimulus within 30 seconds was measured in male mice injected with saline, LPS, and/or NSA after 6 hours using the hot plate test. Immunoblotting studies were performed to determine changes in caspase-11/GSDMD-mediated pyroptosis, receptor-interacting serine/threonine-protein kinase (RIPK) 1/RIPK3/MLKL necrosome-mediated necroptosis, demyelination, and remyelination in the brains and spinal cords of animals. Results: NSA demonstrated significant antinociceptive activity compared with LPS-treated mice. In the tissues of LPS-treated mice, NSA decreased expression of caspase-11 p20, p30-GSDMD, interleukin-1β, high-mobility-group-box 1, and semaphorin 3A, and activity of RIPK1, RIPK3, and MLKL. NSA also increased the expression of myelin proteolipid protein. Conclusion: Therefore, NSA may have therapeutic potential in the treatment of inflammatory painful conditions due to bacterial infections.

Cell type-specific molecular mechanisms and implications of necroptosis in inflammatory respiratory diseases.

Necroptosis is generally considered as an inflammatory cell death form. The core regulators of necroptotic signaling are receptor-interacting serine-threonine protein kinases 1 (RIPK1) and RIPK3, and the executioner, mixed lineage kinase domain-like pseudokinase (MLKL). Evidence demonstrates that necroptosis contributes profoundly to inflammatory respiratory diseases that are common public health problem. Necroptosis occurs in nearly all pulmonary cell types in the settings of inflammatory respiratory diseases. The influence of necroptosis on cells varies depending upon the type of cells, tissues, organs, etc., which is an important factor to consider. Thus, in this review, we briefly summarize the current state of knowledge regarding the biology of necroptosis, and focus on the key molecular mechanisms that define the necroptosis status of specific cell types in inflammatory respiratory diseases. We also discuss the clinical potential of small molecular inhibitors of necroptosis in treating inflammatory respiratory diseases, and describe the pathological processes that engage cross talk between necroptosis and other cell death pathways in the context of respiratory inflammation. The rapid advancement of single-cell technologies will help understand the key mechanisms underlying cell type-specific necroptosis that are critical to effectively treat pathogenic lung infections and inflammatory respiratory diseases.

Role of Necroptosis in Intervertebral Disc Degeneration.

Apoptosis has historically been considered the primary form of programmed cell death (PCD) and is responsible for regulating cellular processes during development, homeostasis, and disease. Conversely, necrosis was considered uncontrolled and unregulated. However, recent evidence has unveiled the significance of necroptosis, a regulated form of necrosis, as an important mechanism of PCD alongside apoptosis. The activation of necroptosis leads to cellular membrane disruption, inflammation, and vascularization. This process is crucial in various pathological conditions, including intervertebral disc degeneration (IVDD), neurodegeneration, inflammatory diseases, multiple cancers, and kidney injury. In recent years, extensive research efforts have shed light on the molecular regulation of the necroptotic pathway. Various stimuli trigger necroptosis, and its regulation involves the activation of specific proteins such as receptor-interacting protein kinase 1 (RIPK1), RIPK3, and the mixed lineage kinase domain-like (MLKL) pseudokinase. Understanding the intricate mechanisms governing necroptosis holds great promise for developing novel therapeutic interventions targeting necroptosis-associated IVDD. The objective of this review is to contribute to the growing body of scientific knowledge in this area by providing a comprehensive overview of necroptosis and its association with IVDD. Ultimately, these understandings will allow the development of innovative drugs that can modulate the necroptotic pathway, offering new therapeutic avenues for individuals suffering from necroptosis.

Telaprevir Improves Memory and Cognition in Mice Suffering Ischemic Stroke via Targeting MALT1-Mediated Calcium Overload and Necroptosis.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) has been confirmed to contribute to brain injury in ischemic stroke via promoting excitotoxicity and necroptosis. Telaprevir, a hepatitis C virus protease inhibitor, is predicted to be a potential MALT1 inhibitor. Here, we showed that telaprevir protected against cerebral ischemic injury via inhibiting MALT1, thereby preventing glutamate receptor ionotropic NMDA 2B (GluN2B) activation, limiting calcium overload, and suppressing necroptosis. In ischemic stroke mice, telaprevir reduced infarct volume, improved the long-term survival rate, and enhanced sensorimotor, memory, and cognitive functions. In hypoxia-treated nerve cells, telaprevir decreased the intracellular calcium concentrations and reduced LDH release. Mechanistically, telaprevir inhibited MALT1 protease activity, thus decreasing the membrane protein level of GluN2B and its phosphorylation through reducing the level of STEP61. Moreover, telaprevir was able to inhibit the levels of necroptosis-associated proteins. According to these results, it can be concluded that telaprevir alleviates neuronal brain injury in stroke mice via restraining GluN2B activation and suppresses the receptor-interacting protein kinase 1 (RIPK1)/receptor-interacting protein kinase 3 (RIPK3)/mixed lineage kinase domain-like pseudokinase (MLKL) pathway through inhibiting MALT1. Thus, telaprevir might have a novel indication for treating patients with ischemic stroke.

PROTACs Targeting MLKL Protect Cells from Necroptosis.

Mixed Lineage Kinase domain-Like pseudokinase (MLKL) is implicated in a broad range of diseases due to its role as the ultimate effector of necroptosis and has therefore emerged as an attractive drug target. Here, we describe the development of PROteolysis TArgeting Chimeras (PROTACs) as a novel approach to knock down MLKL through chemical means. A series of candidate degraders were synthesized from a high-affinity pyrazole carboxamide-based MLKL ligand leading to the identification of a PROTAC molecule that effectively degraded MLKL and completely abrogated cell death in a TSZ model of necroptosis. By leveraging the innate ability of these PROTACs to degrade MLKL in a dose-dependent manner, the quantitative relationship between MLKL levels and necroptosis was interrogated. This work demonstrates the feasibility of targeting MLKL using a PROTAC approach and provides a powerful tool to further our understanding of the role of MLKL within the necroptotic pathway.

Alleviation of hippocampal necroptosis and neuroinflammation by NecroX-7 treatment after acute seizures.

Temporal lobe epilepsy (TLE) is one of the most common neurological disorders, but still one-third of patients cannot be properly treated by current medication. Thus, we investigated the therapeutic effects of a novel small molecule, NecroX-7, in TLE using both a low [Mg2+]o-induced epileptiform activity model and a mouse model of pilocarpine-induced status epilepticus (SE). NecroX-7 post-treatment enhanced the viability of primary hippocampal neurons exposed to low [Mg2+]o compared to controls in an MTT assay. Application of NecroX-7 after pilocarpine-induced SE also reduced the number of degenerating neurons labelled with Fluoro-Jade B. Immunocytochemistry and immunohistochemistry showed that NecroX-7 post-treatment significantly alleviated ionized calcium-binding adaptor molecule 1 (Iba1) intensity and immunoreactive area, while the attenuation of reactive astrocytosis by glial fibrillary acidic protein (GFAP) staining was observed in cultured hippocampal neurons. However, NecroX-7-mediated morphologic changes of astrocytes were seen in both in vitro and in vivo models of TLE. Finally, western blot analysis demonstrated that NecroX-7 post-treatment after acute seizures could decrease the expression of mixed lineage kinase domain-like pseudokinase (MLKL) and phosphorylated MLKL (p-MLKL), markers for necroptosis. Taken all together, NecroX-7 has potential as a novel medication for TLE with its neuroprotective, anti-inflammatory, and anti-necroptotic effects.

Inflammatory Forms of Cardiomyocyte Cell Death in the Rat Model of Isoprenaline-Induced Takotsubo Syndrome.

Takotsubo syndrome (TTS) is associated with inflammatory response, therefore the aim of the study was to evaluate the presence and dynamics of inflammatory-associated forms of cell death, necroptosis, and pyroptosis in the female rat model of isoprenaline (ISO)-induced TTS. TTS was induced in female Sprague Dawley rats (n = 36) by ISO 150 mg/kg intraperitoneally. Animals were divided into four groups: TTSO (TTS+ovariectomy; n = 10), TTSP (TTS+sham operation; n = 10), CO (0.9% NaCl+ovariectomy; n = 8), CP (0.9% NaCl+sham operation; n = 8). Histopathological analysis, evaluation of plasma concentration, and myocardial expression of pyroptosis- and necroptosis-associated proteins were performed. TTSO and TTSP groups had higher plasma concentrations of interleukin-1β in comparison with the controls. Low myocardial protein expression of mixed lineage kinase domain-like pseudokinase (MLKL), caspase-1 (Casp-1), and calcium/calmodulin-dependent kinase type II isoform delta (CAMKIIδ) was visible 6 and/or 12 h post-ISO. Twenty-four hours post-ISO, high myocardial and vascular protein expression of CAMKIIδ was visible in TTSO but not TTSP rats, while high myocardial expression of MLKL and Casp-1 was visible both in TTSO and TTSP rats. The course of TTS is associated with activation of inflammatory-associated programmed cell death, necroptosis, and pyroptosis, therefore inflammation may be a primary response occurring simultaneously with cardiomyocyte death in TTS.

Pharmaceutical Therapies for Necroptosis in Myocardial Ischemia-Reperfusion Injury.

Cardiovascular disease morbidity/mortality are increasing due to an aging population and the rising prevalence of diabetes and obesity. Therefore, innovative cardioprotective measures are required to reduce cardiovascular disease morbidity/mortality. The role of necroptosis in myocardial ischemia-reperfusion injury (MI-RI) is beyond doubt, but the molecular mechanisms of necroptosis remain incompletely elucidated. Growing evidence suggests that MI-RI frequently results from the superposition of multiple pathways, with autophagy, ferroptosis, and CypD-mediated mitochondrial damage, and necroptosis all contributing to MI-RI. Receptor-interacting protein kinases (RIPK1 and RIPK3) as well as mixed lineage kinase domain-like pseudokinase (MLKL) activation is accompanied by the activation of other signaling pathways, such as Ca2+/calmodulin-dependent protein kinase II (CaMKII), NF-κB, and JNK-Bnip3. These pathways participate in the pathological process of MI-RI. Recent studies have shown that inhibitors of necroptosis can reduce myocardial inflammation, infarct size, and restore cardiac function. In this review, we will summarize the molecular mechanisms of necroptosis, the links between necroptosis and other pathways, and current breakthroughs in pharmaceutical therapies for necroptosis.