Discovery of 2,3-Diaminoindole Derivatives as a Novel Class of NOD Antagonists.

NOD1 and NOD2 are members of the pattern recognition receptors involved in the innate immune response. Overactivation of NOD1 is implicated in inflammatory disorders, multiple sclerosis, and cancer cell metastases. NOD1 antagonists would represent valuable pharmacological tools to gain further insight into protein roles, potentially leading to new therapeutic strategies. We herein report the expansion of the chemical space of NOD1 antagonists via a multicomponent synthetic approach affording a novel chemotype, namely, 2,3-diaminoindoles. These efforts resulted in compound 37, endowed with low micromolar affinity toward NOD1. Importantly, a proof-of-evidence of direct binding to NOD1 of Noditinib-1 and derivative 37 is provided here for the first time. Additionally, the combination of computational studies and NMR-based displacement assays enabled the characterization of the binding modality of 37 to NOD1, thus providing key unprecedented knowledge for the design of potent and selective NOD1 antagonists.

Halogenation of common phenolic household and personal care product ingredients enhances their AhR-modulating capacity.

The Aryl Hydrocarbon Receptor (AhR), a ligand-activated transcription factor, orchestrates responses to numerous structurally diverse endogenous and exogenous ligands. In addition to binding various xenobiotics, AhR also recognizes endocrine disruptors, particularly those featuring chlorinated or brominated aromatic structures. There is limited data available on the impact of common household and personal care product ingredients let alone their halogenated transformation products. Herein we bridge this knowledge gap by preparing a library of chlorinated and brominated parabens, bisphenols, UV filters, and nonylphenols. An evaluation of total of 125 compounds for agonistic and antagonistic activity on AhR unveiled a low micromolar agonist, Cl2BPAF with an EC50 of 13 µM. Moreover, our study identified several AhR antagonists, with BrBzP emerging as the most potent with an IC50 of 8.9 µM. To further investigate the functional implications of these compounds, we subjected the most potent agonist and antagonist to a functional assay involving cytokine secretion from peripheral blood mononuclear cells and compared their activity with the commercially available AhR agonist and antagonist. Cl2BPAF exhibited an overall immunosuppressive effect by reducing the secretion of proinflammatory cytokines, including IL-6, IFN-γ, and TNF-α, while BrBzP displayed opposite effects, leading to an increase of those cytokines. Notably, the immunomodulatory effects of Cl2BPAF surpassed those of ITE, a bona fide AhR agonist, while the impact of BrBzP exceeded that of CH223191, a bona fide AhR antagonist. In summary, our study underscores the potential influence of halogenated transformation products on the AhR pathway and, consequently, their role in shaping the immune responses.

In vitro investigation of immunomodulatory activities of selected UV-filters.

Organic UV filters are ubiquitous as they are used in numerous personal care products. Consequently, people constantly come into direct or indirect contact with these chemicals. Albeit studies of the effects of UV filters on human health have been undertaken, their toxicological profiles are not complete. In this work, we investigated the immunomodulatory properties of eight UV filters representing different chemotypes, including benzophenone-1, benzophenone-3, ethylhexyl methoxycinnamate, octyldimethyl-para-aminobenzoic acid, octyl salate, butylmethoxydibenzoylmethane, 3-benzylidenecamphor, and 2,4-di-tert-butyl-6-(5-chlorobenzotriazol-2-yl)phenol. We demonstrated that none of these UV filters were cytotoxic to THP-1 cells at concentrations up to 50 µM. Importantly, our study highlighted the capacity of nontoxic concentrations of avobenzone and 3-benzylidene camphor to increase the secretion of interleukin 8 (IL-8) from both THP-1 cells and THP-1 derived macrophages. Further, they also exhibited a pronounced decrease of IL-6 and IL-10 release from lipopolysaccharide-stimulated peripheral blood mononuclear cells. The observed immune cell alterations suggest that exposure to 3-BC and BMDM could be involved in immune deregulation. Our research thus provided additional insight into UV filter safety profile.

In silico profiling of endocrine-disrupting potential of bisphenol analogues and their halogenated transformation products.

Due to its endocrine-disrupting properties, bisphenol A (BPA) is being phased out from plastics, thermal paper and epoxy resins, and its replacements are being introduced into the market. Bisphenols are released into the environment, where they can undergo halogenation. Unlike BPA, the endocrine-disrupting potential of BPA analogues and their halogenated transformation products has not been extensively studied. The aim of this study was to evaluate the endocrine-disrupting potential of 18 BPA analogues and their halogenated derivatives by calculating affinities for 14 human nuclear receptors utilizing the Endocrine Disruptome and VirtualToxLab™ in silico tools. Our simulations identified AR, ERs, and GR as the most favorable targets of bisphenols and their derivatives. Several BPA analogues displayed a higher predicted potential for endocrine disruption than BPA. Our models highlighted BPZ and BPPH as the most hazardous in terms of predicted endocrine activities. Halogenation, in general, was predicted to increase the binding affinity of bisphenols for AR, ERß, MR, GR, PPARγ, and TRß. Notably, mono- or 2,2'-di-halogenated bisphenols exhibited the highest potential for endocrine disruption. In vitro corroboration of the obtained results should be the next milestone in evaluating the safety of BPA substitutes and their halogenated transformation products.

Lipidation of NOD2 Agonists with Adamantane and Stearoyl Moieties Differentially Regulates Their In Vivo Adjuvant Activity.

NOD2 is an innate immune receptor that constitutes an important target for the development of small molecule immunopotentiators with great potential to be used as vaccine adjuvants. We report here the results of an in vivo study of the adjuvant properties of a desmuramylpeptide NOD2 agonist SG29 and its lipidated analogs featuring an adamantyl moiety or a stearoyl group. These compounds have been synthesized, incorporated into liposomes, and evaluated for their in vivo adjuvant activity. The characterization of liposome formulations of examined compounds revealed that their size increased in comparison to that of empty liposomes. The introduction of a stearoyl or an adamantane lipophilic anchor into the structure of SG29, to produce SG115 and ZSB63, respectively, substantially improved the in vivo adjuvant activity. Of note, the attachment of the stearoyl moiety produced a Th2-biased immune response, while the incorporation of the adamantyl moiety greatly enhanced the production of total IgG but mostly augmented the production of IgG2a antibodies, which indicated a shift toward a Th1 immune response. The identified bona fide capacity of ZSB63 to initiate a cellular immune response thus highlights its untapped potential as an alternative vaccine adjuvant.

Covalently Conjugated NOD2/TLR7 Agonists Are Potent and Versatile Immune Potentiators.

The success of vaccination with subunit vaccines often relies on the careful choice of adjuvants. There is great interest in developing new adjuvants that can elicit a cellular immune response. Here, we address this challenge by taking advantage of the synergistic cross-talk between two pattern recognition receptors: nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) and Toll-like receptor 7 (TLR7). We designed two conjugated NOD2/TLR7 agonists, which showed potent immunostimulatory activities in human primary peripheral blood mononuclear cells and murine bone-marrow-derived dendritic cells. One of these, 4, also generated a strong antigen-specific immune response in vivo, with a Th1-polarized profile. Importantly, our study shows that novel NOD2/TLR7 agonists elicit sophisticated and fine-tuned immune responses that are inaccessible to individual NOD2 and TLR7 agonists.

Environmental contamination status with common ingredients of household and personal care products exhibiting endocrine-disrupting potential.

The continuous use of household and personal care products (HPCPs) produces an immense amount of chemicals, such as parabens, bisphenols, benzophenones and alkylphenol ethoxylates, which are of great concern due to their well-known endocrine-disrupting properties. These chemicals easily enter the environment through man-made activities, thus contaminating the biota, including soil, water, plants and animals. Thus, on top of the direct exposure on account of their presence in HPCPs, humans are also susceptible to secondary indirect exposure attributed to the ubiquitous environmental contamination. The aim of this review was therefore to examine the sources and occurrence of these noteworthy contaminants (i.e. parabens, bisphenols, benzophenones, alkylphenol ethoxylates), to summarise the available research on their environmental presence and to highlight their bioaccumulation potential. The most notable environmental contaminants appear to be MeP and PrP among parabens, BPA and BPS among bisphenols, BP-3 among benzophenones and NP among alkylphenols. Their maximum detected concentrations in the environment are mostly in the range of ng/L, while in human tissues, their maximum concentrations achieved µg/L due to bioaccumulation, with BP-3 and nonylphenol showing the highest potential to bioaccumulate. Finally, of another great concern is the fact that even the unapproved parabens and benzophenones have been detected in the environment.

Nucleotide-Binding Oligomerization Domain 1/Toll-Like Receptor 4 Co-Engagement Promotes Non-Specific Immune Response Against K562 Cancer Cells.

Nucleotide-binding oligomerization domain 1 (NOD1) receptor and Toll-like receptor 4 (TLR4) belong to the family of pattern recognition receptors. Interactions between these receptors profoundly shape the innate immune responses. We previously demonstrated that co-stimulation of peripheral blood mononuclear cells (PBMCs) with D-glutamyl-meso-diaminopimelic acid (iE-DAP)-based NOD1 agonists and lipopolysaccharide (LPS), a TLR4 agonist, synergistically increased the cytokine production. Herein, we postulate that stimulation of NOD1 alone or a combined stimulation of NOD1 and TLR4 could also strengthen PBMC-mediated cytotoxicity against cancer cells. Initially, an in-house library of iE-DAP analogs was screened for NOD1 agonist activity to establish their potency in HEK-Blue NOD1 cells. Next, we showed that our most potent NOD1 agonist SZZ-38 markedly enhanced the LPS-induced cytokine secretion from PBMCs, in addition to PBMC- and natural killer (NK) cell-mediated killing of K562 cancer cells. Activation marker analysis revealed that the frequencies of CD69+, CD107a+, and IFN-γ+ NK cells are significantly upregulated following NOD1/TLR4 co-stimulation. Of note, SZZ-38 also enhanced the IFN-γ-induced PBMC cytotoxicity. Overall, our findings provide further insight into how co-engagement of two pathways boosts the non-specific immune response and attest to the importance of such interplay between NOD1 and TLR4.

Discovery of Desmuramylpeptide NOD2 Agonists with Single-Digit Nanomolar Potency.

The innate immune receptor nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) represents an important target for the development of structurally defined small molecule immunomodulatory compounds that have great potential to be used either as vaccine adjuvants or as general immunostimulatory agents. We report here the investigation of the structure-activity relationship of a series of novel desmuramylpeptide NOD2 agonists. Extensive exploration of chemical space culminated in the discovery of a lipophilic adamantane-moiety-featuring compound 40, the first single-digit nanomolar and the most potent NOD2 agonist in its structural class to date. Moreover, 40 acted synergistically with lipopolysaccharide and interferon-γ to induce the production of cytokines in human peripheral blood mononuclear cells and enhance their nonspecific cytotoxic activity against K562 cancer cells. These findings provide initial insight into its immunostimulatory potential, especially when used in combination with other immunopotentiators.

Novel Scaffolds for Modulation of NOD2 Identified by Pharmacophore-Based Virtual Screening.

Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an innate immune pattern recognition receptor responsible for the recognition of bacterial peptidoglycan fragments. Given its central role in the formation of innate and adaptive immune responses, NOD2 represents a valuable target for modulation with agonists and antagonists. A major challenge in the discovery of novel small-molecule NOD2 modulators is the lack of a co-crystallized complex with a ligand, which has limited previous progress to ligand-based design approaches and high-throughput screening campaigns. To that end, a hybrid docking and pharmacophore modeling approach was used to identify key interactions between NOD2 ligands and residues in the putative ligand-binding site. Following docking of previously reported NOD2 ligands to a homology model of human NOD2, a structure-based pharmacophore model was created and used to virtually screen a library of commercially available compounds. Two compounds, 1 and 3, identified as hits by the pharmacophore model, exhibited NOD2 antagonist activity and are the first small-molecule NOD2 modulators identified by virtual screening to date. The newly identified NOD2 antagonist scaffolds represent valuable starting points for further optimization.

Design and synthesis of novel 3-triazolyl-1-thiogalactosides as galectin-1, -3 and -8 inhibitors.

Galectins are galactoside-binding proteins that play a role in various pathophysiological conditions, making them attractive targets in drug discovery. We have designed and synthesised a focused library of aromatic 3-triazolyl-1-thiogalactosides targeting their core site for binding of galactose and a subsite on its non-reducing side. Evaluation of their binding affinities for galectin-1, -3, and -8N identified acetamide-based compound 36 as a suitable compound for further affinity enhancement by adding groups at the reducing side of the galactose. Synthesis of its dichlorothiophenyl analogue 59 and the thiodigalactoside analogue 62 yielded promising pan-galectin inhibitors.

Halogenated ingredients of household and personal care products as emerging endocrine disruptors.

The everyday use of household and personal care products (HPCPs) generates an enormous amount of chemicals, of which several groups warrant additional attention, including: (i) parabens, which are widely used as preservatives; (ii) bisphenols, which are used in the manufacture of plastics; (iii) UV filters, which are essential components of many cosmetic products; and (iv) alkylphenol ethoxylates, which are used extensively as non-ionic surfactants. These chemicals are released continuously into the environment, thus contaminating soil, water, plants and animals. Wastewater treatment and water disinfection procedures can convert these chemicals into halogenated transformation products, which end up in the environment and pose a potential threat to humans and wildlife. Indeed, while certain parent HPCP ingredients have been confirmed as endocrine disruptors, less is known about the endocrine activities of their halogenated derivatives. The aim of this review is first to examine the sources and occurrence of halogenated transformation products in the environment, and second to compare their endocrine-disrupting properties to those of their parent compounds (i.e., parabens, bisphenols, UV filters, alkylphenol ethoxylates). Albeit previous reports have focused individually on selected classes of such substances, none have considered the problem of their halogenated transformation products. This review therefore summarizes the available research on these halogenated compounds, highlights the potential exposure pathways, and underlines the existing knowledge gaps within their toxicological profiles.

Selective Galectin-8N Ligands: The Design and Synthesis of Phthalazinone-d-Galactals.

Ligand selectivity among the highly conserved galectins has been an ever-challenging objective. For galectin-8, a protein prevalent in both pathology and tissue distribution, we report phthalazinone-galactals that show excellent selectivity for the galectin-8N-terminal domain. A dissection of structure-activity relationships of the phthalazinone and an extensive molecular dynamics meta-analysis accompany the discovery of the selective galectin-8N ligands presented here. These selective compounds will facilitate the study of galectin-8 biology and may have pharmaceutical relevance in the wide range of galectin-8 associated pathologies.

Selective Monovalent Galectin-8 Ligands Based on 3-Lactoylgalactoside.

Galectin-8 has gained attention as a potential new pharmacological target for the treatment of various diseases, including cancer, inflammation, and disorders associated with bone mass reduction. To that end, new molecular probes are needed in order to better understand its role and its functions. Herein we aimed to improve the affinity and target selectivity of a recently published galectin-8 ligand, 3-O-[1-carboxyethyl]-ß-d-galactopyranoside, by introducing modifications at positions 1 and 3 of the galactose. Affinity data measured by fluorescence polarization show that the most potent compound reached a KD of 12 µM. Furthermore, reasonable selectivity versus other galectins was achieved, making the highlighted compound a promising lead for the development of new selective and potent ligands for galectin-8 as molecular probes to examine the protein's role in cell-based and in vivo studies.

Structure-Guided Design of d-Galactal Derivatives with High Affinity and Selectivity for the Galectin-8 N-Terminal Domain.

Galectin-8 is a carbohydrate-binding protein that plays a crucial role in tumor progression and metastasis, antibacterial autophagy, modulation of the immune system, and bone remodeling. The design, synthesis, and protein affinity evaluation of a set of C-3 substituted benzimidazole and quinoline d-galactal derivatives identified a d-galactal-benzimidazole hybrid as a selective ligand for the galectin-8 N-terminal domain (galectin-8N), with a K d of 48 µM and 15-fold selectivity over galectin-3 and even better selectivity over the other mammalian galectins. X-ray structural analysis of galectin-8N in complex with one benzimidazole- and one quinoline-galactal derivative at 1.52 and 2.1 Å together with molecular dynamics simulations and quantum mechanical calculations of galectin-8N in complex with the benzimidazole derivative revealed orbital overlap between a NH LUMO of Arg45 with electron rich HOMOs of the olefin and O4 of the d-galactal. Such overlap is hypothesized to contribute to the high affinity of the d-galactal-derived ligands for galectin-8N. A (3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay evaluation of the d-galactal-benzimidazole hybrid and an analogous galactoside derivative on a panel of cell lines with MTS assay showed no effect on cell viability up to 100 µM concentration. A subsequent functional assay using the MDA-MB-231 cell line demonstrated that the d-galactal-benzimidazole hybrid and the analogous galactoside derivative reduced the secretion of the proinflammatory cytokines interleukin-6 (IL-6) and IL-8 in a dose-dependent manner. Therefore, these compounds represent potential probes for galectin-8N pharmacology investigations and possibly promising leads for the design and synthesis of potent and selective galectin-8 inhibitors as potential antitumor and anti-inflammatory agents.

Benzimidazole-galactosides bind selectively to the Galectin-8 N-Terminal domain: Structure-based design and optimisation.

We have obtained the X-ray crystal structure of the galectin-8 N-terminal domain (galectin-8N) with a previously reported quinoline-galactoside ligand at a resolution of 1.6 Å. Based on this X-ray structure, a collection of galactosides derivatised at O3 with triazole, benzimidazole, benzothiazole, and benzoxazole moieties were designed and synthesised. This led to the discovery of a 3-O-(N-methylbenzimidazolylmethyl)-galactoside with a Kd of 1.8 µM for galectin-8N, the most potent selective synthetic galectin-8N ligand to date. Molecular dynamics simulations showed that benzimidazole-galactoside derivatives bind the non-conserved amino acid Gln47, accounting for the higher selectivity for galectin-8N. Galectin-8 is a carbohydrate-binding protein that plays a key role in pathological lymphangiogenesis, modulation of the immune system, and autophagy. Thus, the benzimidazole-derivatised galactosides represent promising compounds for studies of the pathological implications of galectin-8, as well as a starting point for the development of anti-tumour and anti-inflammatory therapeutics targeting galectin-8.

Structural Fine-Tuning of Desmuramylpeptide NOD2 Agonists Defines Their In Vivo Adjuvant Activity.

We report on the design, synthesis, and biological evaluation of a series of nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) desmuramylpeptide agonists with improved in vitro and in vivo adjuvant properties. We identified two promising compounds: 68, a potent nanomolar in vitro NOD2 agonist, and the more lipophilic 75, which shows superior adjuvant activity in vivo. Both compounds had immunostimulatory effects on peripheral blood mononuclear cells at the protein and transcriptional levels, and augmented dendritic-cell-mediated activation of T cells, while 75 additionally enhanced the cytotoxic activity of peripheral blood mononuclear cells against malignant cells. The C18 lipophilic tail of 75 is identified as a pivotal structural element that confers in vivo adjuvant activity in conjunction with a liposomal delivery system. Accordingly, liposome-encapsulated 75 showed promising adjuvant activity in mice, surpassing that of muramyl dipeptide, while achieving a more balanced Th1/Th2 immune response, thus highlighting its potential as a vaccine adjuvant.

Rational Vaccine Design in Times of Emerging Diseases: The Critical Choices of Immunological Correlates of Protection, Vaccine Antigen and Immunomodulation.

Vaccines are the most effective medical intervention due to their continual success in preventing infections and improving mortality worldwide. Early vaccines were developed empirically however, rational design of vaccines can allow us to optimise their efficacy, by tailoring the immune response. Establishing the immune correlates of protection greatly informs the rational design of vaccines. This facilitates the selection of the best vaccine antigens and the most appropriate vaccine adjuvant to generate optimal memory immune T cell and B cell responses. This review outlines the range of vaccine types that are currently authorised and those under development. We outline the optimal immunological correlates of protection that can be targeted. Finally we review approaches to rational antigen selection and rational vaccine adjuvant design. Harnessing current knowledge on protective immune responses in combination with critical vaccine components is imperative to the prevention of future life-threatening diseases.

Does targeting Arg98 of FimH lead to high affinity antagonists?

Bacterial resistance has become an important challenge in the treatment of urinary tract infections. The underlying resistance mechanisms can most likely be circumvented with an antiadhesive approach, antagonizing the lectin FimH located at the tip of fimbriae of uropathogenic E. coli. Here we report on a novel series of FimH antagonists based on the 1-(α-d-mannopyranosyl)-4-phenyl-1,2,3-triazole scaffold, designed to incorporate carboxylic acid or ester functions to interact with FimH Arg98. The most potent representative of the series, ester 11e, displayed a Kd value of 7.6 nM for the lectin domain of FimH with a general conclusion that all esters outperform carboxylates in terms of affinity. Surprisingly, all compounds from this new series exhibited improved binding affinities also for the R98A mutant, indicating another possible interaction contributing to binding. Our study on 1-(α-d-mannopyranosyl)-4-phenyl-1,2,3-triazole-based FimH antagonists offers proof that targeting Arg98 side chain by a "chemical common sense", i.e. by introduction of the acidic moiety to form ionic bond with Arg98 is most likely unsuitable approach to boost FimH antagonists' potency.

GSK669, a NOD2 receptor antagonist, inhibits thrombosis and oxidative stress via targeting platelet GPVI.

BACKGROUND AND PURPOSE: Previously, we discovered that the activation of nucleotide-binding oligomerization domain 2 (NOD2) enhances platelet activation. We here investigated the antiplatelet and antithrombotic potential of GSK669, a NOD2 antagonist. EXPERIMENTAL APPROACH: Effects of GSK669 on platelet functions, reactive oxygen species (ROS) and proinflammatory cytokine generation were detected. NOD2-/- platelets were used to confirm GSK669 target. The interaction between GSK669 and glycoprotein VI (GPVI) was detected using surface plasmon resonance (SPR) spectroscopy. GPVI downstream signaling was examined by Western blot. The antithrombotic and antioxidative effects were investigated using mouse mesenteric arteriole thrombosis model and pulmonary embolism model. KEY RESULTS: GSK669 significantly inhibits platelet proinflammatory cytokine release induced by muramyl dipeptide, platelet aggregation, ATP release, and ROS generation induced by collagen and collagen related peptide (CRP). Platelet spreading and clot retraction are also inhibited. GSK669 also decreases collagen-induced phosphorylation of Src, Syk, PLCγ2, and Akt. The antiplatelet effect of GSK669 is NOD2-independent and mediated by GPVI antagonism. Consistent with its antiplatelet activity as a GPVI antagonist, GSK669 inhibits platelet adhesion on collagen in flow condition. Notably, GSK669 inhibits mouse mesenteric arteriole thrombosis similarly to aspirin without bleeding. The antithrombotic effect of GSK669 is further confirmed in the pulmonary embolism model; decreased malonaldehyde (MDA) and increased superoxide dismutase (SOD) levels in mouse plasma reveal a significant antioxidant effect of GSK669. CONCLUSION AND IMPLICATIONS: Beyond its anti-inflammatory effect as a NOD2 antagonist, GSK669 is also an efficient and safe antiplatelet agent combined with antioxidant effect by targeting GPVI. An antiplatelet agent bearing antioxidative and anti-inflammatory activities without bleeding risk may have therapeutic advantage over current antiplatelet drugs for atherothrombosis.