Stress-induced activation of the hypothalamo-pituitary-adrenocortical (HPA) axis and high circulating glucocorticoid levels are well known to impair the retrieval of memory. Vasopressin can activate ...the HPA axis by stimulating vasopressin 1b (V1b) receptors located on the pituitary. In the present study, we investigated the effect of A-988315, a selective and highly potent non-peptidergic V1b-receptor antagonist with good pharmacokinetic properties, in blocking stress effects on HPA-axis activity and memory retrieval. To study cognitive performance, male Sprague-Dawley rats were trained on an object-discrimination task during which they could freely explore two identical objects. Memory for the objects and their location was tested 24 h later. A-988315 (20 or 60 mg/kg) or water was administered orally 90 min before retention testing, followed 60 min later by stress of footshock exposure. A-988315 dose-dependently dampened stress-induced increases in corticosterone plasma levels, but did not significantly alter HPA-axis activity of non-stressed control rats. Most importantly, A-988315 administration prevented stress-induced impairment of memory retrieval on both the object-recognition and the object-location tasks. A-988315 did not alter the retention of non-stressed rats and did not influence the total time spent exploring the objects or experimental context in either stressed or non-stressed rats. Thus, these findings indicate that direct antagonism of V1b receptors is an effective treatment to block stress-induced activation of the HPA axis and the consequent impairment of retrieval of different aspects of recognition memory.
Evaluation of drug–target interaction kinetics is becoming increasingly important during the drug-discovery process to investigate selectivity of a drug and predict in vivo target occupancy. To date, ...it remains challenging to obtain kinetic information for interactions between G-protein-coupled receptors (GPCRs) and small-molecule ligands in a label-free manner. Often GPCRs need to be solubilized or even stabilized by mutations which can be difficult and is time consuming. In addition, it is often unclear if the native conformation of the receptors is sustained.
In this study, surface plasmon resonance (SPR) and surface acoustic wave (SAW) technologies have been used to detect ligand binding to the GPCR chemokine (C-X-C motif) receptor 4 (CXCR4) expressed in lipoparticles. We first evaluated different strategies to immobilize CXCR4-expressing lipoparticles. The highest small-molecule binding signal in SPR and SAW was achieved with a matrix-free carboxymethylated sensor chip coated with wheat germ agglutinin for lipoparticle capturing. Next, the binding kinetics of the anti-CXCR4 antibody 12G5 raised against a conformational epitope (kon=1.83×106M−1s−1, koff=2.79×10−4 s−1) and the small molecule AMD3100 (kon=5.46×105M−1s−1, koff=1.01×10−2s−1) were assessed by SAW. Our kinetic and affinity data are consistent with previously published radioligand-binding experiments using cells and label-free experiments with solubilized CXCR4. This is the first study demonstrating label-free kinetic characterization of small-molecule binding to a GPCR in the membrane environment. The presented method holds the potential to greatly facilitate label-free assay development for GPRCs that can be expressed at high levels in lipoparticles.
•Combination of GPCR-expressing lipoparticles with SAW detection technology•Optimization of lipoparticle immobilization for small-molecule binding•First label-free measurement of small-molecule binding to a GPCR in the membrane•Kinetic measurements agree with previous data on cells and solubilized receptors.
In the forebrain, synaptic glycine concentrations are regulated through the glycine transporter GlyT1. Because glycine is
a coagonist of the N -methyl- d -aspartate (NMDA) receptor (NMDAR), which has ...been implicated in schizophrenia, inhibition of GlyT1 is thought to provide
an option for the treatment of schizophrenia. In support of this hypothesis, GlyT1 inhibitors facilitate in vivo NMDAR function
and demonstrate antipsychotic-like effects in animal models. Among the specific GlyT1 inhibitors, substituted N -methyl-glycine (sarcosine) derivatives (e.g., ( R )- N 3-(4â²fluorophenyl)-3-(4â²phenyl-phenoxy)propyl-sarcosine NFPS, ( R )- N 3-phenyl-3-(4â²-(4-toluoyl)phenoxy)-propylsarcosine (R)-NPTS, and ( R , S )-(±) N -methyl- N -(4-trifluoromethyl)phenoxy-3-phenyl-propylglycine Org24589), and non-sarcosine-containing inhibitors, such as 2-chloro- N -( S )-phenyl(2 S )-piperidin-2-yl methyl-3-trifluoromethyl benzamide, monohydrochloride (SSR504734), have been described. In the present
study, we analyzed the mode of interaction of these compounds with GlyT1 by using electrophysiological measurements in Xenopus laevis oocytes, and with two binding assays, using 3 H(R)-NPTS or 2-chloro- N -( S )-phenyl(2 S )- N -methylpiperidin-2-yl-methyl-3-trifluoromethyl benzamide monohydrochloride ( 3 H N -methyl-SSR504734) as radioligands. Inhibition of electrogenic glycine transport by sarcosine-based compounds was apparently
irreversible and independent of glycine concentration. The latter indicates a noncompetitive mode of action. In contrast,
both SSR504734 and N -methyl-SSR504734 exhibited reversible and competitive inhibition of glycine transport. In GlyT1-expressing membranes, the
binding of the novel radioligand 3 H N -methyl-SSR504734 to a single site on GlyT1 was competitively displaced by glycine and SSR504734 but noncompetitively by sarcosine-based
compounds. Inversely, 3 H(R)-NPTS binding was competitively inhibited by sarcosine-based compounds, whereas glycine, SSR504734, and N -methyl-SSR504734 noncompetitively decreased maximal binding. Our data indicate that besides exerting an apparently irreversible
or reversible inhibition, GlyT1 inhibitors differ by exhibiting either a noncompetitive or competitive mode of inhibition.
The divergent modes of inhibition may significantly affect the efficacy and tolerability of these drugs.
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Aberrant activation of calpain has been observed in various pathophysiological disorders including neurodegenerative diseases such as Alzheimer’s Disease. Here we describe our efforts ...on ketoamide-based 1-benzyl-5-oxopyrrolidine-2-carboxamides as a novel series of highly selective calpain inhibitors mitigating the metabolic liability of carbonyl reduction. The most advanced compound from this new series, namely A-1212805 (ABT-957, Alicapistat) proceeded to clinical phase I studies.
Calpain overactivation has been implicated in a variety of pathological disorders including ischemia/reperfusion injury, cataract formation, and neurodegenerative diseases such as Alzheimer’s disease ...(AD). Herein we describe our efforts leading to the identification of ketoamide-based 2-(3-phenyl-1H-pyrazol-1-yl)nicotinamides as potent and reversible inhibitors of calpain with high selectivity versus related cysteine protease cathepsins, other proteases, and receptors. Broad efficacy in a set of preclinical models relevant to AD suggests that inhibition of calpain represents an attractive approach with potential benefit for the treatment of AD.
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•Novel V1b antagonists.•Markedly improved selectivity and pharmacokinetic profiles.•Improved half-life and brain penetration (rat PK).•In vivo efficacy at 3 mg/kg (ip, rat forced swim ...test).•High volume of distribution to mitigate high in vivo plasma clearance.
Herein we report the discovery of a novel oxindole-based series of vasopressin 1b (V1b) receptor antagonists. Introducing a substituted piperazine moiety and optimizing the southern and the northern aromatic rings resulted in potent, selective and brain penetrant V1b receptor antagonists. Compound 9c was found to be efficacious in a rat model of anti-depressant activity (3 mg/kg, ip). Interestingly, both moderate terminal half-life and moderate bioavailability could be achieved despite sub-optimal microsomal stability.
The development of glycine transporter 1 (GlyT1) inhibitors may offer putative treatments for schizophrenia and other disorders associated with hypofunction of the glutaminergic N-methyl-d-aspartate ...(NMDA) receptor. Herein, we describe the synthesis and biological evaluation of a series of 3,4-disubstituted pyrrolidine sulfonamides as competitive GlyT1 inhibitors that arose from de novo scaffold design. Relationship of chemical structure to drug–drug interaction (DDI) and bioactivation was mechanistically investigated. Murine studies were strategically incorporated into the screening funnel to provide early assessments of in vivo target occupancy (TO) by ex vivo binding studies. Advanced compounds derived from iterative structure–activity relationship (SAR) studies possessed high potency in ex vivo binding studies and good brain penetration, promising preliminary in vivo efficacy, acceptable preclinical pharmacokinetics, and manageable DDI and bioactivation liabilities.
The glycine transporter 1 (GlyT1) has emerged as a key novel target for the treatment of schizophrenia. Herein, we report the synthesis and biological evaluation of aminotetralines and aminochromanes ...as novel classes of competitive GlyT1 inhibitors. Starting from a high-throughput screening hit, structure–activity relationship studies led first to the discovery of aminotetralines displaying high GlyT1 potency and selectivity, with favorable pharmacokinetic properties. Systematic investigations of various parameters (e.g., topological polar surface area, number of hydrogen bond donors) guided by ex vivo target occupancy evaluation resulted in lead compounds possessing favorable brain penetration properties as for (7S,8R)-27a. Further optimization revealed compounds with reduced efflux liabilities as for aminochromane 51b. In an in vivo efficacy model (7S,8R)-27a, dose-dependently reversed L-687,414 induced hyperlocomotion in mice with an ED50 of 0.8 mg/kg. All these results suggest (7S,8R)-27a and 51b as new GlyT1 inhibitors worthy of further profiling.
Improved stability in rat and human liver microsomes. Improved rat PK profile. Efficacy in rat model of anti-depressant activity.
Herein we report the discovery of a novel series of vasopressin 1b ...(V1b) receptor antagonists, starting from potent but metabolically labile oxindole SSR149415. Masking the proline
N,
N-dimethyl amide moiety as an oxazole and attaching a benzylic amine moiety to the northern phenyl ring resulted in potent and selective V1b receptor antagonists with improved metabolic stability and improved pharmacokinetic properties in rat. Compound
18c was found to be efficacious in a rat model of anti-depressant activity.
Dysregulation of calpains 1 and 2 has been implicated in a variety of pathological disorders including ischemia/reperfusion injuries, kidney diseases, cataract formation, and neurodegenerative ...diseases such as Alzheimer’s disease (AD). 2-(3-Phenyl-1H)-pyrazol-1-yl)nicotinamides represent a series of novel and potent calpain inhibitors with high selectivity and in vivo efficacy. However, carbonyl reduction leading to the formation of the inactive hydroxyamide was identified as major metabolic liability in monkey and human, a pathway not reflected by routine absorption, distribution, metabolism, and excretion (ADME) assays. Using cytosolic clearance as a tailored in vitro ADME assay coupled with in vitro hepatocyte metabolism enabled the identification of analogues with enhanced stability against carbonyl reduction. These efforts led to the identification of P1′ modified calpain inhibitors with significantly improved pharmacokinetic profile including P1′ N-methoxyamide 23 as potential candidate compound for non-central nervous system indications.
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