fluorescence/luminescence-based techniques play an increasingly important role in the development of test systems for the characterization of future drug candidates, especially in terms of receptor binding in the field of G protein-coupled receptors (GPCRs). In this article, we present the establishment of a homogeneous live cell-based BRet binding assay for the histamine H 2 receptor with different fluorescently labeled squaramide-type compounds synthesized in the course of this study. Py-1-labeled ligand 8 (UR-KAT478) was found to be most suitable in BRET saturation binding experiments with respect to receptor affinity (pK d = 7.35) and signal intensity. Real-time kinetic experiments showed a full association of 8 within approximately 30 min and a slow dissociation of the ligand from the receptor. Investigation of reference compounds in BRET-based competition binding with 8 yielded pK i values in agreement with radioligand binding data. This study shows that the BRET binding assay is a versatile test system for the characterization of putative new ligands at the histamine H 2 receptor and represents a valuable fluorescence-based alternative to canonical binding assays.
Alzheimer's disease (AD) was first described by Alois Alzheimer over 100 years ago, but there is still no overarching theory that can explain its cause in detail. There are also no effective therapies to treat either the cause or the associated symptoms of this devastating disease. A potential approach to better understand the pathogenesis of AD could be the development of selective caspase-2 (Casp2) probes, as we have shown that a Casp2-mediated cleavage product of tau (Δtau314) reversibly impairs cognitive and synaptic function in animal models of tauopathies. In this article, we map out the Casp2 binding site through the preparation and assay of a series of 35 pentapeptide inhibitors with the goal of gaining selectivity against caspase-3 (Casp3). We also employed computational docking methods to understand the key interactions in the binding pocket of Casp2 and the differences predicted for binding at Casp3. Moreover, we crystallographically characterized the binding of selected pentapeptides with Casp3. Furthermore, we engineered and expressed a series of recombinant tau mutants and investigated them in an in vitro cleavage assay. These studies resulted in simple peptidic inhibitors with nanomolar affinity, for example, AcVDV(Dab)D-CHO (24) with up to 27.7-fold selectivity against Casp3. Our findings provide a good basis for the future development of selective Casp2 probes and inhibitors that can serve as pharmacological tools in planned in vivo studies and as lead compounds for the design of bioavailable and more drug-like small molecules.
In an integrative approach, we studied cardiac effects of recently published novel H 2 receptor agonists in the heart of mice that overexpress the human H 2 receptor (H 2 -TG), littermate wild type control mice (WT) and in isolated electrically driven muscle preparations from patients undergoing cardiac surgery. Under our experimental conditions, the H 2 receptor agonists UR-Po563, UR-MB-158 and UR-MB-159 increased force of contraction in left atrium from H 2 -TG with pEC 50 values of 8.27, 9.38, and 8.28, respectively, but not in WT. Likewise, UR-Po563, UR-MB-158 and UR-MB-159increased the beating rate in right atrium from H 2 -TG with pEC 50 values of 9.01, 9.24, and 7.91, respectively, but not in WT. These effects could be antagonized by famotidine, a H 2 receptor antagonist. UR-Po563 (1 µM) increased force of contraction in Langendorff perfused hearts from H 2 -TG but not WT. Similarly, UR-Po563, UR-MB-158 or UR-MB-159 increased the left ventricular ejection fraction in echocardiography of H 2 -TG. Finally, UR-Po563 increased force of contraction in isolated human right atrial muscle strips. The contractile effects of UR-Po563 in H 2 -TG were accompanied by an increase in the phosphorylation state of phospholamban. In summary, we report here three recently developed agonists functionally stimulating human cardiac H 2 receptors in vitro and in vivo.We speculate that these compounds might be of some merit to treat neurological disorders if their cardiac effects are blocked by concomitantly applied receptor antagonists that cannot pass through the blood-brain barrier or might be useful to treat congestive heart failure in patients.
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