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Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a key regulator in inflammation and cell death and is involved in mediating a variety of inflammatory or degenerative diseases. A number of allosteric RIPK1 inhibitors (RIPK1i) have been developed, and some of them have already advanced into clinical evaluation. Recently, selective RIPK1i that interact with both the allosteric pocket and the ATP-binding site of RIPK1 have started to emerge. Here, we report the rational development of a new series of type-II RIPK1i based on the rediscovery of a reported but mechanistically atypical RIPK3i. We also describe the structure-guided lead optimization of a potent, selective, and orally bioavailable RIPK1i, 62, which exhibits extraordinary efficacies in mouse models of acute or chronic inflammatory diseases. Collectively, 62 provides a useful tool for evaluating RIPK1 in animal disease models and a promising lead for further drug development.

The formation of neural circuits requires extensive interactions of cell-surface proteins to guide axons to their correct target neurons. Trans-cellular interactions of the adhesion G protein-coupled receptor latrophilin-2 (Lphn2) with its partner teneurin-3 instruct the precise assembly of hippocampal networks by reciprocal repulsion. Lphn2 acts as a repulsive receptor in distal CA1 neurons to direct their axons to the proximal subiculum, and as a repulsive ligand in the proximal subiculum to direct proximal CA1 axons to the distal subiculum. It remains unclear if Lphn2-mediated intracellular signaling is required for its role in either context. Here, we show that Lphn2 couples to Gα12/13 in heterologous cells; this coupling is increased by constitutive exposure of the tethered agonist. Specific mutations of Lphn2’s tethered agonist region disrupt its G protein coupling and autoproteolytic cleavage, whereas mutating the autoproteolytic cleavage site alone prevents cleavage but preserves a functional tethered agonist. Using an in vivo misexpression assay, we demonstrate that wild-type Lphn2 misdirects proximal CA1 axons to the proximal subiculum and that Lphn2 tethered agonist activity is required for its role as a repulsive receptor in axons. By contrast, neither tethered agonist activity nor autoproteolysis were necessary for Lphn2’s role as a repulsive ligand in the subiculum target neurons. Thus, tethered agonist activity is required for Lphn2-mediated neural circuit assembly in a context-dependent manner.

Abstract RIPK1, a death domain-containing kinase, has been recognized as an important therapeutic target for inhibiting apoptosis, necroptosis, and inflammation under pathological conditions. RIPK1 kinase inhibitors have been advanced into clinical studies for the treatment of various human diseases. One of the current bottlenecks in developing RIPK1 inhibitors is to discover new approaches to inhibit this kinase as only limited chemotypes have been developed. Here we describe Necrostatin-34 (Nec-34), a small molecule that inhibits RIPK1 kinase with a mechanism distinct from known RIPK1 inhibitors such as Nec-1s. Mechanistic studies suggest that Nec-34 stabilizes RIPK1 kinase in an inactive conformation by occupying a distinct binding pocket in the kinase domain. Furthermore, we show that Nec-34 series of compounds can synergize with Nec-1s to inhibit RIPK1 in vitro and in vivo. Thus, Nec-34 defines a new strategy to target RIPK1 kinase and provides a potential option of combinatorial therapy for RIPK1-mediated diseases.

Summary: In developing pro-myelination treatment, an important hurdle is the lack of reliable animal models for assessing de novo myelination in disease settings. We recently showed that regenerated axons in injured optic nerves fail to be myelinated, providing an animal model for this purpose. Here, we describe procedures to promote axonal regeneration, administer optic nerve crush, and assess oligodendrocyte differentiation and maturation into myelination-competent oligodendrocytes. This protocol allows for testing the efficacy of remyelination treatments in an in vivo central nervous system (CNS).For complete details on the use and execution of this protocol, please refer to Wang et al. (2020) and Bei et al. (2016).

Background: The D-dimer-to-fibrinogen ratio (DFR) is a good indicator of thrombus activity in thrombotic diseases, but its clinical role in acute ischaemic stroke (AIS) patients with different etiologies has not been studied. We evaluated the diagnostic value of the DFR for different subtypes of AIS., Methods: We conducted a single-center retrospective study of 269 patients with AIS who were referred to our stroke center within 4.5 h from Jan 2017 to Oct 2019. Coagulation data including DFRs were compared among the different stroke subtypes, and a separate retrospective validation sample was utilized to evaluate the prediction efficiency of the DFR for subtype diagnosis., Results: A higher DFR was observed in patients with cardioembolism than in those with large artery atherosclerosis (LAA) (odds ratio (OR) per 0.1 increase of the DFR: 1.49 [1.01-2.18]) after we adjusted for vascular risk factors. The diagnostic value of the DFR for detecting cardioembolism (AUC = 0.722, 95 % CI = 0.623-0.820) exceeded that of isolated D-dimer or fibrinogen. The validation sample (n = 117) further supported the notion that a diagnosis of cardioembolism was more common in patients with a DFR > 0.11 (multivariable risk ratio = 3.11[1.33-7.31], P = 0.009)., Conclusion: High DFRs were associated with cardioembolism in patients with AIS. The utilization of DFR can be beneficial for distinguishing a cardiac embolic source from atherosclerotic stroke., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Objective: To analyze the influencing factors of futile recanalization after endovascular therapy (EVT) in acute ischemic stroke patients with large vessel occlusions (AIS-LVO). Methods: AIS-LVO patients who underwent EVT with successful recanalization between January 2019 and December 2021 in Neurovascular Center of Changhai Hospital of Naval Medical University were retrospectively selected. Modified Rankin scale (mRS) score 3 months after EVT was used as the prognostic evaluation index, and patients with mRS scores≤2 were classified as the meaningful recanalization group and mRS scores 3-6 as the futile recanalization group. The risk factors, National Institutes of Health stroke scale (NIHSS) score, Glasgow coma scale (GCS) score, Alberta Stroke Program Early CT (ASPECT) score, core infarct volume, etc. in both groups were analyzed, and the influencing factors of futile recanalization after EVT were analyzed by multivariate logistic regression. Continuous variables that do not conform to the normal distribution are represented by [ M ( Q 1 ,Q 3 )]. Results: A total of 368 patients meeting the inclusion criteria were collected, including 228 males and 140 females, and aged 68 (61, 77) years. There are 196 patients and 172 patients in the meaningful recanalization and futile recanalization groups, respectively, with the rate of futile recanalization 3 months after EVT of 46.74% (172/368). Comparing the general information and risk factors between the two groups found that the age of patients in the futile recanalization group [71 (65, 79) years] was higher than that in the meaningful recanalization group [65 (59, 72) years]. The baseline NIHSS score [18 (14, 22)] and the rate of not achieving modified Thrombolysis in Cerebral Ischemia grade 3 (mTICI 3) reperfusion (36.1%) were higher in the futile recanalization group than those in the meaningful recanalization group [12 (7, 17) and 19.9%]. The baseline GCS score [11 (9, 13)] was lower in the futile recanalization group than that in the meaningful recanalization group [14 (11, 15)]. The core infarct volume in the futile recanalization group [28 (7, 65) ml] was larger than that in the meaningful recanalization group [6 (0, 17) ml]. The ASPECT score [7 (5, 9)] was lower in the futile recanalization group than that in the meaningful recanalization group [9 (7, 10)]. In addition, the proportion of hypertension, atrial fibrillation, general anesthesia, and symptomatic intracranial hemorrhage was higher in the futile recanalization group (all P <0.05). The time from symptom onset to puncture and from symptom onset to reperfusion was longer in the futile recanalization group (both P <0.05). There were statistically significant differences in trial of Org 10172 in acute stroke treatment (TOAST) classification and the site of occluded blood vessels between the two groups (both P <0.05). Multivariate logistic regression indicated that age ≥80 years( OR =1.935,95% CI : 1.168-3.205), baseline NIHSS score ( OR =1.999,95% CI : 1.202-3.325), GCS score ( OR =2.299,95% CI : 1.386-3.814), previous stroke history ( OR =1.977,95% CI : 1.085-3.604), general anesthesia ( OR =1.981,95% CI : 1.143-3.435), not achieving grade 3 recanalization ( OR =2.846, 95% CI : 1.575-5.143), ASPECT score<6 ( OR= 2.616, 95% CI : 1.168-5.857), and core infarct volume>70 ml ( OR =2.712, 95% CI : 1.130-6.505) were risk factors for futile recanalization. Conclusion: Age≥80 years, previous stroke history, baseline NIHSS score≥20, GCS score≤8, general anesthesia, ASPECT score<6, core infarct volume>70 ml, and failure to achieve Grade 3 recanalization are independent influencing factors for futile recanalization after endovascular therapy in AIS-LVO patients.