Discovery and SAR of cinnolines/quinolines as liver X receptor (LXR) agonists with binding selectivity for LXRβ

A series of cinnolines/quinolines was prepared and it was found that 4-phenyl-cinnoline/quinolines with either a 2′,3′ or 2′,5′-disubstituted benzyloxy moiety or the 1-Me-7-indole methoxy moiety on the meta position of the 4-phenyl ring showed good binding selectivity for LXRβ over LXRα. The LXRβ binding selective modulators displayed good activity for inducing ABCA1 gene expression in J774 macrophage cell line and poor efficacy in the LXRα Gal4 functional assay. 26, 37 and 41 were examined for their ability to induce SREBP-1c gene expression in Huh-7 liver cell line and they were weak partial agonists.     

Discovery, synthesis and SAR of azinyl- and azolylbenzamides antagonists of the P2X₇ receptor

The discovery, of a series of 2-Cl-5-heteroaryl-benzamide antagonists of the P2X(7) receptor via parallel medicinal chemistry is described. Initial analogs suffered from poor metabolic stability and low Vd(ss). Multi parametric optimization led to identification of pyrazole 39 as a viable lead with excellent potency and oral bioavailability. Further attempts to improve the low Vd(ss) of 39 via introduction of amines led to analogs 40 and 41 which maintained the favorable pharmacology profile of 39 and improved Vd(ss) after iv dosing. But these analogs suffered from poor oral absorption, probably driven by poor permeability.     

Discovery of olodaterol,a novel inhaled β2-adrenoceptor agonist with a 24 h bronchodilatory efficacy

Compound 4p was identified from a series of 6-hydroxy-4H-benzo[1,4]oxazin-3-ones as potent agonist of the human β₂-adrenoceptor with a high β₁/β₂-selectivity. A complete reversal of acetylcholine-induced bronchoconstriction which lasted over the whole study period of 5 h was demonstrated for 4p in a guinea pig in vivo model without any signs of cardiovascular effects up to 10-fold above the first dose reaching 100% bronchoprotection. The enantiomerically pure (R)-form of 4p exerted a bronchodilatory efficacy over 24 h in dogs and guinea pigs in the absence of systemic pharmacodynamic effects. Formoterol which was tested as comparator in the same in vivo models of acetylcholine-induced bronchoconstriction did not retain efficacy after 24 h. In summary, the preclinical profile of compound (R)-4p (olodaterol, also known as BI 1744 CL) suggests a potential for once-daily dosing in man accompanied with an improved safety profile.     

Structure–activity relationship of bile alcohols as human farnesoid X receptor agonist

FXR (farnesoid X receptor) is a bile acid-activated nuclear receptor that regulates not only the biosynthesis and enterohepatic circulation of bile acids, but also triglyceride, cholesterol and glucose metabolism. FXR-mediated signaling pathways have become promising novel drug targets for the treatment of common metabolic and hepatic diseases. With the aim of uncovering novel modulators of FXR and further elucidating the molecular basis of FXR activation, we investigated the structure–activity relationships of a variety of naturally occurring sterols structurally related to bile acids in terms of their FXR agonist activity. Here, we report that the ability of bile alcohols to activate FXR varied with the position and number of hydroxyl groups existing in the steroid side chain of bile alcohols. In addition, we showed that the shortening of the steroid side chain of bile acids as well as bile alcohols resulted in a decline of the ability of these agents to activate FXR. Thus, we provide new insights into the structure–activity relationships of bile acids and bile alcohols as FXR agonists.     

Analysis of L-DOPA and droxidopa binding to human β2-adrenergic receptor

Over the last two decades, an increasing number of studies has been devoted to a deeper understanding of the molecular process involved in the binding of various agonists and antagonists to active and inactive conformations of β₂-adrenergic receptor (β₂AR). The 3.2 Å x-ray crystal structure of human β₂AR active state in combination with the endogenous low affinity agonist adrenaline offers an ideal starting structure for studying the binding of various catecholamines to adrenergic receptors. We show that molecular docking of levodopa (L-DOPA) and droxidopa into rigid and flexible β₂AR models leads for both ligands to binding anchor sites comparable to those experimentally reported for adrenaline, namely D113/N312 and S203/S204/S207 side chains. Both ligands have a hydrogen bond network that is extremely similar to those of noradrenaline and dopamine. Interestingly, redocking neutral and protonated versions of adrenaline to rigid and flexible β₂AR models results in binding poses that are more energetically stable and distinct from the x-ray crystal structure. Similarly, lowest energy conformations of noradrenaline and dopamine generated by docking into flexible β₂AR models had binding free energies lower than those of best poses in rigid receptor models. Furthermore, our findings show that L-DOPA and droxidopa molecules have binding affinities comparable to those predicted for adrenaline, noradrenaline, and dopamine, which are consistent with previous experimental and computational findings and supported by the molecular dynamics simulations of β₂AR-ligand complexes performed here.     

Discovery and structure–activity relationships of a series of pyroglutamic acid amide antagonists of the P2X7 receptor

A computational lead-hopping exercise identified compound 4 as a structurally distinct P2X₇ receptor antagonist. Structure–activity relationships (SAR) of a series of pyroglutamic acid amide analogues of 4 were investigated and compound 31 was identified as a potent P2X₇ antagonist with excellent in vivo activity in animal models of pain, and a profile suitable for progression to clinical studies.     

Discovery of a cyclopentylamine as an orally active dual NK1 receptor antagonist–serotonin reuptake transporter inhibitor

Cyclopentylamine 4 was identified as a potent dual NK₁R antagonist–SERT inhibitor. This compound demonstrated significant oral activity in the gerbil forced swimming test, suggesting that dual NK₁R antagonists–SERT inhibitors may be useful in treating depression disorders.     

AS1907417, a novel GPR119 agonist, as an insulinotropic and β-cell preservative agent for the treatment of type 2 diabetes

G-protein-coupled receptor (GPR) 119 is involved in glucose-stimulated insulin secretion (GSIS) and represents a promising target for the treatment of type 2 diabetes as it is highly expressed in pancreatic β-cells. Although a number of oral GPR119 agonists have been developed, their inability to adequately directly preserve β-cell function limits their effectiveness. Here, we evaluated the therapeutic potential of a novel small-molecule GPR119 agonist, AS1907417, which represents a modified form of a 2,4,6-tri-substituted pyrimidine core agonist, AS1269574, we previously identified. The exposure of HEK293 cells expressing human GPR119, NIT-1 cells expressing human insulin promoter, and the pancreatic β-cell line MIN-6-B1 to AS1907417, enhanced intracellular cAMP, GSIS, and human insulin promoter activity, respectively. In in vivo experiments involving fasted normal mice, a single dose of AS1907417 improved glucose tolerance, but did not affect plasma glucose or insulin levels. Twice-daily doses of AS1907417 for 4 weeks in diabetic db/db, aged db/db mice, ob/ob mice, and Zucker diabetic fatty rats reduced hemoglobin A1c levels by 1.6%, 0.8%, 1.5%, and 0.9%, respectively. In db/db mice, AS1907417 improved plasma glucose, plasma insulin, pancreatic insulin content, lipid profiles, and increased pancreatic insulin and pancreatic and duodenal homeobox 1 (PDX-1) mRNA levels. These data demonstrate that novel GPR119 agonist AS1907417 not only effectively controls glucose levels, but also preserves pancreatic β-cell function. We therefore propose that AS1907417 represents a new type of antihyperglycemic agent with promising potential for the effective treatment of type 2 diabetes.     

Age-related increase of β1-adrenergic receptor gene expression in rat liver: a potential mechanism contributing to increased β-adrenergic receptor density and responsiveness during aging

In this study we examined whether the levels of gene expressions of the three β- adrenergic receptor (βAR) subtypes, β₁, β₂, and β₃, contribute to age-related increase in βAR density. Liver membranes and total RNA were prepared from young (4- to 6-month-old) and old (24-month-old) male Fischer 344 rats. βAR density (B_max) in liver membranes was measured by a radioligand receptor binding assay using the receptor subtype nonselective βAR antagonist ¹²⁵I-pindolol as the radioligand. Steady-state levels of β₂AR mRNA in rat liver were measured by Northern blot analysis; because of the low abundance of β₁AR and β₃AR mRNA in rat liver, the expressions of these genes were measured by a semiquantitative RT-PCR or an RT-PCR. Scatchard analysis of saturation binding curves of the binding assay confirmed an age-related increase in B_max (young: 7.1?±?0.8?fmol/mg protein vs. old: 18.1?±?4.3?fmol/mg protein). No age-related differences were found in the levels of β₂AR mRNA. However, semiquantitative RT-PCR revealed an approximately twofold increase in β₁AR mRNA level between young and old rats (P?<?0.05). β₁AR mRNA levels were also correlated with B_max values for ¹²⁵I-pindolol binding sites in individual rats (r = 0.67; P?=?0.012). β₃AR mRNA, which was demonstrable in rat white adipose tissue by RT-PCR, was generally not detected in livers from young or old rats, with the exception of two old rats with the highest B_max. These results suggest that an age-related increase of β₁AR gene expression contributes to increased βAR density and β adrenergic responsiveness in rat liver during aging.     

Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling

Activated G_q protein–coupled receptors (G_qPCRs) can be desensitized by phosphorylation and β-arrestin binding. The kinetics and individual contributions of these two mechanisms to receptor desensitization have not been fully distinguished. Here, we describe the shut off of protease-activated receptor 2 (PAR2). PAR2 activates G_q and phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP₂) into diacylglycerol and inositol trisphosphate (IP₃). We used fluorescent protein–tagged optical probes to monitor several consequences of PAR2 signaling, including PIP₂ depletion and β-arrestin translocation in real time. During continuous activation of PAR2, PIP₂ was depleted transiently and then restored within a few minutes, indicating fast receptor activation followed by desensitization. Knockdown of β-arrestin 1 and 2 using siRNA diminished the desensitization, slowing PIP₂ restoration significantly and even adding a delayed secondary phase of further PIP₂ depletion. These effects of β-arrestin knockdown on PIP₂ recovery were prevented when serine/threonine phosphatases that dephosphorylate GPCRs were inhibited. Thus, PAR2 may continuously regain its activity via dephosphorylation when there is insufficient β-arrestin to trap phosphorylated receptors. Similarly, blockers of protein kinase C (PKC) and G protein–coupled receptor kinase potentiated the PIP₂ depletion. In contrast, an activator of PKC inhibited receptor activation, presumably by augmenting phosphorylation of PAR2. Our interpretations were strengthened by modeling. Simulations supported the conclusions that phosphorylation of PAR2 by protein kinases initiates receptor desensitization and that recruited β-arrestin traps the phosphorylated state of the receptor, protecting it from phosphatases. Speculative thinking suggested a sequestration of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) to the plasma membrane by β-arrestin to explain why knockdown of β-arrestin led to secondary depletion of PIP₂. Indeed, artificial recruitment of PIP5K removed the secondary loss of PIP₂ completely. Altogether, our experimental and theoretical approaches demonstrate roles and dynamics of the protein kinases, β-arrestin, and PIP5K in the desensitization of PAR2.     

Synthesis and evaluation of a 18F-labeled spirocyclic piperidine derivative as promising σ1 receptor imaging agent

Several spirocyclic piperidine derivatives were designed and synthesized as σ₁ receptor ligands. In vitro competition binding assays showed that the fluoroalkoxy analogues with small substituents possessed high affinity towards σ₁ receptors and subtype selectivity. Particularly for ligand 1′-((6-(2-fluoroethoxy)pyridin-3-yl)methyl)-3H-spiro[2-benzofuran-1,4′-piperidine] (2), high σ₁ receptor affinity (K_i = 2.30 nM) and high σ₁/σ₂ subtype selectivity (142-fold) as well as high σ₁/VAChT selectivity (234-fold) were observed. [¹⁸F]2 was synthesized using an efficient one-pot, two-step reaction method in a home-made automated synthesis module, with an overall isolated radiochemical yield of 8–10%, a radiochemical purity of higher than 99%, and specific activity of 56–78 GBq/μmol. Biodistribution studies of [¹⁸F]2 in ICR mice indicated high initial brain uptake and a relatively fast washout. Administration of haloperidol, compound 1 and different concentrations of SA4503 (3, 5, or 10 μmol/kg) 5 min prior to injection of [¹⁸F]2 significantly decreased the accumulation of radiotracer in organs known to contain σ₁ receptors. Ex vivo autoradiography in Sprague–Dawley rats demonstrated high accumulation of radiotracer in brain areas with high expression of σ₁ receptors. These encouraging results prove that [¹⁸F]2 is a suitable candidate for σ₁ receptor imaging with PET in humans.     

The synthetic peptide octraphin TPLVTLFK is a selective agonist of nonopioid β-endorphin receptor

The peptide TPLVTLFK (coined by the authors “octarphin”), corresponding to the amino acid sequence of β-endorphin fragment 12–19, and its analogs (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, TPLVTLFL) were synthesized. The peptide octarphin was labeled with tritium (specific activity, 28 Ci/mol) and its binding to the rat brain cortex membranes and mouse peritoneal macrophages was studied. [³H]Octarphin was found to bind to brain membranes and macrophages with high affinity (K _d = 2.6 ±0.2 and 2.3 +0.2 nM, respectively) and specificity. The specific binding of [³H]octarphin with rat brain membranes and mouse macrophages was inhibited by unlabeled β-endorphin (K _i = 2.4 +0.2 and 2.7 +0.2 nM, respectively) and selective agonist of nonopioid β-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (K _i = 2.9 +0.2 and 2.4 +0.2 nM, respectively) and was not inhibited by unlabeled naloxone, α-endorphin, γ-endorphin and [Met⁵]enkephalin (K _i > 10 mM). Inhibiting activity of unlabeled analogs of octarphin was more than 100 times lower than that of the unlabeled octarphin. Octarphin was shown to stimulate activity of mouse immunocompetent cells in vitro: at the concentration of 1 nM it enhanced the capacity of peritoneal macrophages to digest bacteria Salmonella typhimurium virulent strain 415 in vitro. Thus, octarphin is a selective agonist of nonopioid (insensitive to the opioid antagonist naloxone) β-endorphin receptor of rat brain cortex membranes and mouse peritoneal macrophages.     

Synthetic peptide immunorphin as an instrument of the study of nonopioid β-endorphin receptor

Research results of the synthetic decapeptide SLTCLVKGFY (the author’s term is immunorphin) corresponding to the 364–373 sequence of G heavy-chain human immunoglobulin are summarized. Special attention is paid to the interaction between immunorphin and nonopioid (insensitive to the opioid antagonist naloxone) β-endorphin receptor. Using radioligand analysis, data were found regarding the distribution and functions of nonopioid β-endorphin receptor in human and animal bodies and the binding characteristics of immunorphin with nonopioid receptor.     

Long time molecular dynamic simulation on the agonist binding and activation of the β2-adrenergic receptor

β₂-Adrenergic receptor (β₂AR) plays a critical role in mediating the effects of catecholamine hormones. Due to the flexibility of the structure of its active state, study of agonist–β2AR is usually performed by molecular dynamic (MD) simulation. In this study we show the representative characteristics of agonist binding and activation on β₂AR by MD simulation. The binding site and the conformational changes in the specific regions of β2AR are reasonable which confirmed the conclusion that agonist–β2AR reached its active-like state. We have studied the disruption of non-covalent intramolecular interactions, including the conserved DRY motif, the rotamer toggle switch and the ionic lock, the cytoplasmic ends of transmembranes 5 and 6, and some water-mediated hydrogen bond network regions. We conclude that agonist induced β₂AR to its active conformation, or at least the active-like conformation.     

Discovery and optimization of a series of 2-aminothiazole-oxazoles as potent phosphoinositide 3-kinase γ inhibitors

A novel series of 2-aminothiazole-oxazoles was designed and synthesized as part of efforts to develop potent phosphoinositide 3-kinase γ (PI3Kγ) inhibitors. The modification of a high-throughput screening hit, compound 1, resulted in the identification of compounds 10 and 15, which displayed potent inhibitory activities in enzyme-based and cell-based assays.     

Discovery of atorvastatin as a tetramer stabilizer of nuclear receptor RXRα through structure-based virtual screening

Retinoid X receptor alpha (RXRα), a central member of the nuclear receptor superfamily and a key regulator of many signal transduction pathways, has been an attractive drug target. We previously discovered that an N-terminally truncated form of RXRα can be induced by specific ligands to form homotetramers, which, as a result of conformational selection, forms the basis for inhibiting the nongenomic activation of RXRα. Here, we report the identification and characterization of atorvastatin as a new RXRα tetramer stabilizer by using structure-based virtual screening and demonstrate that virtual library screening can be used to aid in identifying RXRα ligands that can induce its tetramerization. In this study, docking was applied to screen the FDA-approved small molecule drugs in the DrugBank 4.0 collection. Two compounds were selected and purchased for testing. We showed that the selected atorvastatin could bind to RXRα to promote RXRα-LBD tetramerization. We also showed that atorvastatin possessed RXRα-dependent apoptotic effects. In addition, we used a chemical approach to aid in the studies of the binding mode of atorvastatin.     

Kindlin 2 forms a transcriptional complex with β-catenin and TCF4 to enhance Wnt signalling

Kindlin 2, as a focal adhesion protein, controls integrin activation. However, the association of Kindlin 2 with cancer-related signalling pathways is unknown. Here we identified a new direct interaction between Kindlin 2 and the active β-catenin. Importantly, Kindlin 2 forms a tripartite complex with β-catenin and TCF4. Mechanistically, Kindlin 2 selectively strengthens the occupancy of β-catenin on the Wnt target gene Axin2 and enhances Axin2 gene expression. Functionally, the β-catenin-Axin2-Snail cascade is required for Kindlin 2-induced tumour cell invasion. Our data indicate that Kindlin 2 is a new regulator of Wnt signalling, providing a mechanistic insight into the role of Kindlin 2 in cancer progression.