No abstract

Recently regulators of G protein signalling (RGS) proteins have emerged as potential therapeutic targets since they provide an alternative method of modulating the activity of GPCRs, the target of so many drugs. Inhibitors of RGS proteins must block a protein-protein interaction (RGS-Gα), but also be cell and, depending on the therapeutic target, blood brain barrier permeable. A lead compound (1a) was identified as an inhibitor of RGS4 in a screening assay and this has now been optimised for activity, selectivity and solubility. The newly developed ligands (11b, 13) display substantial selectivity over the closely related RGS8 protein, lack the off-target calcium mobilisation activity of the lead 1a and have excellent aqueous solubility. They are currently being evaluated in vivo in rodent models of depression.

Many current therapies target G protein coupled receptors (GPCR), transporters, or ion channels. In addition to directly targeting these proteins, disrupting the protein-protein interactions that localize or regulate their function could enhance selectivity and provide unique pharmacologic actions. Regulators of G protein signaling (RGS) proteins, especially RGS4, play significant roles in epilepsy and Parkinson's disease. Thiadiazolidinone (TDZD) inhibitors of RGS4 are nanomolar potency blockers of the biochemical actions of RGS4 in vitro. Here, we demonstrate the substantial selectivity (8- to >5000-fold) of CCG-203769 for RGS4 over other RGS proteins. It is also 300-fold selective for RGS4 over GSK-3β, another target of this class of chemical scaffolds. It does not inhibit the cysteine protease papain at 100 μM. CCG-203769 enhances Gαq-dependent cellular Ca(2+) signaling in an RGS4-dependent manner. TDZD inhibitors also enhance Gαi-dependent δ-OR inhibition of cAMP production in SH-SY-5Y cells, which express endogenous receptors and RGS4. Importantly, CCG-203769 potentiates the known RGS4 mechanism of Gαi-dependent muscarinic bradycardia in vivo. Furthermore, it reverses raclopride-induced akinesia and bradykinesia in mice, a model of some aspects of the movement disorder in Parkinson's disease. A broad assessment of compound effects revealed minimal off-target effects at concentrations necessary for cellular RGS4 inhibition. These results expand our understanding of the mechanism and specificity of TDZD RGS inhibitors and support the potential for therapeutic targeting of RGS proteins in Parkinson's disease and other neural disorders.

Changes in the density of imidazoline-I 2 binding sites have been observed in a range of neurologic disorders including Alzheimer's disease, Huntington's chorea, and glial tumor; however, the precise function of these sites remains unclear. A PET probe for I 2 binding sites would further our understanding of the target and may find application as a biomarker for early disease diagnosis. Compound BU99008 has previously been identified as a promising I 2 ligand from autoradiography studies, displaying high affinity and good selectivity toward the target. In this study, BU99008 was radiolabeled with 11 C in order to image the I 2 binding sites in vivo using PET. Methods: 11 C-BU99008 was radiolabeled by N-alkylation of the desmethyl precursor using 11 C-methyl iodide. A series of PET experiments was performed to investigate the binding of 11 C-BU99008 in porcine brains, in the presence or absence of a nonradiolabeled, competing I 2 ligand, BU224. Results: 11 C-BU99008 was obtained in good yield and specific activity. In vivo, 11 C-BU99008 displayed good brain penetration and gave a heterogeneous distribution with high uptake in the thalamus and low uptake in the cortex and cerebellum. 11 C-BU99008 brain kinetics were well described by the 1-tissue-compartment model, which was used to provide estimates for the total volume of distribution (V T ) across brain regions of interest. Baseline V T values were ranked in the following order: thalamus . striatum . hippocampus . frontal cortex $ cerebellum, consistent with the known distribution and concentration of I 2 binding sites. Administration of a selective I 2 binding site ligand, BU224, reduced the V T to near-homogeneous levels in all brain regions. Conclusion: 11 C-BU99008 appears to be a suitable PET radioligand for imaging the I 2 binding sites in vivo.

The density of the Imidazoline₂ binding site (I₂BS) has been shown to change in psychiatric conditions such as depression and addiction, along with neurodegenerative disorders such as Alzheimer's disease and Huntington's chorea. The presence of I₂BS on glial cells and the possibility that they may in some way regulate glial fibrillary acidic protein has led to increased interest into the role of I₂BS and I₂BS ligands in conditions characterized by marked gliosis. In addition, it has been suggested that I₂BS may be a marker for human glioblastomas. Therefore, the development of a positron emission tomography (PET) radioligand for the I₂BS would be of major benefit in our understanding of these conditions. We now report the successful synthesis and initial pharmacological evaluation of potential PET radioligands for the I₂BS as well as the tritiation and characterization of the most favorable of the series, BU99008 (6), both in vitro and ex vivo in rat. The series as a whole demonstrated excellent affinity and selectivity for the I₂BS, with BU99008 (6) selected as the lead candidate to be taken forward for in vivo assessment. BU99008 (6) showed very good affinity for the I₂BS (K(i) of 1.4 nM; K(d) = 1.3 nM), good selectivity compared with the α₂ -adrenoceptor (909-fold). In addition, following peripheral administration, [³H]BU99008 demonstrated a heterogenous uptake into the rat brain consistent with the known distribution of the I₂BS in vivo. This, and the amenability of BU99008 (6) to radiolabeling with a positron-emitting radioisotope, indicates its potential as a PET radioligand for imaging the I₂BS in vivo.