Probing the Existence of a Metastable Binding Site at the ß2-Adrenergic Receptor with Homobivalent Bitopic Ligands.

Herein, we report the development of bitopic ligands aimed at targeting the orthosteric binding site (OBS) and a metastable binding site (MBS) within the same receptor unit. Previous molecular dynamics studies on ligand binding to the ß2-adrenergic receptor (ß2AR) suggested that ligands pause at transient, less-conserved MBSs. We envisioned that MBSs can be regarded as allosteric binding sites and targeted by homobivalent bitopic ligands linking two identical pharmacophores. Such ligands were designed based on docking of the antagonist (S)-alprenolol into the OBS and an MBS and synthesized. Pharmacological characterization revealed ligands with similar potency and affinity, slightly increased ß2/ß1AR-selectivity, and/or substantially slower ß2AR off-rates compared to (S)-alprenolol. Truncated bitopic ligands suggested the major contribution of the metastable pharmacophore to be a hydrophobic interaction with the ß2AR, while the linkers alone decreased the potency of the orthosteric fragment. Altogether, the study underlines the potential of targeting MBSs for improving the pharmacological profiles of ligands.

Ethylene glycol: Evidence of glucuronidation in vivo shown by analysis of clinical toxicology samples.

In the search for improved laboratory methods for the diagnosis of ethylene glycol poisoning, the in vivo formation of a glucuronide metabolite of ethylene glycol was hypothesized. Chemically pure standards of the ß-O-glucuronide of ethylene glycol (EG-GLUC) and a deuterated analog (d4 -EG-GLUC) were synthesized. A high-performance liquid chromatography and tandem mass spectrometry method for determination of EG-GLUC in serum after ultrafiltration was validated. Inter-assay precision (%RSD) was 3.9% to 15.1% and inter-assay %bias was -2.8% to 12.2%. The measuring range was 2-100 µmol/L (0.48-24 mg/L). Specificity testing showed no endogenous amounts in routine clinical samples (n = 40). The method was used to analyze authentic, clinical serum samples (n = 31) from patients intoxicated with ethylene glycol. EG-GLUC was quantified in 15 of these samples, with a mean concentration of 6.5 µmol/L (1.6 mg/L), ranging from 2.3 to 15.6 µmol/L (0.55 to 3.7 mg/L). In five samples, EG-GLUC was detected below the limit of quantification (2 µmol/L) and it was below the limit of detection in 11 samples (1 µmol/L). Compared to the millimolar concentrations of ethylene glycol present in blood after intoxications and potentially available for conjugation, the concentrations of EG-GLUC found in clinical serum samples are very low, but comparable to concentrations of ethyl glucuronide after medium dose ethanol intake. In theory, EG-GLUC has a potential value as a biomarker for ethylene glycol intake, but the pharmacokinetic properties, in vivo/vitro stability and the biosynthetic pathways of EG-GLUC must be further studied in a larger number of patients and other biological matrices.

Structure-activity-relationship study of N-acyl-N-phenylpiperazines as potential inhibitors of the Excitatory Amino Acid Transporters (EAATs): improving the potency of a micromolar screening Hit is not truism.

The excitatory amino acid transporters (EAATs) are transmembrane proteins responsible for the uptake of (S)-glutamate from the synaptic cleft. To date, five subtypes EAAT1-5 have been identified for which selective inhibitors have been discovered for EAAT1 and EAAT2. By screening of a commercially available compound library consisting of 4,000 compounds, N-acyl-N-phenylpiperazine analog (±)- exo -1 was identified to be a non-selective inhibitor at EAAT1-3 displaying IC50 values in the mid-micromolar range (10 µM, 40 µM and 30 µM at EAAT1, 2 and 3, respectively). Subsequently, we designed and synthesized a series of analogs to explore the structure-activity-relationship of this scaffold in the search for analogs characterized by increased inhibitory potency and/or EAAT subtype selectivity. Despite extensive efforts, all analogs of (±)- exo -1 proved to be either inactive or to have least 3-fold lower inhibitory potency than the lead, and furthermore none of the active analogs displayed selectivity for a particular subtype amongst the EAAT1-3. On the basis of our findings, we speculate that (±)- exo -1 binds to a recess (deepening) on the EAAT proteins than a well-defined pocket.