Enhancement of efferocytosis through biased FPR2 signaling attenuates intestinal inflammation.

Efficient clearance of dying cells (efferocytosis) is an evolutionarily conserved process for tissue homeostasis. Genetic enhancement of efferocytosis exhibits therapeutic potential for inflammation resolution and tissue repair. However, pharmacological approaches to enhance efferocytosis remain sparse due to a lack of targets for modulation. Here, we report the identification of columbamine (COL) which enhances macrophage-mediated efferocytosis and attenuates intestinal inflammation in a murine colitis model. COL enhances efferocytosis by promoting LC3-associated phagocytosis (LAP), a non-canonical form of autophagy. Transcriptome analysis and pharmacological characterization revealed that COL is a biased agonist that occupies a part of the ligand binding pocket of formyl peptide receptor 2 (FPR2), a G-protein coupled receptor involved in inflammation regulation. Genetic ablation of the Fpr2 gene or treatment with an FPR2 antagonist abolishes COL-induced efferocytosis, anti-colitis activity and LAP. Taken together, our study identifies FPR2 as a potential target for modulating LC3-associated efferocytosis to alleviate intestinal inflammation and highlights the therapeutic value of COL, a natural and biased agonist of FPR2, in the treatment of inflammatory bowel disease.

Icaritin Derivative IC2 Induces Cytoprotective Autophagy of Breast Cancer Cells via SCD1 Inhibition.

Breast cancer is one of the most prevalent malignancies and the leading cause of cancer-associated mortality in China. Icaritin (ICT), a prenyl flavonoid derived from the Epimedium Genus, has been proven to inhibit the proliferation and stemness of breast cancer cells. Our previous study demonstrated that IC2, a derivative of ICT, could induce breast cancer cell apoptosis by Stearoyl-CoA desaturase 1 (SCD1) inhibition. The present study further investigated the mechanism of the inhibitory effects of IC2 on breast cancer cells in vitro and in vivo. Our results proved that IC2 could stimulate autophagy in breast cancer cells with the activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling and mitogen-activated protein kinase (MAPK) signaling. Combination treatment of the AMPK inhibitor decreased IC2-induced autophagy while it markedly enhanced IC2-induced apoptosis. In common with IC2-induced apoptosis, SCD1 overexpression or the addition of exogenous oleic acid (OA) could also alleviate IC2-induced autophagy. In vivo assays additionally demonstrated that IC2 treatment markedly inhibited tumor growth in a mouse breast cancer xenograft model. Overall, our study was the first to demonstrate that IC2 induced cytoprotective autophagy by SCD1 inhibition in breast cancer cells and that the autophagy inhibitor markedly enhanced the anticancer activity of IC2. Therefore, IC2 was a potential candidate compound in combination therapy for breast cancer.

Structural basis for recognition of N-formyl peptides as pathogen-associated molecular patterns.

The formyl peptide receptor 1 (FPR1) is primarily responsible for detection of short peptides bearing N-formylated methionine (fMet) that are characteristic of protein synthesis in bacteria and mitochondria. As a result, FPR1 is critical to phagocyte migration and activation in bacterial infection, tissue injury and inflammation. How FPR1 distinguishes between formyl peptides and non-formyl peptides remains elusive. Here we report cryo-EM structures of human FPR1-Gi protein complex bound to S. aureus-derived peptide fMet-Ile-Phe-Leu (fMIFL) and E. coli-derived peptide fMet-Leu-Phe (fMLF). Both structures of FPR1 adopt an active conformation and exhibit a binding pocket containing the R2015.38XXXR2055.42 (RGIIR) motif for formyl group interaction and receptor activation. This motif works together with D1063.33 for hydrogen bond formation with the N-formyl group and with fMet, a model supported by MD simulation and functional assays of mutant receptors with key residues for recognition substituted by alanine. The cryo-EM model of agonist-bound FPR1 provides a structural basis for recognition of bacteria-derived chemotactic peptides with potential applications in developing FPR1-targeting agents.

Anti-inflammatory signaling through G protein-coupled receptors.

G protein-coupled receptors (GPCRs) play important roles in human physiology. GPCRs are involved in immunoregulation including regulation of the inflammatory response. Chemotaxis of phagocytes and lymphocytes is mediated to a great extent by the GPCRs for chemoattractants including myriads of chemokines. Accumulation and activation of phagocytes at the site of inflammation contribute to local inflammatory response. A handful of GPCRs have been found to transduce anti-inflammatory signals that promote resolution of inflammation. These GPCRs interact with selected metabolites of arachdonic acid, such as lipoxins, and of omega-3 essential fatty acids, such as resolvins and protectins. Despite mounting evidence for the in vivo functions of these anti-inflammatory and pro-resolving ligands paired with their respective GPCRs, the underlying signaling mechanisms have not been fully delineated. The present review summarizes what we have learned about these GPCRs, their structures and signaling pathways and the prospect of targeting these receptors for novel anti-inflammatory therapies.

Biased allosteric modulation of formyl peptide receptor 2 leads to distinct receptor conformational states for pro- and anti-inflammatory signaling.

BACKGROUND AND PURPOSE: Formyl peptide receptor 2 (FPR2) is a Class A G protein-coupled receptor (GPCR) that interacts with multiple ligands and transduces both proinflammatory and anti-inflammatory signals. These ligands include weak agonists and modulators that are produced during inflammation. The present study investigates how prolonged exposure to FPR2 modulators influence receptor signaling. EXPERIMENTAL APPROACH: Fluorescent biosensors of FPR2 were constructed based on single-molecule fluorescent resonance energy transfer (FRET) and used for measurement of ligand-induced receptor conformational changes. These changes were combined with FPR2-mediated signaling events and used as parameters for the conformational states of FPR2. Ternary complex models were developed to interpret ligand concentration-dependent changes in FPR2 conformational states. KEY RESULTS: Incubation with Ac2-26, an anti-inflammatory ligand of FPR2, decreased FRET intensity at picomolar concentrations. In comparison, WKYMVm (W-pep) and Aß42, both proinflammatory agonists of FPR2, increased FRET intensity. Preincubation with Ac2-26 at 10 pM diminished W-pep-induced Ca2+ flux but potentiated W-pep-stimulated ß-arrestin2 membrane translocation and p38 MAPK phosphorylation. The opposite effects were observed with 10 pM of Aß42. Neither Ac2-26 nor Aß42 competed for W-pep binding at the picomolar concentrations. CONCLUSIONS AND IMPLICATIONS: The results support the presence of two allosteric binding sites on FPR2, each for Ac2-26 and Aß42, with high and low affinities. Sequential binding of the two allosteric ligands at increasing concentrations induce different conformational changes in FPR2, providing a novel mechanism by which biased allosteric modulators alter receptor conformations and generate pro- and anti-inflammatory signals.

Dual modulation of formyl peptide receptor 2 by aspirin-triggered lipoxin contributes to its anti-inflammatory activity.

The eicosanoid lipoxin A4 and aspirin-triggered 15-epi-lipoxin A4 (ATL) are potent anti-inflammatory agents. How their anti-inflammatory effects are mediated by receptors such as the formyl peptide receptor 2 (FPR2/ALX) remains incompletely understood. In the present study, fluorescent biosensors of FPR2/ALX were prepared and ATL-induced conformational changes were recorded. A biphasic dose curve consisting of a descending phase and an ascending phase was observed, with the descending phase corresponding to diminished FPR2 response such as Ca2+ mobilization induced by the potent synthetic agonist WKYMVm. Preincubation of FPR2-expressing cells with 100 pM of ATL also lowered the threshold for WKYMVm to induce ß-arrestin-2 membrane translocation, and inhibited WKYMVm-induced interleukin 8 secretion, suggesting signaling bias favoring anti-inflammatory activities. At 100 pM and above, ATL-induced receptor conformational changes resembling that of the WKYMVm along with a weak but measurable inhibition of forskolin-induced cAMP accumulation. However, no Ca2+ mobilization was induced by ATL until its concentration reached 1 µM. Taken together, these results suggest a dual regulatory mechanism by which ATL exerts anti-inflammatory effects through FPR2/ALX.

A Ganoderma-Derived Compound Exerts Inhibitory Effect Through Formyl Peptide Receptor 2.

Formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) widely expressed in neutrophils and other phagocytes. FPRs play important roles in host defense, inflammation, and the pathogenesis of infectious and inflammatory diseases. Because of these functions, FPRs are potential targets for anti-inflammatory therapies. In order to search for potentially novel anti-inflammatory agents, we examined Ganoderma (Lingzhi), a Chinese medicinal herbs known for its anti-inflammatory effects, and found that compound 18 (C18) derived from Ganoderma cochlear could limit the inflammatory response through FPR-related signaling pathways. Further studies showed that C18 could bind to FPR2 and induce conformation change of the receptor that differed from the conformational change induced by the pan-agonist, WKYMVm. C18 inhibited at the receptor level and blocked WKYMVm signaling through FPR2, resulting in reduced superoxide production and compromised cell chemotaxis. These results identified for the first time that a Ganoderma-derived component with inhibitory effects that acts through a G protein-coupled receptor FPR2. Considering its less than optimal IC50 value, further optimization of C18 would be necessary for future applications.

Structural basis of ligand binding modes at the human formyl peptide receptor 2.

The human formyl peptide receptor 2 (FPR2) plays a crucial role in host defense and inflammation, and has been considered as a drug target for chronic inflammatory diseases. A variety of peptides with different structures and origins have been characterized as FPR2 ligands. However, the ligand-binding modes of FPR2 remain elusive, thereby limiting the development of potential drugs. Here we report the crystal structure of FPR2 bound to the potent peptide agonist WKYMVm at 2.8 Å resolution. The structure adopts an active conformation and exhibits a deep ligand-binding pocket. Combined with mutagenesis, ligand binding and signaling studies, key interactions between the agonist and FPR2 that govern ligand recognition and receptor activation are identified. Furthermore, molecular docking and functional assays reveal key factors that may define binding affinity and agonist potency of formyl peptides. These findings deepen our understanding about ligand recognition and selectivity mechanisms of the formyl peptide receptor family.

4-Aroyl-3-hydroxy-5-phenyl-1H-pyrrol-2(5H)-ones as N-formyl peptide receptor 1 (FPR1) antagonists.

Formyl peptide receptors (FPRs) are expressed on a variety of leukocytes and play important roles in inflammation. Thus, FPR antagonists may represent novel therapeutics for modulating innate immunity and treating inflammatory diseases. Previously, 1H-pyrrol-2(5H)-ones were reported to be potent and competitive FPR1 antagonists. In the present studies, 42 additional 1H-pyrrol-2(5H)-one analogs were evaluated for FPR1 antagonist activity. We identified a number of novel competitive FPR1 antagonists that inhibited N-formylmethionyl-leucyl-phenylalanine (fMLF)-induced intracellular Ca2+ mobilization in FPR1-transfected HL60 cells and effectively competed with WKYMVm-FITC for binding to FPR1 in FPR1-transfected RBL cells. The most active pyrroles inhibited human neutrophil Ca2+ flux, chemotaxis, and adhesion to human epithelial cells, with the most potent being compounds 14 (4-benzoyl-1-hexyl-3-hydroxy-5-(4-hydroxy-3-methoxyphenyl)-2,5-dihydro-1H-pyrrol-2-one) and 17 (4-benzoyl-5-(2,5-dimethoxyphenyl)-3-hydroxy-1-(2-methoxyethyl)-2,5-dihydro-1H-pyrrol-2-one). In addition, these FPR1 antagonists inhibited fMLF-induced phosphorylation of extracellular signal-regulated kinases (ERK1/2) in FPR1-RBL cells, differentiated HL-60 cells, and human neutrophils. Most of the antagonists were specific for FPR1 and did not inhibit WKYMVM/WKYMVm-induced intracellular Ca2+ mobilization in FPR2-HL60 cells, FPR3-HL60 cells, or interleukin 8-induced Ca2+ flux in human neutrophils. Moreover, molecular modeling showed that the active pyrroles had a significantly higher degree of similarity with the FPR1 antagonist pharmacophore template as compared to inactive analogs. Thus, the 4-aroyl-3-hydroxy-5-phenyl-1H-pyrrol-2(5H)-one scaffold represents an important backbone for the development of novel FPR1 antagonists and could provide important clues for understanding the molecular structural requirements of FPR1 antagonists.

The putative signal peptide of glucagon-like peptide-1 receptor is not required for receptor synthesis but promotes receptor expression.

GLP-1R (glucagon-like peptide-1 receptor) mediates the 'incretin effect' and many other anti-diabetic actions of its cognate ligand, GLP-1 (glucagon-like peptide-1). It belongs to the class B family of GPCRs (G protein-coupled receptors) and possesses an N-terminal putative SP (signal peptide). It has been reported that this sequence is required for the synthesis of GLP-1R and is cleaved after receptor synthesis. In the present study, we conducted an in-depth exploration towards the role of the putative SP in GLP-1R synthesis. A mutant GLP-1R without this sequence was expressed in HEK293 cells (human embryonic kidney 293 cells) and displayed normal functionality with respect to ligand binding and activation of adenylate cyclase. Thus the putative SP does not seem to be required for receptor synthesis. Immunoblotting analysis shows that the amount of GLP-1R synthesized in HEK293 cells is low when the putative SP is absent. This indicates that the role of the sequence is to promote the expression of GLP-1R. Furthermore, epitopes tagged at the N-terminal of GLP-1R are detectable by immunofluorescence and immunoblotting in our experiments. In conclusion, the present study points to different roles of SP in GLP-1R expression which broadens our understanding of the functionality of this putative SP of GLP-1R and possibly other Class B GPCRs.

Development of ß-amino-carbonyl compounds as androgen receptor antagonists.

AIM: Androgen receptor (AR) antagonists have proven to be useful in the early control of prostate cancer. The aim of this study was to identify and characterize a novel ß-amino-carbonyl-based androgen receptor antagonist. METHODS: Different isomers of the ß-amino-carbonyl compounds were obtained by chiral separation. The bioactivities of the isomers were evaluated by AR nuclear translocation, mammalian two-hybrid, competitive receptor binding and cell proliferation assays. The expression of genes downstream of AR was analyzed with real-time PCR. The therapeutic effects on tumor growth in vivo were observed in male SCID mice bearing LNCaP xenografts. RESULTS: Compound 21 was previously identified as an AR modulator by the high-throughput screening of a diverse compound library. In the present study, the two isomers of compound 21, termed compounds 21-1 and 21-2, were characterized as partial AR agonists in terms of androgen-induced AR nuclear translocation, prostate-specific antigen expression and cell proliferation. Further structural modifications led to the discovery of a androgen receptor antagonist (compound 6012), which blocked androgen receptor nuclear translocation, androgen-responsive gene expression and androgen-dependent LNCaP cell proliferation. Four stereoisomers of compound 6012 were isolated, and their bioactivities were assessed. The pharmacological effects of 6012, including AR binding, androgen-induced AR translocation, NH2- and COOH-terminal interaction, growth inhibition of LNCaP cells in vitro and LNCaP xenograft growth in nude mice, were mainly restricted to isomer 6012-4 (1R, 3S). CONCLUSION: Compound 6012-4 was determined to be a novel androgen receptor antagonist with prostate cancer inhibitory activities comparable to bicalutamide both in vitro and in vivo.