Development of Receptor for Advanced Glycation End Products (RAGE) ligands through target directed dynamic combinatorial chemistry: a novel class of possible antagonists.

RAGE is a transmembrane receptor of immunoglobulin family that can bind various endogenous and exogenous ligands, initiating the inflammatory downstream signaling pathways, including inflammaging. Therefore, RAGE represents an attractive drug target for age-related diseases. For the development of small-molecule RAGE antagonists, we employed protein-templated dynamic combinatorial chemistry (ptDCC) using RAGE's VC1 domain as a template, the first application of this approach in the context of RAGE. The affinities of DCC hits were validated using microscale thermophoresis. Subsequent screening against AGE2 (glyceraldehyde-modified AGE)-sRAGE (solubleRAGE) (AGE2-BSA/sRAGE) interaction using ELISA tests led to the identification of antagonists with micromolar potency. Our findings not only demonstrate the successful application of ptDCC on RAGE but also highlight its potential to address the pressing need for alternative strategies for the development of small-molecule RAGE antagonists, an area of research that has experienced a slowdown in recent years.

Relevance of inflammatory and complement activation biomarkers profiling in antiphospholipid syndrome patients outside acute thrombosis.

OBJECTIVES: To evaluate whether inflammatory and complement biomarkers are associated with specific characteristics of antiphospholipid syndrome (APS). METHODS: Serum levels of interleukin (IL)-1ß (IL-1ß), IL-6, IL-8, IL-10, tumour necrosis factor (TNF)-α, interferon-α (IFN)-α, IFN-γ, vascular endothelial growth factor (VEGF), intercellular adhesion molecule 1 (ICAM-1), E-selectin, and vascular cell adhesion molecule (VCAM)-1, and plasma levels of soluble C5b-9 (sC5b-9), C3a, C4a, Bb fragment were measured in unselected APS patients. Twenty-five healthy blood donors were included as controls. RESULTS: Between January 2020 and April 2021, 98 APS patients were included outside acute thrombosis (median time from the last APS manifestation: 60 (23;132) months). Levels of IL6, VCAM-1, sC5b-9, C3a, C4a, and Bb were significantly increased in APS patients compared to controls. A cluster analysis allowed to divide patients into two clusters: "inflammatory" (higher levels of IL-6 and VCAM-1) and "complement". In APS, elevated IL-6 was associated with hypertension, diabetes, BMI, and hypertriglyceridaemia. 85% of our APS patients had elevated levels of at least one complement biomarker. Elevated Bb (34%) was associated with aPL positivities, especially with triple aPL positivity (50% vs. 18%, p<0.001). 7/8 patients with history of catastrophic APS had elevated levels of complement biomarkers. CONCLUSIONS: Our findings suggested that APS patients outside acute thrombosis might be divided into two clusters: "inflammatory" and "complement". Elevated IL-6 was associated with cardiovascular risk factors and metabolic parameters, whereas Bb fragments, a marker of alternative pathway complement activation, was strongly associated with aPL profile at highest risk of severe disease.

Hemolysis is associated with altered heparan sulfate of the endothelial glycocalyx and with local complement activation in thrombotic microangiopathies.

The complement system plays a key role in the pathophysiology of kidney thrombotic microangiopathies (TMA), as illustrated by atypical hemolytic uremic syndrome. But complement abnormalities are not the only drivers of TMA lesions. Among other potential pathophysiological actors, we hypothesized that alteration of heparan sulfate (HS) in the endothelial glycocalyx could be important. To evaluate this, we analyzed clinical and histological features of kidney biopsies from a monocentric, retrospective cohort of 72 patients with TMA, particularly for HS integrity and markers of local complement activation. The role of heme (a major product of hemolysis) as an HS-degrading agent in vitro, and the impact of altering endothelial cell (ECs) HS on their ability to locally activate complement were studied. Compared with a positive control, glomerular HS staining was lower in 57 (79%) patients with TMA, moderately reduced in 20 (28%), and strongly reduced in 37 (51%) of these 57 cases. Strongly reduced HS density was significantly associated with both hemolysis at the time of biopsy and local complement activation (C3 and/or C5b-9 deposits). Using primary endothelial cells (HUVECs, Glomerular ECs), we observed decreased HS expression after short-term exposure to heme, and that artificial HS degradation by exposure to heparinase was associated with local complement activation. Further, prolonged exposure to heme modulated expression of several key genes of glycocalyx metabolism involved in coagulation regulation (C5-EPI, HS6ST1, HS3ST1). Thus, our study highlights the impact of hemolysis on the integrity of endothelial HS, both in patients and in endothelial cell models. Hence, acute alteration of HS may be a mechanism of heme-induced complement activation.

Sepsis-like Energy Deficit Is Not Sufficient to Induce Early Muscle Fiber Atrophy and Mitochondrial Dysfunction in a Murine Sepsis Model.

Sepsis-induced myopathy is characterized by muscle fiber atrophy, mitochondrial dysfunction, and worsened outcomes. Whether whole-body energy deficit participates in the early alteration of skeletal muscle metabolism has never been investigated. Three groups were studied: "Sepsis" mice, fed ad libitum with a spontaneous decrease in caloric intake (n = 17), and "Sham" mice fed ad libitum (Sham fed (SF), n = 13) or subjected to pair-feeding (Sham pair fed (SPF), n = 12). Sepsis was induced by the intraperitoneal injection of cecal slurry in resuscitated C57BL6/J mice. The feeding of the SPF mice was restricted according to the food intake of the Sepsis mice. Energy balance was evaluated by indirect calorimetry over 24 h. The tibialis anterior cross-sectional area (TA CSA), mitochondrial function (high-resolution respirometry), and mitochondrial quality control pathways (RTqPCR and Western blot) were assessed 24 h after sepsis induction. The energy balance was positive in the SF group and negative in both the SPF and Sepsis groups. The TA CSA did not differ between the SF and SPF groups, but was reduced by 17% in the Sepsis group compared with the SPF group (p < 0.05). The complex-I-linked respiration in permeabilized soleus fibers was higher in the SPF group than the SF group (p < 0.05) and lower in the Sepsis group than the SPF group (p < 0.01). Pgc1α protein expression increased 3.9-fold in the SPF mice compared with the SF mice (p < 0.05) and remained unchanged in the Sepsis mice compared with the SPF mice; the Pgc1α mRNA expression decreased in the Sepsis compared with the SPF mice (p < 0.05). Thus, the sepsis-like energy deficit did not explain the early sepsis-induced muscle fiber atrophy and mitochondrial dysfunction, but led to specific metabolic adaptations not observed in sepsis.

Electrophoretic separation of multiple chiral center analyte with a three cyclodextrins mixture.

A capillary electrokinetic chromatography method (CEKC) was developed for complete stereoisomeric separation of a neutral, hydrophobic, multiple chiral center dihydropyridone analogue, a drug candidate proposed in type 2 diabetes treatment. A background electrolyte comprising three cyclodextrins was found to successfully separate the eight isomers. First an anionic cyclodextrin, the SBE-ß-CD, was selected to allow the chiral separation of our neutral compound and partial resolutions of the eight isomers were obtained. Then, the effects of different parameters such as the nature and concentration of the other cyclodextrins added and pH of the buffer were examined. Finally, a triple CD-system consisted of 15 mM SBE-ß-CD plus 15 mM Î³-CD and 40 mM HP-γ-CD in a 50 mM borate background electrolyte at pH 10, was found to successfully separate the eight isomers. Last, the selectivity and limits of detection and quantification were evaluated for this optimized method.