Investigation of N-Glycan Functions in Receptor for Advanced Glycation End Products V Domain through Chemical Glycoprotein Synthesis.

The receptor for advanced glycation end products (RAGE) plays a crucial role in inflammation-related pathways and various chronic diseases. Despite the recognized significance of N-glycosylation in the ligand-binding V domain (VD) of RAGE, a comprehensive understanding of the site-activity and structure-activity relationships is lacking due to the challenges in obtaining homogeneous glycoprotein samples through biological expression. Here, we combined chemical and chemoenzymatic approaches to synthesize RAGE-VD and its congeners with Asn3-glycosylation by incorporating precise N-glycan structures. Evaluation of these samples revealed that, in comparison to other RAGE-VD forms, α2,6-sialylated N-glycosylation at the Asn3 site results in more potent inhibition of HMGB1-induced nuclear factor-κB (NF-κB) expression in RAGE-overexpressing cells. Hydrogen/deuterium exchange-mass spectrum analysis revealed a sialylated RAGE-VD-induced interaction region within HMGB1. Conversely, Asn3 N-glycosylation in VD has negligible effects on RAGE-VD/S100B interactions. This study established an approach for accessing homogeneously glycosylated RAGE-VD and explored the modulatory effects of N-glycosylation on the interactions between RAGE-VD and its ligand proteins.

Understanding the interactions between repurposed drugs sertindole and temoporfin with receptor for advanced glycation endproducts: Therapeutic implications in cancer and metabolic diseases.

CONTEXT: In the pursuit of novel therapeutic possibilities, repurposing existing drugs has gained prominence as an efficient strategy. The findings from our study highlight the potential of repurposed drugs as promising candidates against receptor for advanced glycation endproducts (RAGE) that offer therapeutic implications in cancer, neurodegenerative conditions and metabolic syndromes. Through careful analyses of binding affinities and interaction patterns, we identified a few promising candidates, ultimately focusing on sertindole and temoporfin. These candidates exhibited exceptional binding affinities, efficacy, and specificity within the RAGE binding pocket. Notably, they displayed a pronounced propensity to interact with the active site of RAGE. Our investigation further revealed that sertindole and temoporfin possess desirable pharmacological properties that highlighted them as attractive candidates for targeted drug development. Overall, our integrated computational approach provides a comprehensive understanding of the interactions between repurposed drugs, sertindole and temoporfin and RAGE that pave the way for future experimental validation and drug development endeavors. METHODS: We present an integrated approach utilizing molecular docking and extensive molecular dynamics (MD) simulations to evaluate the potential of FDA-approved drugs, sourced from DrugBank, against RAGE. To gain deeper insights into the binding mechanisms of the elucidated candidate repurposed drugs, sertindole and temoporfin with RAGE, we conducted extensive all-atom MD simulations, spanning 500 nanoseconds (ns). These simulations elucidated the conformational dynamics and stability of the RAGE-sertindole and RAGE-temoporfin complexes.

Computational Deciphering of the Role of S100A8 and S100A9 Proteins and Their Changes in the Structure Assembly Influences Their Interaction with TLR4, RAGE, and CD36.

S100A8 and S100A9 belong to the calcium-binding, damage associated molecular pattern (DAMP) proteins shown to aggravate the pathogenesis of rheumatoid arthritis (RA) through their interaction with the TLR4, RAGE and CD36 receptors. S100A8 and S100A9 proteins tend to exist in monomeric, homo and heterodimeric forms, which have been implicated in the pathogenesis of RA, via interacting with Pattern Recognition receptors (PRRs). The study aims to assess the influence of changes in the structure and biological assembly of S100A8 and S100A9 proteins as well as their interaction with significant receptors in RA through computational methods and surface plasmon resonance (SPR) analysis. Molecular docking analysis revealed that the S100A9 homodimer and S100A8/A9 heterodimer showed higher binding affinity towards the target receptors. Most S100 proteins showed good binding affinity towards TLR4 compared to other receptors. Based on the 50 ns MD simulations, TLR4, RAGE, and CD36 formed stable complexes with the monomeric and dimeric forms of S100A8 and S100A9 proteins. However, SPR analysis showed that the S100A8/A9 heterodimers formed stable complexes and exhibited high binding affinity towards the receptors. SPR data also indicated that TLR4 and its interactions with S100A8/A9 proteins may play a primary role in the pathogenesis of RA, with additional contributions from CD36 and RAGE interactions. Subsequent in vitro and in vivo investigations are warranted to corroborate the involvement of S100A8/A9 and the expression of TLR4, RAGE, and CD36 in the pathophysiology of RA.

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.

Generation, biochemical characterizations and validation of potent nanobodies derived from alpaca specific for human receptor of advanced glycation end product.

A detrimental role of the receptor for the advanced glycation end product (RAGE) has been identified in the immune response, and various pathological conditions and its V and C1 domains in the extracellular region of RAGE are believed to be the main ligand-binding domains. Consequently, specific inhibitors targeting those domains could be of clinical value in fighting against the pathological condition associated with RAGE over-activation. Single-domain antibodies, also called nanobodies (Nbs), are antibody fragments engineered from the heavy-chain only antibodies found in camelids, which offer a range of advantages in therapy. In this study, we report the development and characterization of the V-C1 domain-specific Nbs. Three Nbs (3CNB, 4BNB, and 5ENB) targeting V-C1 domain of human RAGE were isolated from an immunized alpaca using a phage display. All of these Nbs revealed high thermostability. 3CNB, 4BNB, and 5ENB bind to V-C1 domain with a dissociation constant (KD) of 27.25, 39.37, and 47.85 nM, respectively, using Isothermal Titration Calorimetry (ITC). After homodimerization using human IgG1-Fc fusion, their binding affinity improved to 0.55, 0.62, and 0.41 nM, respectively, using Surface Plasmon Resonance (SPR). Flow cytometry showed all the Fc fusions Nbs can bind to human RAGE expressed on the cell surface. Competitive ELISA further confirmed their V-C1-hS100B blocking ability in solution, providing insights into the applicability of Nbs in treating RAGE-associated diseases.

Impact of licochalcone A on the progression of diabetic nephropathy in type 2 diabetes mellitus of C57BL/6 mice.

Diabetic nephropathy (DN) is the most common chronic microvascular complication of diabetes. Therefore, it is of great significance to effectively prevent and treat DN. Licochalcone A (LicA) is a flavonoid found in licorice; previous studies have shown that LicA can reduce blood glucose, blood lipids and improve insulin resistance. There has been no research on whether LicA can prevent and treat DN. In this study, an animal model of type 2 diabetes mellitus (T2DM) mice induced by high fat diet/streptozotocin was established, and the intervention of LicA was applied to investigate the protective effect of LicA on the kidneys of DN mice. After 4 weeks of intervention, LicA could effectively reduce blood glucose and alleviate the phenomenon of weight loss in mice. Meanwhile, the levels of MDA, SOD and GSH-Px in the kidney tissue and serum were recovered to different degrees. Besides, LicA decreased the levels of TC, TG and LDL-C in the kidney tissue and increased the level of HDL-C in the kidney tissue. The 24 h urinary protein, blood urea nitrogen (BUN) and serum creatinine (SCr) levels of mice in the treatment group of LicA were significantly lower than those in the model group. Furthermore, HE staining, PAS staining and Masson staining indicated that LicA improved the pathological damage of kidneys, and the kidney index of mice also decreased. Western blotting results indicated that LicA could significantly down-regulate the protein expression of AGEs/RAGE, TGF-ß1, HIF-1α and GLUT1, and up-regulate the protein expression of Nrf2. It provides a theoretical basis for the further development and utilization of LicA.

Molecular Characteristics of RAGE and Advances in Small-Molecule Inhibitors.

Receptor for advanced glycation end-products (RAGE) is a member of the immunoglobulin superfamily. RAGE binds and mediates cellular responses to a range of DAMPs (damage-associated molecular pattern molecules), such as AGEs, HMGB1, and S100/calgranulins, and as an innate immune sensor, can recognize microbial PAMPs (pathogen-associated molecular pattern molecules), including bacterial LPS, bacterial DNA, and viral and parasitic proteins. RAGE and its ligands stimulate the activations of diverse pathways, such as p38MAPK, ERK1/2, Cdc42/Rac, and JNK, and trigger cascades of diverse signaling events that are involved in a wide spectrum of diseases, including diabetes mellitus, inflammatory, vascular and neurodegenerative diseases, atherothrombosis, and cancer. Thus, the targeted inhibition of RAGE or its ligands is considered an important strategy for the treatment of cancer and chronic inflammatory diseases.

A fragment-based approach to discovery of Receptor for Advanced Glycation End products inhibitors.

The Receptor for Advanced Glycation End products (RAGE) is a pattern recognition receptor that signals for inflammation via the NF-κB pathway. RAGE has been pursued as a potential target to suppress symptoms of diabetes and is of interest in a number of other diseases associated with chronic inflammation, such as inflammatory bowel disease and bronchopulmonary dysplasia. Screening and optimization have previously produced small molecules that inhibit the activity of RAGE in cell-based assays, but efforts to develop a therapeutically viable direct-binding RAGE inhibitor have yet to be successful. Here, we show that a fragment-based approach can be applied to discover fundamentally new types of RAGE inhibitors that specifically target the ligand-binding surface. A series of systematic assays of structural stability, solubility, and crystallization were performed to select constructs of the RAGE ligand-binding domain and optimize conditions for NMR-based screening and co-crystallization of RAGE with hit fragments. An NMR-based screen of a highly curated ~14 000-member fragment library produced 21 fragment leads. Of these, three were selected for elaboration based on structure-activity relationships generated through cycles of structural analysis by X-ray crystallography, structure-guided design principles, and synthetic chemistry. These results, combined with crystal structures of the first linked fragment compounds, demonstrate the applicability of the fragment-based approach to the discovery of RAGE inhibitors.

A model of full-length RAGE in complex with S100B.

The receptor for advanced glycation end products (RAGE) is an immunoglobulin-type multiligand transmembrane protein expressed in numerous cell types, including the central nervous system cells. RAGE interaction with S100B, released during brain tissue damage, leads to RAGE upregulation and initialization of a spiral proinflammatory associated with different neural disorders. Here, we present the structural characterization of the hetero-oligomeric complex of the full-length RAGE with S100B, obtained by a combination of mass spectrometry-based methods and molecular modeling. We predict that RAGE functions as a tightly packed tetramer exposing a positively charged surface formed by V domains for S100B binding. Based on HDX results we demonstrate an allosteric coupling of the distal extracellular V domains and the transmembrane region, indicating a possible mechanism of signal transmission by RAGE across the membrane. Our model provides an insight into RAGE-ligand interactions, providing a basis for the rational design of the therapeutic modifiers of its activity.

Activation of RAGE leads to the release of glutamate from astrocytes and stimulates calcium signal in neurons.

The receptor for advanced glycation end products (RAGE) is a signal receptor first shown to be activated by advanced glycation end products, but also by a variety of signal molecules, including pathological advanced oxidation protein products and ß-amyloid. However, most of the RAGE activators have multiple intracellular targets, making it difficult to unravel the exact pathway of RAGE activation. Here, we show that the cell-impermeable RAGE fragment sequence (60-76) of the V-domain of the receptor is able to activate RAGE present on the plasma membrane of neurons and, preferentially, astrocytes. This leads to the exocytosis of vesicular glutamate transporter vesicles and the release of glutamate from astrocytes, which stimulate NMDA and AMPA/kainate receptors, resulting in calcium signals predominantly in neurons. Thus, we show a specific mechanism of RAGE activation by the RAGE fragment and propose a mechanism by which RAGE activation can contribute to the neuronal-astrocytic communication in physiology and pathology.

Targeting adaptor protein SLP76 of RAGE as a therapeutic approach for lethal sepsis.

Accumulating evidence shows that RAGE has an important function in the pathogenesis of sepsis. However, the mechanisms by which RAGE transduces signals to downstream kinase cascades during septic shock are not clear. Here, we identify SLP76 as a binding partner for the cytosolic tail of RAGE both in vitro and in vivo and demonstrate that SLP76 binds RAGE through its sterile α motif (SAM) to mediate downstream signaling. Genetic deficiency of RAGE or SLP76 reduces AGE-induced phosphorylation of p38 MAPK, ERK1/2 and IKKα/ß, as well as cytokine release. Delivery of the SAM domain into macrophages via the TAT cell-penetrating peptide blocks proinflammatory cytokine production. Furthermore, administration of TAT-SAM attenuates inflammatory cytokine release and tissue damage in mice subjected to cecal ligation and puncture (CLP) and protects these mice from the lethality of sepsis. These findings reveal an important function for SLP76 in RAGE-mediated pro-inflammatory signaling and shed light on the development of SLP76-targeted therapeutics for sepsis.

Molecular investigation of glycated insulin-induced insulin resistance via insulin signaling and AGE-RAGE axis.

Hyperglycemic condition in diabetes promotes glycation of various plasma proteins including insulin. Glycation of insulin has been reported to reduce its biological activity. Reduced biological activity of glycated insulin could be either due to reduced affinity for the insulin receptor and impaired insulin signaling, or it can act as a ligand for the receptor for advanced glycation end products (RAGE) and activates oxidative stress and pro-inflammatory pathways leading to insulin resistance. This study investigates the effect of glycated insulin on both insulin and RAGE signaling. Glycated insulin treatment to Chinese hamster ovary (CHO-IR-GLUT4) cells stably expressing insulin receptor (IR) and glucose transporter fused with a green fluorescent protein (GLUT4-GFP) resulted in the impairment of insulin signaling, as the phosphorylation of IR and AKT significantly reduced, which affected GLUT4 translocation and glucose uptake. Moreover, it also activated RAGE signaling as observed by increased expression of NADPH oxidase accompanied by an increase in reactive oxygen species (ROS). Immunofluorescence study indicated the translocation of NF-κB to the nucleus upon treatment of glycated insulin. This was associated with increased RAGE expression, Caspase 3, and cell death. Downregulation of RAGE with the losartan treatment restored the impaired insulin signaling and glucose uptake. Additionally, in silico study demonstrated that glycated insulin has reduced binding affinity to insulin receptor and increased binding affinity to RAGE. Overall, this study demonstrates the role of glycated insulin in exacerbating insulin resistance by impairing insulin signaling as well as stimulating AGE-RAGE signaling.

RAGE Signaling in Melanoma Tumors.

Despite recent progresses in its treatment, malignant cutaneous melanoma remains a cancer with very poor prognosis. Emerging evidences suggest that the receptor for advance glycation end products (RAGE) plays a key role in melanoma progression through its activation in both cancer and stromal cells. In tumors, RAGE activation is fueled by numerous ligands, S100B and HMGB1 being the most notable, but the role of many other ligands is not well understood and should not be underappreciated. Here, we provide a review of the current role of RAGE in melanoma and conclude that targeting RAGE in melanoma could be an approach to improve the outcomes of melanoma patients.

Circulating Ligands of the Receptor for Advanced Glycation End Products and the Soluble Form of the Receptor Modulate Cardiovascular Cell Apoptosis in Diabetes.

We determined whether plasma concentrations of the receptor for advanced glycation end products (RAGE) and the soluble (s) form of RAGE (sRAGE) in healthy individuals and patients with type 2 diabetes (T2D) modulate vascular remodeling. Healthy individuals and patients with T2D were divided into two age groups: young = <35 years old or middle-aged (36-64 years old) and stratified based on normal glucose tolerance (NGT), impaired (IGT), and T2D. Plasma titers of sRAGE, the RAGE ligands, AGEs, S100B, S100A1, S100A6, and the apoptotic marker Fas ligand Fas(L) were measured by enzyme-linked immunosorbent assay (ELISA). The apoptotic potential of the above RAGE ligands and sRAGE were assessed in cultured adult rat aortic smooth muscle cells (ASMC). In NGT individuals, aging increased the circulating levels of AGEs and S100B and decreased sRAGE, S100A1 and S100A6. Middle-aged patients with T2D presented higher levels of circulating S100B, AGEs and FasL, but lower levels of sRAGE, S100A1 and S100A6 than individuals with NGT or IGT. Treatment of ASMC with either AGEs or S100B at concentrations detected in T2D patients increased markers of inflammation and apoptosis. Responses attenuated by concomitant administration of sRAGE. In middle-aged patients with T2D, lower circulating plasma levels of sRAGE may limit decoy and exogenous trapping of deleterious pro-apoptotic/pro-inflammatory RAGE ligands AGEs and S100B, increasing the risk for diabetic complications.

Blocking the interface region amongst S100A6 and RAGE V domain via S100B protein.

The Ca2+-mediated S100 family protein S100A6 has a crucial task in various intracellular and extracellular activities thereby demonstrating a possible involvement in the advancement and development of malignant tumors. S100A6 has been found to associate with receptor for advanced glycation end products, RAGE, through its extracellular extension. This extension is famously identified as a prominent receptor for many S100 family associates. Additionally, S100A6 binds to S100B protein and forms a heterodimer. Thus, we consider the S100B protein to be a prospective drug molecule to obstruct the interacting regions amongst S100A6 and RAGE V domain. We applied the NMR spectroscopy method to locate the binding area amid the S100A6m (mutant S100A6, cysteine at 3rd position of S100A6 is replaced with serine, C3S) and S100B proteins. The 1H-15N HSQC NMR titrations revealed the probable requisite dynamics of S100A6m and S100B interfaces. Utilizing data from the NMR titrations as input parameters, we ran the HADDOCK program and created a S100A6m-S100B heterodimer complex. The obtained complex was then superimposed with the reported complex of S100A6m-RAGE V domain. This superimposition displayed the possibility of S100B to be a potential antagonist that can block the interface area of the S100A6m and the RAGE V domain. Moreover, an in vitro cancer model using SW480 cells in water-soluble tetrazolium-1 assay (WST-1) showed a noticeable change in the cell proliferation as an effect of these proteins. Our study indicates the possibility to develop a S100B-like competitor that could play a key role in the treatment of S100- and RAGE-mediated human diseases.

Synthetic fragment (60-76) of RAGE improves brain mitochondria function in olfactory bulbectomized mice.

The receptor for advanced glycation end products (RAGE) is considered to contribute to the pathogenesis of Alzheimer's disease (AD), mediating amyloid beta (Aß) accumulation, mitochondrial damage, and neuroinflammation. Previously, we have synthesized small peptides corresponding to the fragments (60-76) (P1) and (60-62) (P2) of the RAGE extracellular domain, and have shown that administration of P1 fragment but not P2 results in restoration of the spatial memory and decreases the brain Aß (1-40) level in olfactory bulbectomized (OBX) mice demonstrating main features of Alzheimer's type neurodegeneration. In the present study, we have investigated the supposed mechanism of the therapeutic efficacy of P1 RAGE fragment and compared it to P2 short fragment. We have found that P1 restored activities of the respiratory chain in the Complexes I and IV in both cortical and hippocampal mitochondria of the OBX mice while P2 had no effect. Besides, fluorescein-labeled analog Flu-P1 bound to Aß (1-40) and Aß (1-42) with high affinity (Kd in the nanomolar range) whereas Flu-P2 revealed low affinity with tenfold higher Kd value for Aß (1-40) and did not bind to Aß (1-42). However, neither of the peptides had a notable impact on inflammation, estimated as mRNA expression of proinflammatory cytokines in the brain tissues of OBX mice. Taken together, our results suggest that direct Aß-P1 interaction is one of the molecular events mediating the protection of the mitochondria in OBX animals from Aß toxic effect. The RAGE fragment P1 would be the soluble decoy for Aßs and serve as a promising therapeutic agent against neurodegeneration accompanied by mitochondrial dysfunction.

Prothrombin is a binding partner of the human receptor of advanced glycation end products.

The receptor for advanced glycation end products (RAGE) plays a key role in mammal physiology and in the etiology and progression of inflammatory and oxidative stress-based diseases. In adults, RAGE expression is normally high only in the lung where the protein concentrates in the basal membrane of alveolar Type I epithelial cells. In diseases, RAGE levels increase in the affected tissues and sustain chronic inflammation. RAGE exists as a membrane glycoprotein with an ectodomain, a transmembrane helix, and a short carboxyl-terminal tail, or as a soluble ectodomain that acts as a decoy receptor (sRAGE). VC1 domain is responsible for binding to the majority of RAGE ligands including advanced glycation end products (AGEs), S100 proteins, and HMGB1. To ascertain whether other ligands exist, we analyzed by MS the material pulled down by VC1 from human plasma. Twenty of 295 identified proteins were selected and associated to coagulation and complement processes and to extracellular matrix. Four of them contained a γ-carboxyl glutamic acid (Gla) domain, a calcium-binding module, and prothrombin (PT) was the most abundant. Using MicroScale thermophoresis, we quantified the interaction of PT with VC1 and sRAGE in the absence or presence of calcium that acted as a competitor. PT devoid of the Gla domain (PT des-Gla) did not bind to sRAGE, providing further evidence that the Gla domain is critical for the interaction. Finally, the presence of VC1 delayed plasma clotting in a dose-dependent manner. We propose that RAGE is involved in modulating blood coagulation presumably in conditions of lung injury.

Unveiling the molecular mechanisms underpinning biorecognition of early-glycated human serum albumin and receptor for advanced glycation end products.

Serum levels of early-glycated albumin are significantly increased in patients with diabetes mellitus and may play a role in worsening inflammatory status and sustaining diabetes-related complications. To investigate possible pathological recognition involving early-glycated albumin and the receptor for advanced glycation end products (RAGE), an early-glycated human serum albumin (HSAgly), with a glycation pattern representative of the glycated HSA form abundant in diabetic patients, and the recombinant human RAGE ectodomain (VC1) were used. Biorecognition between the two interactants was investigated by combining surface plasmon resonance (SPR) analysis and affinity chromatography coupled with mass spectrometry (affinity-MS) for peptide extraction and identification. SPR analysis proved early-glycated albumin could interact with the RAGE ectodomain with a steady-state affinity constant of 6.05 ± 0.96 × 10-7 M. Such interaction was shown to be specific, as confirmed by a displacement assay with chondroitin sulfate, a known RAGE binder. Affinity-MS studies were performed to map the surface area involved in the recognition. These studies highlighted that a region surrounding Lys525 and part of subdomain IA were involved in VC1 recognition. Finally, an in silico analysis highlighted (i) a key role for glycation at Lys525 (the most commonly glycated residue in HSA in diabetic patients) through a triggering mechanism similar to that previously observed for AGEs or advanced lipoxidation end products and (ii) a stabilizing role for subdomain IA. Albeit a moderate affinity for complex formation, the high plasma levels of early-glycated albumin and high percentage of glycation at Lys525 in diabetic patients make this interaction of possible pathological relevance. Graphical abstract.

The Trp triad within the V-domain of the receptor for advanced glycation end products modulates folding, stability and ligand binding.

The receptor for advanced glycation end products (RAGE) recognizes damage-associated molecular patterns (DAMPs) and plays a critical role for the innate immune response and sterile tissue inflammation. RAGE overexpression is associated with diabetic complications, neurodegenerative diseases and certain cancers. Yet, the molecular mechanism of ligand recognition by RAGE is insufficiently understood to rationalize the binding of diverse ligands. The N-terminal V-type Ig-domain of RAGE contains a triad of tryptophan residue; Trp51, Trp61 and Trp72. The role of these three Trp residues for domain folding, stability and binding of the RAGE ligand S100B was investigated through site-directed mutagenesis, UV/VIS, CD and fluorescence spectrometry, protein-protein interaction studies, and X-ray crystallography. The data show that the Trp triad stabilizes the folded V-domain by maintaining a short helix in the structure. Mutation of any Trp residue increases the structural plasticity of the domain. Residues Trp61 and Trp72 are involved in the binding of S100B, yet they are not strictly required for S100B binding. The crystal structure of the RAGE-derived peptide W72 in complex with S100B showed that Trp72 is deeply buried in a hydrophobic depression on the S100B surface. The studies suggest that multiple binding modes between RAGE and S100B exist and point toward a not previously recognized role of the Trp residues for RAGE-ligand binding. The Trp triad of the V-domain appears to be a suitable target for novel RAGE inhibitors, either in the form of monoclonal antibodies targeting this epitope, or small organic molecules.

The receptor for advanced glycation end-products (RAGE) is an important pattern recognition receptor (PRR) for inflammaging.

The receptor for advanced glycation end-products (RAGE) was initially characterized and named for its ability to bind to advanced glycation end-products (AGEs) that form upon the irreversible and non-enzymatic interaction between nucleophiles, such as lysine, and carbonyl compounds, such as reducing sugars. The concentrations of AGEs are known to increase in conditions such as diabetes, as well as during ageing. However, it is now widely accepted that RAGE binds with numerous ligands, many of which can be defined as pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). The interaction between RAGE and its ligands mainly results in a pro-inflammatory response, and can lead to stress events often favouring mitochondrial dysfunction or cellular senescence. Thus, RAGE should be considered as a pattern recognition receptor (PRR), similar to those that regulate innate immunity. Innate immunity itself plays a central role in inflammaging, the chronic low-grade and sterile inflammation that increases with age and is a potentially important contributory factor in ageing. Consequently, and in addition to the age-related accumulation of PAMPs and DAMPs and increases in pro-inflammatory cytokines from senescent cells and damaged cells, PRRs are therefore important in inflammaging. We suggest here that, through its interconnection with immunity, senescence, mitochondrial dysfunction and inflammasome activation, RAGE is a key contributor to inflammaging and that the pro-longevity effects seen upon blocking RAGE, or upon its deletion, are thus the result of reduced inflammaging.