Antibody-mediated neutralization of galectin-3 as a strategy for the treatment of systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune, inflammatory and fibrotic disease with limited treatment options. Developing new therapies is therefore crucial to address patient needs. To this end, we focused on galectin-3 (Gal-3), a lectin known to be associated with several pathological processes seen in SSc. Using RNA sequencing of whole-blood samples in a cross-sectional cohort of 249 patients with SSc, Gal-3 and its interactants defined a strong transcriptomic fingerprint associated with disease severity, pulmonary and cardiac malfunctions, neutrophilia and lymphopenia. We developed new Gal-3 neutralizing monoclonal antibodies (mAb), which were then evaluated in a mouse model of hypochlorous acid (HOCl)-induced SSc. We show that two of these antibodies, D11 and E07, reduced pathological skin thickening, lung and skin collagen deposition, pulmonary macrophage content, and plasma interleukin-5 and -6 levels. Moreover, E07 changed the transcriptional profiles of HOCl-treated mice, resulting in a gene expression pattern that resembled that of control mice. Similarly, pathological pathways engaged in patients with SSc were counteracted by E07 in mice. Collectively, these findings demonstrate the translational potential of Gal-3 blockade as a therapeutic option for SSc.

Targeting galectin-3 in inflammatory and fibrotic diseases.

Galectin (Gal)-3 is a ß-galactoside-binding lectin emerging as a key player in cardiac, hepatic, renal, and pulmonary fibrosis and inflammation, respiratory infections caused by COVID-19, and neuroinflammatory disorders. Here, we review recent information highlighting Gal-3 as a relevant therapeutic target in these specific disease conditions. While a causal link was difficult to establish until now, we discuss how recent strategic breakthroughs allowed us to identify new-generation Gal-3 inhibitors with improved potency, selectivity, and bioavailability, and report their usefulness as valuable tools for proof-of-concept studies in various preclinical models of the aforementioned diseases, with emphasis on those actually in clinical stages. We also address critical views and suggestions intended to expand the therapeutic opportunities provided by this complex target.

Design, synthesis and biological evaluation of conformationnally-restricted analogues of E7010 as inhibitors of tubulin assembly (ITA) and vascular disrupting agents (VDA).

Vascular-disrupting agents (VDA) specifically target established neovasculature which results in vascular shutdown. This therapeutic strategy could improve the outcome of pathologies involving aberrant angiogenesis. Although several classes of VDA exist, inhibitors of tubulin assembly (ITA) represent the main category. A series of 21 conformationnally-restricted analogues of E7010, a known ITA-VDA, were designed and synthesised as novel inhibitors of tubulin assembly (ITA) and vascular-disrupting agents (VDA). Among them, indole 4j exhibited good potency against HUVEC and HIG-82 cell lines, as well as a good ability to inhibit tubulin assembly. Furthermore, indole 4j reduced HUVEC migration in a dose-dependent manner, indicating a vascular disrupting activity comparable to that of the gold standard, Combretastatin A4 (CA4).

Gremlin-1 and BMP-4 Overexpressed in Osteoarthritis Drive an Osteochondral-Remodeling Program in Osteoblasts and Hypertrophic Chondrocytes.

Osteoarthritis (OA) is a whole joint disease characterized by an important remodeling of the osteochondral junction. It includes cartilage mineralization due to chondrocyte hypertrophic differentiation and bone sclerosis. Here, we investigated whether gremlin-1 (Grem-1) and its BMP partners could be involved in the remodeling events of the osteochondral junction in OA. We found that Grem-1, BMP-2, and BMP-4 immunostaining was detected in chondrocytes from the deep layer of cartilage and in subchondral bone of knee OA patients, and was positively correlated with cartilage damage. ELISA assays showed that bone released more Grem-1 and BMP-4 than cartilage, which released more BMP-2. In vitro experiments evidenced that compression stimulated the expression and the release of Grem-1 and BMP-4 by osteoblasts. Grem-1 was also overexpressed during the prehypertrophic to hypertrophic differentiation of murine articular chondrocytes. Recombinant Grem-1 stimulated Mmp-3 and Mmp-13 expression in murine chondrocytes and osteoblasts, whereas recombinant BMP-4 stimulated the expression of genes associated with angiogenesis (Angptl4 and osteoclastogenesis (Rankl and Ccl2). In conclusion, Grem-1 and BMP-4, whose expression at the osteochondral junction increased with OA progression, may favor the pathological remodeling of the osteochondral junction by inducing a catabolic and tissue remodeling program in hypertrophic chondrocytes and osteoblasts.

Neuromedin B promotes chondrocyte differentiation of mesenchymal stromal cells via calcineurin and calcium signaling.

BACKGROUND: Articular cartilage is a complex tissue with poor healing capacities. Current approaches for cartilage repair based on mesenchymal stromal cells (MSCs) are often disappointing because of the lack of relevant differentiation factors that could drive MSC differentiation towards a stable mature chondrocyte phenotype. RESULTS: We used a large-scale transcriptomic approach to identify genes that are modulated at early stages of chondrogenic differentiation using the reference cartilage micropellet model. We identified several modulated genes and selected neuromedin B (NMB) as one of the early and transiently modulated genes. We found that the timely regulated increase of NMB was specific for chondrogenesis and not observed during osteogenesis or adipogenesis. Furthermore, NMB expression levels correlated with the differentiation capacity of MSCs and its inhibition resulted in impaired chondrogenic differentiation indicating that NMB is required for chondrogenesis. We further showed that NMB activated the calcineurin activity through a Ca2+-dependent signaling pathway. CONCLUSION: NMB is a newly described chondroinductive bioactive factor that upregulates the key chondrogenic transcription factor Sox9 through the modulation of Ca2+ signaling pathway and calcineurin activity.

Pyrroline-5-Carboxylate Reductase 1 Directs the Cartilage Protective and Regenerative Potential of Murphy Roths Large Mouse Mesenchymal Stem Cells.

Murphy Roths Large (MRL) mice possess outstanding capacity to regenerate several tissues. In the present study, we investigated whether this regenerative potential could be associated with the intrinsic particularities possessed by their mesenchymal stem cells (MSCs). We demonstrated that MSCs derived from MRL mice (MRL MSCs) display a superior chondrogenic potential than do C57BL/6 MSC (BL6 MSCs). This higher chondrogenic potential of MRL MSCs was associated with a higher expression level of pyrroline-5-carboxylate reductase 1 (PYCR1), an enzyme that catalyzes the biosynthesis of proline, in MRL MSCs compared with BL6 MSCs. The knockdown of PYCR1 in MRL MSCs, using a specific small interfering RNA (siRNA), abolishes their chondrogenic potential. Moreover, we showed that PYCR1 silencing in MRL MSCs induced a metabolic switch from glycolysis to oxidative phosphorylation. In two in vitro chondrocyte models that reproduce the main features of osteoarthritis (OA) chondrocytes including a downregulation of chondrocyte markers, a significant decrease of PYCR1 was observed. A downregulation of chondrocyte markers was also observed by silencing PYCR1 in freshly isolated healthy chondrocytes. Regarding MSC chondroprotective properties on chondrocytes with OA features, we showed that MSCs silenced for PYCR1 failed to protect chondrocytes from a reduced expression of anabolic markers, while MSCs overexpressing PYCR1 exhibited an increased chondroprotective potential. Finally, using the ear punch model, we demonstrated that MRL MSCs induced a regenerative response in non-regenerating BL6 mice, while BL6 and MRL MSCs deficient for PYCR1 did not. In conclusion, our results provide evidence that MRL mouse regenerative potential is, in part, attributed to its MSCs that exhibit higher PYCR1-dependent glycolytic potential, differentiation capacities, chondroprotective abilities, and regenerative potential than BL6 MSCs.

IFN-α: A key therapeutic target for multiple autoimmune rheumatic diseases.

Interferon (IFN)-α has emerged as a major therapeutic target for several autoimmune rheumatic diseases. In this review, we focus on clinical and preclinical advances in anti-IFN-α treatments in systemic lupus erythematosus (SLE), primary Sjögren syndrome (pSS), systemic sclerosis (SSc), and dermatomyositis (DM), for which a high medical need persists. Promising achievements were obtained following direct IFN-α neutralization, targeting its production through the cytosolic nucleic acid sensor pathways or by blocking its downstream effects through the type I IFN receptor. We further focus on molecular profiling and data integration approaches as crucial steps to select patients most likely to benefit from anti-IFN-α therapies within a precision medicine approach.

S95021, a novel selective and pan-neutralizing anti interferon alpha (IFN-α) monoclonal antibody as a candidate treatment for selected autoimmune rheumatic diseases.

Increased interferon-α (IFN-α) production is a critical component in the pathophysiology of systemic lupus erythematosus (SLE) and other rheumatic autoimmune diseases. Herein, we report the characterization of S95021, a fully human IgG1 anti-IFN-α monoclonal antibody (mAb) as a novel therapeutic candidate for targeted patient populations. S95021 was expressed in CHOZN GS-/- cells, purified by chromatography and characterized by using electrophoresis, size exclusion chromatography and liquid chromatography-mass spectrometry. High purity S95021 was obtained as a monomeric entity comprising different charge variants mainly due to N-glycosylation. Surface plasmon resonance kinetics experiments showed strong association rates with all IFN-α subtypes and estimated KDs below picomolar values. Pan-IFN-α-binding properties were confirmed by immunoprecipitation assays and neutralization capacity with reporter HEK-Blue IFN-α/ß cells. S95021 was IFN-α-selective and exhibited superior potency and broader neutralization profile when compared with the benchmark anti-IFN-α mAbs rontalizumab and sifalimumab. STAT-1 phosphorylation and the type I IFN gene signature induced in human peripheral blood mononuclear cells by recombinant IFN-α subtypes or plasmas from selected autoimmune patients were efficiently reduced by S95021 in a dose-dependent manner. Together, our results show that S95021 is a new potent, selective and pan IFN-α-neutralizing mAb. It is currently further evaluated as a valid therapeutic candidate in selected autoimmune diseases in which the IFN-α pro-inflammatory pathway is dysregulated.

Establishment of a collection of human pluripotent stem cell lines (iPSC) from mesenchymal stem cells (MSC) from three healthy elderly donors.

The study of molecular mechanism driving osteoarticular diseases like osteoarthritis or osteoporosis is impaired by the low accessibility to mesenchymal stem cells (MSC) from healthy donors (HD) for differential multi-omics analysis. Advances in cell reprogramming have, however, provided both a new source of human cells for laboratory research and a strategy to erase epigenetic marks involved in cell identity and the development of diseases. To unravel the pathological signatures on the MSC at the origin of cellular drifts during the formation of bone and cartilage, we previously developed iPSC from MSC of osteoarthritis donors. Here we present the derivation of three iPSCs from healthy age matched donors to model the disease and further identify (epi)genomic signatures of the pathology.

Discovery of GLPG1972/S201086, a Potent, Selective, and Orally Bioavailable ADAMTS-5 Inhibitor for the Treatment of Osteoarthritis.

There are currently no approved disease-modifying osteoarthritis (OA) drugs (DMOADs). The aggrecanase ADAMTS-5 is key in the degradation of human aggrecan (AGC), a component of cartilage. Therefore, ADAMTS-5 is a promising target for the identification of DMOADs. We describe the discovery of GLPG1972/S201086, a potent and selective ADAMTS-5 inhibitor obtained by optimization of a promising hydantoin series following an HTS. Biochemical activity against rat and human ADAMTS-5 was assessed via a fluorescence-based assay. ADAMTS-5 inhibitory activity was confirmed with human aggrecan using an AGC ELISA. The most promising compounds were selected based on reduction of glycosaminoglycan release after interleukin-1 stimulation in mouse cartilage explants and led to the discovery of GLPG1972/S201086. The anticatabolic activity was confirmed in mouse cartilage explants (IC50 < 1.5 µM). The cocrystal structure of GLPG1972/S201086 with human recombinant ADAMTS-5 is discussed. GLPG1972/S201086 has been investigated in a phase 2 clinical study in patients with knee OA (NCT03595618).

Applications of transcriptomics in support of drug development for osteoarthritis.

Objective: Understanding the heterogeneity and pathophysiology of osteoarthritis (OA) is critical to support the development of tailored disease-modifying treatments. To this aim, transcriptomics tools are highly relevant to delineate dysregulated molecular pathways and identify new therapeutic targets. Methods: We review the methodology and outcomes of transcriptomics studies conducted in OA, based on a comprehensive literature search of the PubMed and Google Scholar databases using the terms "osteoarthritis", "OA", "knee OA", "hip OA", "genes", "RNA-seq", "microarray", "transcriptomic" and "PCR" as key words. Beyond target-focused RT-qPCR, more comprehensive techniques include microarrays, RNA sequencing (RNA-seq) and single cell RNA-seq analyses. Results: The standardization of those methods to ensure the quality of both RNA extraction and sequencing is critical to get meaningful insights. Transcriptomics studies have been conducted in various tissues involved in the pathogenesis of OA, including articular cartilage, subchondral bone and synovium, as well as in the blood of patients. Molecular pathways dysregulated in OA relate to cartilage degradation, matrix and bone remodeling, neurogenic pain, inflammation, apoptosis and angiogenesis. This knowledge has direct application to patient stratification and further, to the identification of candidate therapeutic targets and biomarkers intended to monitor OA progression. Conclusion: In light of its high-throughput capabilities and ability to provide comprehensive information on major biological processes, transcriptomics represents a powerful method to support the development of new disease-modifying drugs in OA.

Access to Galectin-3 Inhibitors from Chemoenzymatic Synthons.

Chemoenzymatic strategies are useful for providing both regio- and stereoselective access to bioactive oligosaccharides. We show herein that a glycosynthase mutant of a Thermus thermophilus α-glycosidase can react with unnatural glycosides such as 6-azido-6-deoxy-d-glucose/glucosamine to lead to ß-d-galactopyranosyl-(1→3)-d-glucopyranoside or ß-d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-d-glucopyranoside derivatives bearing a unique azide function. Taking advantage of the orthogonality between the azide and the hydroxyl functional groups, the former was next selectively reacted to give rise to a library of galectin-3 inhibitors. Combining enzyme substrate promiscuity and bioorthogonality thus appears as a powerful strategy to rapidly access to sugar-based ligands.

Generation of human pluripotent stem cell lines (iPSCs) from mesenchymal stem cells (MSCs) from three elderly patients with osteoarthritis.

Mesenchymal stem cells (MSCs) are a unique population of adult stem cells that can differentiate into many cell types. As such, MSCs represent an interesting source of stem cells for use in the clinical treatment of a variety of disorders involving tissue regeneration. It is therefore crucial to investigate further, whether MSCs from patients with bone or cartilage diseases are able to provide iPSCs lines with efficient differentiation ability into MSC derivatives. For this purpose, we derived 3 stable iPSC lines from the MSCs of 3 elderly patients with osteoarthritis (OA) able to re-differentiate into MSC to make bone, cartilage and adipose tissue.

Structure-Activity Relationship of Azaindole-Based Glucokinase Activators.

7-Azaindole has been identified as a novel bidentate anchor point for allosteric glucokinase activators. A systematic investigation around three principal parts of the new small molecule glucokinase activators led to a robust SAR in agreement with structural data that also helped to assess the conformational flexibility of the allosteric activation site. The increase in glucose uptake resulting from glucokinase activation in hepatocytes in vitro translated into the efficient lowering of glucose levels in vivo with the best compounds.

Bearing arms against osteoarthritis and sarcopenia: when cartilage and skeletal muscle find common interest in talking together.

Osteoarthritis, a disease characterized by cartilage degradation, abnormal subchondral bone remodelling and some grade of inflammation, and sarcopenia, a condition of pathological muscle weakness associated with altered muscle mass, strength, and function, are prevalent disorders in elderly people. There is increasing evidence that decline in lower limb muscle strength is associated with knee or hip osteoarthritis in a context of pain, altered joint stability, maladapted postures and defective neuromuscular communication. At the cellular and molecular levels, chondrocytes and myoblasts share common pathological targets and pathways, and the close anatomical location of both cell types suggest a possibility of paracrine communication. In this review, we examine the relationship between osteoarthritis and sarcopenia in the musculoskeletal field, and discuss the potential advantage of concomitant therapies, or how each disorder may benefit from treatment of the other.

S 50131 and S 51434, two novel small molecule glucokinase activators, lack chronic efficacy despite potent acute antihyperglycaemic activity in diabetic mice.

BACKGROUND AND PURPOSE: Small molecule glucokinase activators (GKAs) have been associated with potent antidiabetic efficacy and hepatic steatosis in rodents. This study reports the discovery of S 50131 and S 51434, two novel GKAs with an original scaffold and an atypical pharmacological profile. EXPERIMENTAL APPROACH: Activity of the compounds was assessed in vitro by measuring activation of recombinant glucokinase, stimulation of glycogen synthesis in rat hepatocytes and increased insulin secretion from rat pancreatic islets of Langerhans. Efficacy and safety in vivo were evaluated after oral administration in db/db mice by measuring glycaemia, HbA1c and dyslipidaemia-associated events. KEY RESULTS: S 50131 and S 51434 activated GK and stimulated glycogen synthesis in hepatocytes and insulin secretion from pancreatic islets. Unexpectedly, while both compounds effectively lowered glycaemia after acute oral administration, they did not decrease HbA1c after a 4-week treatment in db/db mice. This lack of antidiabetic efficacy was associated with increased plasma free fatty acids (FFAs), contrasting with the effect of GKA50 and N00236460, two GKAs with sustained HbA1c lowering activity but neutral regarding plasma FFAs. S 50131, but not S 51434, also induced hepatic steatosis, as did GKA50 and N00236460. However, a shorter, 4-day treatment resulted in increased hepatic triglycerides without changing the plasma FFA levels, demonstrating dynamic alterations in the lipid profile over time. CONCLUSIONS AND IMPLICATIONS: In addition to confirming the occurrence of dyslipidaemia with GKAs, these findings provide new insights into understanding how such compounds may sustain or lose efficacy over time.

Small molecule glucokinase activators disturb lipid homeostasis and induce fatty liver in rodents: a warning for therapeutic applications in humans.

BACKGROUND AND PURPOSE: Small-molecule glucokinase activators (GKAs) are currently being investigated as therapeutic options for the treatment of type 2 diabetes (T2D). Because liver overexpression of glucokinase is thought to be associated with altered lipid profiles, this study aimed at assessing the potential lipogenic risks linked to oral GKA administration. EXPERIMENTAL APPROACH: Nine GKA candidates were qualified for their ability to activate recombinant glucokinase and to stimulate glycogen synthesis in rat hepatocytes and insulin secretion in rat INS-1E cells. In vivo activity was monitored by plasma glucose and HbA1c measurements after oral administration in rodents. Risk-associated effects were assessed by measuring hepatic and plasma triglycerides and free fatty acids, as well as plasma aminotransferases, and alkaline phosphatase. KEY RESULTS: GKAs, while efficiently decreasing glycaemia in acute conditions and HbA1c levels after chronic administration in hyperglycemic db/db mice, were potent inducers of hepatic steatosis. This adverse outcome appeared as soon as 4 days after daily oral administration at pharmacological doses and was not transient. GKA treatment similarly increased hepatic triglycerides in diabetic and normoglycaemic rats, together with a pattern of metabolic phenotypes including different combinations of increased plasma triglycerides, free fatty acids, alanine and aspartyl aminotransferases, and alkaline phosphatase. GKAs belonging to three distinct structural families induced hepatic steatosis in db/db mice, arguing in favour of a target-mediated, rather than a chemical class-mediated, effect. CONCLUSION AND IMPLICATIONS: Given the risks associated with fatty liver disease in the general population and furthermore in patients with T2D, these findings represent a serious warning for the use of GKAs in humans. LINKED ARTICLE: This article is commented on by Rees and Gloyn, pp. 335-338 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.02201.x.

Metabolic imbalance of the insulin-like growth factor-I axis in Zucker diabetic fatty rats.

In healthy conditions, insulin-like growth factor-I (IGF-I) acts in a coordinated fashion with insulin to lower glycemia, mainly by increasing insulin sensitivity in peripheral tissues. The aim of this study was to explore the relationship between glucose homeostasis and the endocrine IGF-I axis in Zucker diabetic fatty (ZDF) rats. The plasma levels of glucose, insulin, growth hormone, free IGF-I, total IGF-I (associated to insulin-like growth factor binding proteins plus free), and corticosterone were measured in 13-week-old ZDF rats and in age-matched controls under fasting and postprandial conditions. The plasma IGF-I binding capacity was measured by radioligand binding. In ZDF rats, fasting total and free IGF-I levels were reduced by 22% and 92%, respectively, compared with controls. Postprandial free IGF-I was reduced by 35%, whereas total IGF-I was unaffected. The plasma IGF-I binding capacity in ZDF rats was reduced by 24% after fasting and by 13% under postprandial conditions. A clear correlation between free IGF-I and insulin was observed in postprandial controls but not in ZDF rats. A principal component analysis clearly separated ZDF and control rats into 2 main components under both fasting and postprandial conditions. The first component was determined equally by total IGF-I, bound IGF-I, the free to total IGF-I ratio, and the IGF-I binding capacity. The second component was determined mostly by glucose and insulin. Our results show a marked alteration of the plasma IGF-I levels and of the capacity of plasma to bind IGF-I, and a disturbed relationship between IGF-I and postprandial insulinemia in a rat model of type 2 diabetes mellitus.

Recent progress toward biomarker identification in osteoarthritis.

Osteoarthritis (OA), the most common and disabling form of arthritic disease, is characterized by a slow and progressive degeneration of articular cartilage. Its etiology is multifactorial and includes genetic predisposition, obesity and aging. In addition to the cartilage itself, OA also involves the surrounding tissues, including the synovium and the subchondral bone. This clinical heterogeneity complicates the identification of biomarkers that are crucial for prompt pharmacological intervention at the early stages of the disease and for monitoring treatment efficacy with higher sensitivity than existing imaging methods. In this review, we highlight the difficulties associated with OA diagnosis and discuss the most recent research efforts and successes for the identification of reliable OA biomarkers.

Proteomics: addressing the challenges of osteoarthritis.

Osteoarthritis (OA) is the most frequent rheumatic disease and is the leading cause of disability in developed countries. Its poorly understood pathophysiology limits the discovery of targets for pharmacological intervention and there are few effective medical treatments beyond pain control and surgery. Proteomic technologies may help identifying new targets of OA as well as diagnostic or therapeutic biomarkers, which has stimulated interest in this field. In this review, we discuss the most recent findings arising from the use of proteomics for the identification of OA biomarkers in synovial fluid and serum, and for the discovery of possible therapeutic targets in cartilage or by using chondrocyte culture systems.