A Selective Neutraligand for CXCL12/SDF-1α With Beneficial Regulatory Functions in MRL/Lpr Lupus Prone Mice.

Dysregulation of CXCL12/SDF-1-CXCR4/CD184 signaling is associated with inflammatory diseases and notably with systemic lupus erythematosus. Issued from the lead molecule chalcone-4, the first neutraligand of the CXCL12 chemokine, LIT-927 was recently described as a potent analogue with improved solubility and stability. We aimed to investigate the capacity of LIT-927 to correct immune alterations in lupus-prone MRL/lpr mice and to explore the mechanism of action implemented by this small molecule in this model. We found that in contrast to AMD3100, an antagonist of CXCR4 and agonist of CXCR7, LIT-927 reduces the excessive number of several B/T lymphocyte subsets occurring in the blood of sick MRL/lpr mice (including CD3+/CD4-/CD8-/B220+ double negative T cells). In vitro, LIT-927 downregulated the overexpression of several activation markers on splenic MRL/lpr lymphocytes. It exerted effects on the CXCR4 pathway in MRL/lpr CD4+ T spleen cells. The results underline the importance of the CXCL12/CXCR4 axis in lupus pathophysiology. They indicate that neutralizing CXCL12 by the neutraligand LIT-927 can attenuate hyperactive lymphocytes in lupus. This mode of intervention might represent a novel strategy to control a common pathophysiological mechanism occurring in inflammatory diseases.

Nile Red-Based GPCR Ligands as Ultrasensitive Probes of the Local Lipid Microenvironment of the Receptor.

The local lipid microenvironment of transmembrane receptors is an essential factor in G protein coupled receptor (GPCR) signaling. However, tools are currently missing for studying endogenously expressed GPCRs in primary cells and tissues. Here, we introduce fluorescent environment-sensitive GPCR ligands for probing the microenvironment of the receptor in living cells using fluorescence microscopy under no-wash conditions. We designed and synthesized antagonist ligands of the oxytocin receptor (OTR) by conjugating a high-affinity nonpeptidic OTR ligand PF-3274167 to the environment-sensitive fluorescent dye Nile Red. The length of the polar PEG spacer between the pharmacophore and the fluorophore was adjusted to lower the nonspecific interactions of the probe while preserving a strong fluorogenic response. We demonstrated that the new probes embed into the lipid bilayer in the vicinity of the receptor and convey information about the local polarity and the lipid order via the wavelength-shifting emission of the Nile Red fluorophore.

Neutralization of CXCL12 attenuates established pulmonary hypertension in rats.

AIMS: The progressive accumulation of cells in pulmonary vascular walls is a key pathological feature of pulmonary arterial hypertension (PAH) that results in narrowing of the vessel lumen, but treatments targeting this mechanism are lacking. The C-X-C motif chemokine 12 (CXCL12) appears to be crucial in these processes. We investigated the activity of two CXCL12 neutraligands on experimental pulmonary hypertension (PH), using two complementary animal models. METHODS AND RESULTS: Male Wistar rats were injected with monocrotaline (MCT) or were subjected to SU5416 followed by 3-week hypoxia to induce severe PH. After PH establishment, assessed by pulsed-wave Doppler echocardiography, MCT-injected or SU5416 plus chronic hypoxia (SuHx) rats were randomized to receive CXCL12 neutraligands chalcone 4 or LIT-927 (100 mg/kg/day), the C-X-C motif chemokine receptor 4 (CXCR4) antagonist AMD3100 (5 mg/kg/day), or vehicle, for 2 or 3 weeks, respectively. At the end of these treatment periods, echocardiographic and haemodynamic measurements were performed and tissue samples were collected for protein expression and histological analysis. Daily treatment of MCT-injected or SuHx rats with established PH with chalcone 4 or LIT-927 partially reversed established PH, reducing total pulmonary vascular resistance, and remodelling of pulmonary arterioles. Consistent with these observations, we found that neutralization of CXCL12 attenuates right ventricular hypertrophy, pulmonary vascular remodelling, and decreases pulmonary artery smooth muscle cell (PA-SMC) proliferation in lungs of MCT-injected rats and SuHx rats. Importantly, CXCL12 neutralization with either chalcone 4 or LIT-927 inhibited the migration of PA-SMCs and pericytes in vitro with a better efficacy than AMD3100. Finally, we found that CXCL12 neutralization decreases vascular pericyte coverage and macrophage infiltration in lungs of both MCT-injected and SuHx rats. CONCLUSION: We report here a greater beneficial effect of CXCL12 neutralization vs. the conventional CXCR4 blockade with AMD3100 in the MCT and SuHx rat models of severe PH, supporting a role for CXCL12 in the progression of vascular complications in PH and opening to new therapeutic options.

LIT-001, the First Nonpeptide Oxytocin Receptor Agonist that Improves Social Interaction in a Mouse Model of Autism.

Oxytocin (OT) and its receptor (OT-R) are implicated in the etiology of autism spectrum disorders (ASD), and OT-R is a potential target for therapeutic intervention. Very few nonpeptide oxytocin agonists have currently been reported. Their molecular and in vivo pharmacology remain to be clarified, and none of them has been shown to be efficient in improving social interaction in animal models relevant to ASD. In an attempt to rationalize the design of centrally active nonpeptide full agonists, we studied in a systematic way the structural determinants of the affinity and efficacy of representative ligands of the V1a and V2 vasopressin receptor subtypes (V1a-R and V2-R) and of the oxytocin receptor. Our results confirm the subtlety of the structure-affinity and structure-efficacy relationships around vasopressin/oxytocin receptor ligands and lead however to the first nonpeptide OT receptor agonist active in a mouse model of ASD after peripheral ip administration.

Discovery of a Locally and Orally Active CXCL12 Neutraligand (LIT-927) with Anti-inflammatory Effect in a Murine Model of Allergic Airway Hypereosinophilia.

We previously reported Chalcone-4 (1) that binds the chemokine CXCL12, not its cognate receptors CXCR4 or CXCR7, and neutralizes its biological activity. However, this neutraligand suffers from limitations such as poor chemical stability, solubility, and oral activity. Herein, we report on the discovery of pyrimidinone 57 (LIT-927), a novel neutraligand of CXCL12 which displays a higher solubility than 1 and is no longer a Michael acceptor. While both 1 and 57 reduce eosinophil recruitment in a murine model of allergic airway hypereosinophilia, 57 is the only one to display inhibitory activity following oral administration. Thereby, we here describe 57 as the first orally active CXCL12 neutraligand with anti-inflammatory properties. Combined with a high binding selectivity for CXCL12 over other chemokines, 57 represents a powerful pharmacological tool to investigate CXCL12 physiology in vivo and to explore the activity of chemokine neutralization in inflammatory and related diseases.

Structure-activity relationship studies toward the discovery of selective apelin receptor agonists.

Apelin is the endogenous ligand for the previously orphaned G protein-coupled receptor APJ. Apelin and its receptor are widely distributed in the brain, heart, and vasculature, and are emerging as an important regulator of body fluid homeostasis and cardiovascular functions. To further progress in the pharmacology and the physiological role of the apelin receptor, the development of small, bioavailable agonists and antagonists of the apelin receptor, is crucial. In this context, E339-3D6 (1) was described as the first nonpeptidic apelin receptor agonist. We show here that 1 is actually a mixture of polymethylated species, and we describe an alternative and versatile solid-phase approach that allows access to highly pure 27, the major component of 1. This approach was also applied to prepare a series of derivatives in order to identify the crucial structural determinants required for the ligand to maintain its affinity for the apelin receptor as well as its capacity to promote apelin receptor signaling and internalization. The study of the structure-activity relationships led to the identification of ligands 19, 21, and 38, which display an increased affinity compared to that of 27. The latter and 19 behave as full agonists with regard to cAMP production and apelin receptor internalization, whereas 21 is a biased agonist toward cAMP production. Interestingly, the three ligands display a much higher stability in mouse plasma (T1/2 > 10 h) than the endogenous apelin-17 peptide 2 (T1/2 < 4 min).

Homodimerization of the death-associated protein kinase catalytic domain: development of a new small molecule fluorescent reporter.

BACKGROUND: Death-Associated Protein Kinase (DAPK) is a member of the Ca2+/calmodulin regulated serine/threonine protein kinases. Its biological function has been associated with induced cell death, and in vivo use of selective small molecule inhibitors of DAPK catalytic activity has demonstrated that it is a potential therapeutic target for treatment of brain injuries and neurodegenerative diseases. METHODOLOGY/PRINCIPAL FINDINGS: In the in vitro study presented here, we describe the homodimerization of DAPK catalytic domain and the crucial role played by its basic loop structure that is part of the molecular fingerprint of death protein kinases. Nanoelectrospray ionization mass spectrometry of DAPK catalytic domain and a basic loop mutant DAPK protein performed under a variety of conditions was used to detect the monomer-dimer interchange. A chemical biological approach was used to find a fluorescent probe that allowed us to follow the oligomerization state of the protein in solution. CONCLUSIONS/SIGNIFICANCE: The use of this combined biophysical and chemical biology approach facilitated the elucidation of a monomer-dimer equilibrium in which the basic loop plays a key role, as well as an apparent allosteric conformational change reported by the fluorescent probe that is independent of the basic loop structure.

On the enantioselective olefin epoxidation by doubly bridged biphenyl azepine derivatives--mixed tropos/atropos chiral biaryls.

Diastereomeric doubly bridged biphenyl azepines, atropos at 20 degrees C and tropos at 80 degrees C, are precursors to effective iminium organocatalysts that are employed in the enantioselective epoxidation of prochiral olefins (up to 85% ee).

Biphasic enantioselective olefin epoxidation using Tropos dibenzoazepinium catalysts.

Several novel chiral iminium TRISPHAT [tris(tetrachlorobenzenediolato)phosphate(V)] salts combining a diphenylazepinium core, chiral exocyclic appendages, and lipophilic counterions have been prepared and tested in biphasic enantioselective olefin epoxidation conditions. Interestingly, the iminium salts derived from commercially available (S)- or (R)-1,2,2-trimethylpropylamine can display efficiency similar to those made from L-acetonamine. Variable-temperature NMR spectroscopy (VT-NMR) and circular dichroism (CD) experiments were performed in search of a correlation between good enantioselectivity in the products and high diastereomeric control of the biphenyl axial chirality of the catalysts.