Barbara Zarzycka

Blocking CD40-TRAF6 signaling is a therapeutic target in obesity-associated insulin resistance.

Significance Inflammation is a critical contributor to the pathogenesis of metabolic disorders associated with obesity. A group of molecules crucial in regulating the immune system are costimulatory molecules, including CD40. Our current study shows that CD40 acts as a double-edged sword in the metabolic syndrome through the initiation of differential signaling cascades. The CD40-TNF receptor-associated factor (TRAF) 2/3/5 signaling pathway protects against metabolic dysfunction and inflammation associated with obesity; conversely, the CD40-TRAF6 pathway contributes to the detrimental consequences of obesity. In the present study, we therefore designed, validated, and used a small-molecule inhibitor that blocks CD40-TRAF6 interactions. The improvement of insulin resistance by this specific CD40-TRAF6 inhibitor could represent a therapeutic breakthrough in the field of immunometabolism. The immune system plays an instrumental role in obesity and insulin resistance. Here, we unravel the role of the costimulatory molecule CD40 and its signaling intermediates, TNF receptor-associated factors (TRAFs), in diet-induced obesity (DIO). Although not exhibiting increased weight gain, male CD40−/− mice in DIO displayed worsened insulin resistance, compared with wild-type mice. This worsening was associated with excessive inflammation of adipose tissue (AT), characterized by increased accumulation of CD8+ T cells and M1 macrophages, and enhanced hepatosteatosis. Mice with deficient CD40-TRAF2/3/5 signaling in MHCII+ cells exhibited a similar phenotype in DIO as CD40−/− mice. In contrast, mice with deficient CD40-TRAF6 signaling in MHCII+ cells displayed no insulin resistance and showed a reduction in both AT inflammation and hepatosteatosis in DIO. To prove the therapeutic potential of inhibition of CD40-TRAF6 in obesity, DIO mice were treated with a small-molecule inhibitor that we designed to specifically block CD40-TRAF6 interactions; this compound improved insulin sensitivity, reduced AT inflammation, and decreased hepatosteatosis. Our study reveals that the CD40-TRAF2/3/5 signaling pathway in MHCII+ cells protects against AT inflammation and metabolic complications associated with obesity whereas CD40-TRAF6 interactions in MHCII+ cells aggravate these complications. Inhibition of CD40-TRAF6 signaling by our compound may provide a therapeutic option in obesity-associated insulin resistance.

Comparative analysis of pharmacophore screening tools.

The pharmacophore concept is of central importance in computer-aided drug design (CADD) mainly because of its successful application in medicinal chemistry and, in particular, high-throughput virtual screening (HTVS). The simplicity of the pharmacophore definition enables the complexity of molecular interactions between ligand and receptor to be reduced to a handful set of features. With many pharmacophore screening softwares available, it is of the utmost interest to explore the behavior of these tools when applied to different biological systems. In this work, we present a comparative analysis of eight pharmacophore screening algorithms (Catalyst, Unity, LigandScout, Phase, Pharao, MOE, Pharmer, and POT) for their use in typical HTVS campaigns against four different biological targets by using default settings. The results herein presented show how the performance of each pharmacophore screening tool might be specifically related to factors such as the characteristics of the binding pocket, the use of specific pharmacophore features, and the use of these techniques in specific steps/contexts of the drug discovery pipeline. Algorithms with rmsd-based scoring functions are able to predict more compound poses correctly as overlay-based scoring functions. However, the ratio of correctly predicted compound poses versus incorrectly predicted poses is better for overlay-based scoring functions that also ensure better performances in compound library enrichments. While the ensemble of these observations can be used to choose the most appropriate class of algorithm for specific virtual screening projects, we remarked that pharmacophore algorithms are often equally good, and in this respect, we also analyzed how pharmacophore algorithms can be combined together in order to increase the success of hit compound identification. This study provides a valuable benchmark set for further developments in the field of pharmacophore search algorithms, e.g., by using pose predictions and compound library enrichment criteria.

Blocking CD40-TRAF6 interactions by small-molecule inhibitor 6860766 ameliorates the complications of diet-induced obesity in mice

Background:Immune processes contribute to the development of obesity and its complications, such as insulin resistance, type 2 diabetes mellitus and cardiovascular disease. Approaches that target the inflammatory response are promising therapeutic strategies for obesity. In this context, we recently demonstrated that the interaction between the costimulatory protein CD40 and its downstream adaptor protein tumor necrosis factor receptor-associated factor 6 (TRAF6) promotes adipose tissue inflammation, insulin resistance and hepatic steatosis in mice in the course of diet-induced obesity (DIO).Methods:Here we evaluated the effects of a small-molecule inhibitor (SMI) of the CD40-TRAF6 interaction, SMI 6860766, on the development of obesity and its complications in mice that were subjected to DIO.Results:Treatment with SMI 6860766 did not result in differences in weight gain, but improved glucose tolerance. Moreover, SMI 6860766 treatment reduced the amount of CD45+ leucocytes in the epididymal adipose tissue by 69%. Especially, the number of adipose tissue CD4+ and CD8+ T cells, as well as macrophages, was significantly decreased.Conclusions:Our results indicate that small-molecule-mediated inhibition of the CD40-TRAF6 interaction is a promising therapeutic strategy for the treatment of metabolic complications of obesity by improving glucose tolerance, by reducing the accumulation of immune cells to the adipose tissue and by skewing of the immune response towards a more anti-inflammatory profile.

Discovery of Small Molecule CD40–TRAF6 Inhibitors

The CD154-CD40 receptor complex plays a pivotal role in several inflammatory pathways. Attempts to inhibit the formation of this complex have resulted in systemic side effects. Downstream inhibition of the CD40 signaling pathway therefore seems a better way to ameliorate inflammatory disease. To relay a signal, the CD40 receptor recruits adapter proteins called tumor necrosis factor receptor-associated factors (TRAFs). CD40-TRAF6 interactions are known to play an essential role in several inflammatory diseases. We used in silico, in vitro, and in vivo experiments to identify and characterize compounds that block CD40-TRAF6 interactions. We present in detail our drug docking and optimization pipeline and show how we used it to find lead compounds that reduce inflammation in models of peritonitis and sepsis. These compounds appear to be good leads for drug development, given the observed absence of side effects and their demonstrated efficacy for peritonitis and sepsis in mouse models.

iPPI-DB: an online database of modulators of protein–protein interactions

In order to boost the identification of low-molecular-weight drugs on protein–protein interactions (PPI), it is essential to properly collect and annotate experimental data about successful examples. This provides the scientific community with the necessary information to derive trends about privileged physicochemical properties and chemotypes that maximize the likelihood of promoting a given chemical probe to the most advanced stages of development. To this end we have developed iPPI-DB (freely accessible at http://www.ippidb.cdithem.fr), a database that contains the structure, some physicochemical characteristics, the pharmacological data and the profile of the PPI targets of several hundreds modulators of protein–protein interactions. iPPI-DB is accessible through a web application and can be queried according to two general approaches: using physicochemical/pharmacological criteria; or by chemical similarity to a user-defined structure input. In both cases the results are displayed as a sortable and exportable datasheet with links to external databases such as Uniprot, PubMed. Furthermore each compound in the table has a link to an individual ID card that contains its physicochemical and pharmacological profile derived from iPPI-DB data. This includes information about its binding data, ligand and lipophilic efficiencies, location in the PPI chemical space, and importantly similarity with known drugs, and links to external databases like PubChem, and ChEMBL.

Stabilization of protein-protein interaction complexes through small molecules.

Most of the small molecules that have been identified thus far to modulate protein-protein interactions (PPIs) are inhibitors. Another promising way to interfere with PPI-associated biological processes is to promote PPI stabilization. Even though PPI stabilizers are still scarce, stabilization of PPIs by small molecules is gaining momentum and offers new pharmacological options. Therefore, we have performed a literature survey of PPI stabilization using small molecules. From this, we propose a classification of PPI stabilizers based on their binding mode and the architecture of the complex to facilitate the structure-based design of stabilizers.

Targeting CD40-Induced TRAF6 Signaling in Macrophages Reduces Atherosclerosis

Background Disrupting the costimulatory CD40-CD40L dyad reduces atherosclerosis, but can result in immune suppression. The authors recently identified small molecule inhibitors that block the interaction between CD40 and tumor necrosis factor receptor-associated factor (TRAF) 6 (TRAF-STOPs), while leaving CD40-TRAF2/3/5 interactions intact, thereby preserving CD40-mediated immunity. Objectives This study evaluates the potential of TRAF-STOP treatment in atherosclerosis. Methods The effects of TRAF-STOPs on atherosclerosis were investigated in apolipoprotein E deficient (Apoe−/−) mice. Recombinant high-density lipoprotein (rHDL) nanoparticles were used to target TRAF-STOPs to macrophages. Results TRAF-STOP treatment of young Apoe−/− mice reduced atherosclerosis by reducing CD40 and integrin expression in classical monocytes, thereby hampering monocyte recruitment. When Apoe−/− mice with established atherosclerosis were treated with TRAF-STOPs, plaque progression was halted, and plaques contained an increase in collagen, developed small necrotic cores, and contained only a few immune cells. TRAF-STOP treatment did not impair “classical” immune pathways of CD40, including T-cell proliferation and costimulation, Ig isotype switching, or germinal center formation, but reduced CD40 and β2-integrin expression in inflammatory monocytes. In vitro testing and transcriptional profiling showed that TRAF-STOPs are effective in reducing macrophage migration and activation, which could be attributed to reduced phosphorylation of signaling intermediates of the canonical NF-κB pathway. To target TRAF-STOPs specifically to macrophages, TRAF-STOP 6877002 was incorporated into rHDL nanoparticles. Six weeks of rHDL-6877002 treatment attenuated the initiation of atherosclerosis in Apoe−/− mice. Conclusions TRAF-STOPs can overcome the current limitations of long-term CD40 inhibition in atherosclerosis and have the potential to become a future therapeutic for atherosclerosis.

Targeting the adaptive immune system: new strategies in the treatment of atherosclerosis.

Atherosclerosis is a lipid-driven chronic inflammatory disease of the arterial wall. Current treatment of atherosclerosis is focused on limiting its risk factors, such as hyperlipidemia or hypertension. However, treatments that target the inflammatory nature of atherosclerosis are still under development. Discovery of novel targets involved in the inflammation of the arterial wall creates opportunities to design new therapeutics that successfully modulate atherosclerosis. Here, we review drug targets that have proven to play pivotal roles in the adaptive immune system in atherosclerosis, and we discuss their potential as novel therapeutics.

CD40-TRAF6 pathway as a therapeutic target in obesity-associated metabolic dysfunction

Objective: Inflammation contributes to the pathogenesis of obesity-associated metabolic dysregulation and co-stimulatory molecules, including CD40, are important mediators of this process. This study aimed to reveal the role of CD40 and its signaling intermediates, TNF-Receptor-Associated-Factors (TRAFs), in diet-induced obesity (DIO). Methods: The DIO model was performed in CD40-deficient or -sufficient mice as well as in mice with deficient CD40-TRAF2/3/5- or CD40-TRAF6-signaling in MHCII+ cells. Several metabolic parameters were measured and subcutaneous and gonadal adipose tissues (AT) and liver were isolated. FACS analysis of the stromal vascular fraction from AT as well as qPCR-analysis and histochemistry of the isolated tissues were performed. Virtual Ligand Screening, TRAF6 C-domain expression, purification and binding analyses were also performed to construct a CD40-TRAF6 signalling inhibitor that was used as therapy in DIO mice. Results: CD40ko mice were characterized by worsened insulin resistance linked to excessive AT inflammation, increased accumulation of CD8+ T-cells and M1 macrophages in AT, and enhanced hepatosteatosis. In contrast, mice with deficient CD40-TRAF6 signaling in MHCII+ cells displayed no insulin resistance and showed a reduction in both AT inflammation and hepatosteatosis in DIO, whereas the mice with deficient CD40-TRAF2/3/5 signaling excibited a phenotype similar to the CD40ko mice. Treatment of DIO mice with a small-compound that blocks CD40-TRAF6 interactions displayed improved insulin sensitivity, reduced AT inflammation and decreased hepatosteatosis. Conclusions: The CD40-TRAF6 pathway contributes whereas the CD40-TRAF2/3/5 pathway protects from obesity-associated metabolic dysfunction. Improvement of metabolic dysregulation by a newly-developed CD40-TRAF6-pathway inhibitor may represent a therapeutic innovation in the field of immunometabolism.