Identification of potential glutaminyl cyclase inhibitors from lead-like libraries by in silico and in vitro fragment-based screening.

A glutaminyl cyclase (QC) fragment library was in silico selected by disconnection of the structure of known QC inhibitors and by lead-like 2D virtual screening of the same set. The resulting fragment library (204 compounds) was acquired from commercial suppliers and pre-screened by differential scanning fluorimetry followed by functional in vitro assays. In this way, 10 fragment hits were identified ([Formula: see text]5 % hit rate, best inhibitory activity: 16 [Formula: see text]). The in vitro hits were then docked to the active site of QC, and the best scoring compounds were analyzed for binding interactions. Two fragments bound to different regions in a complementary manner, and thus, linking those fragments offered a rational strategy to generate novel QC inhibitors. Based on the structure of the virtual linked fragment, a 77-membered QC target focused library was selected from vendor databases and docked to the active site of QC. A PubChem search confirmed that the best scoring analogues are novel, potential QC inhibitors.

The influence of 5-HT(2A) activity on a 5-HT(2C) specific in vivo assay used for early identification of multiple acting SERT and 5-HT(2C) receptor ligands.

As a result of our exploratory programme aimed at elaborating dually acting compounds towards the serotonin (5-HT) transporter (SERT) and the 5-HT2C receptor a novel series of 3-amino-1-phenylpropoxy substituted diphenylureas was identified. From that collection two promising compounds (2 and 3) exhibiting highest 5-HT2C receptor affinity strongly inhibited the 5-HT2C receptor agonist 1-(3-chlorophenyl)piperazine (mCPP) induced hypomotility in mice. In further pursuance of that objective (2-aminoethyl)(benzyl)sulfamoyl diphenylureas and diphenylpiperazines have also been elaborated. Herein we report the synthesis of potent multiple-acting compounds from this new class. However, when two optimized representatives (6 and 14) possessing the desired in vitro profile were tested neither reduced the motor activity of mCPP treated animals. Comparative albeit limited in vitro structure-activity relationship (SAR) analysis and detailed in vivo studies are discussed and explanation for their intricate behaviour is proposed.

Analysis of functional selectivity through G protein-dependent and -independent signaling pathways at the adrenergic α(2C) receptor.

Although G protein-coupled receptors (GPCRs) are traditionally categorized as Gs-, Gq-, or Gi/o-coupled, their signaling is regulated by multiple mechanisms. GPCRs can couple to several effector pathways, having the capacity to interact not only with more than one G protein subtype but also with alternative signaling or effector proteins such as arrestins. Moreover, GPCR ligands can have different efficacies for activating these signaling pathways, a characteristic referred to as biased agonism or functional selectivity. In this work our aim was to detect differences in the ability of various agonists acting at the α2C type of adrenergic receptors (α2C-ARs) to modulate cAMP accumulation, cytoplasmic Ca(2+) release, ß-arrestin recruitment and receptor internalization. A detailed comparative pharmacological characterization of G protein-dependent and -independent signaling pathways was carried out using adrenergic agonists (norepinephrine, phenylephrine, brimonidine, BHT-920, oxymetazoline, clonidine, moxonidine, guanabenz) and antagonists (MK912, yohimbine). As initial analysis of agonist Emax and EC50 values suggested possible functional selectivity, ligand bias was quantified by applying the relative activity scale and was compared to that of the endogenous agonist norepinephrine. Values significantly different from 0 between pathways indicated an agonist that promoted different level of activation of diverse effector pathways most likely due to the stabilization of a subtly different receptor conformation from that induced by norepinephrine. Our results showed that a series of agonists acting at the α2C-AR displayed different degree of functional selectivity (bias factors ranging from 1.6 to 36.7) through four signaling pathways. As signaling via these pathways seems to have distinct functional and physiological outcomes, studying all these stages of receptor activation could have further implications for the development of more selective therapeutics with improved efficacy and/or fewer side effects.

Interaction of SSR161421, a novel specific adenosine A(3) receptor antagonist with adenosine A(3) receptor agonists both in vitro and in vivo.

A novel adenosine A(3) receptor antagonist (SSR161421) was characterized by both receptor binding assays and pharmacological tests. Binding studies on cloned human adenosine receptors showed that SSR161421 has high affinity for adenosine hA(3) receptors (K(i)=0.37 nM) with at least 1000-fold selectivity compared to hA(1), hA(2A) and hA(2B) receptors. The receptor antagonist nature of SSR161421 was determined in a functional study on Chinese hamster ovarian cells (CHO) cells expressing human adenosine A(3) receptors. SSR161421 competitively antagonized the effect of 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (Cl-IB-MECA) on cAMP production with a pA2 value in a luciferase reporter gene construct. In mice, intravenously administered SSR161421 inhibited the N6-(4-aminobenzyl)-adenosine-5'-N-methyl-uronamide dihydrochloride (AB-MECA) induced increase in plasma histamine levels (ED(50)=2.0mg/kg) and the Cl-IB-MECA evoked plasma extravasation (ID(50)=2.9 mg/kg) and oedema formation (ID(50)=4.6 mg/kg) in mouse ear.

A novel orally active inhibitor of HLE.

Human leukocyte elastase (HLE) is a serine proteinase, capable of degrading a variety of structural matrix proteins. SSR69071 2-[(4-isopropyl-6-methoxy-1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)methoxy]-9-(2-piperidin-1-ylethoxy)-4H-pyrido[1,2-a]pyrimidin-4-one was selected as a novel orally active HLE inhibitor for treatment of chronic obstructive pulmonary diseases, asthma, emphysema, cystic fibrosis and several inflammatory diseases (WO 01/44245 A1) (J. Pharm. Exp. Ther., submitted for publication).

Biochemical and pharmacological characterization of 2-(9-(2-piperidinoethoxy)-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yloxymethyl)-4-(1-methylethyl)-6-methoxy-1,2-benzisothiazol-3(2H)-one-1,1-dioxide (SSR69071), a novel, orally active elastase inhibitor.

Human leukocyte elastase (HLE) is a proteinase capable of degrading a variety of proteins. Under normal circumstances, the proteolytic activity of HLE is effectively controlled by its natural inhibitors. However, an imbalance between elastase and its endogenous inhibitors may result in several pathophysiological states such as chronic obstructive pulmonary disease, asthma, emphysema, cystic fibrosis, and chronic inflammatory diseases. It is anticipated that an orally active HLE inhibitor could be useful for the treatment of these diseases. 2-(9-(2-Piperidinoethoxy)-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yloxymethyl)-4-(1-methylethyl)-6-methoxy-1,2-benzisothiazol-3(2H)-one-1,1-dioxide (SSR69071) is a potent inhibitor of HLE, with the inhibition constant (K(i)) and the constant for inactivation process (k(on)) being 0.0168 +/- 0.0014 nM and 0.183 +/- 0.013 10(6)/mol sr, respectively. The dissociation rate constant, k(off), was 3.11 + 0.37 10(-6)/s. SSR69071 displays a higher affinity for human elastase than for rat (K(i) = 3 nM), mouse (K(i) = 1.8 nM), and rabbit (K(i) = 58 nM) elastases. Bronchoalveolar lavage fluid from mice orally treated with SSR69071 inhibits HLE (ex vivo), and in this model, SSR69071 has a dose-dependent efficacy with an ED(50) = 10.5 mg/kg p.o. SSR69071 decreases significantly the acute lung hemorrhage induced by HLE (ED(50) = 2.8 mg/kg p.o.) in mice. Furthermore, SSR69071 prevents carrageenan- (ED(30) = 2.2 mg/kg) and HLE-induced (ED(30) = 2.7 mg/kg) paw edema in rats after p.o. administration. In conclusion, SSR69071 is a selective, orally active, and potent inhibitor of HLE with good penetration in respiratory tissues.

SSR69071, an elastase inhibitor, reduces myocardial infarct size following ischemia-reperfusion injury.

Neutrophil elastase contributes to the severity of cardiac damage following coronary ischemia and reperfusion. We evaluated the effects of 2-(9-(2-piperidinoethoxy)-4-oxo-4H-pyridol[1,2-a]pyrimidin-2-yloxymethyl)-4-(1-methyethyl)-6-methoxy-1,2-benzisothiazol-3(2H)-one-1,1-dioxide hemihydrate (SSR69071), a novel, potent and selective inhibitor of neutrophil elastase, on infarct size in anaesthetized rabbits subjected to coronary artery occlusion for 30 min followed by reperfusion for 120 min. SSR69071 (3 mg/kg i.v.) reduced cardiac infarct size when administered before ischemia (-39%, P<0.05) or just prior to reperfusion (-37%, P<0.05). Subsequent experiments using the latter administration protocol confirmed the ability of SSR69071 (1 and 3 mg/kg i.v.) to reduce infarct size. This cardioprotective activity was associated with inhibition of cardiac elastase.