Small molecule TSHR agonists and antagonists.

TSH activates the TSH receptor (TSHR) thereby stimulating the function of thyroid follicular cells (thyrocytes) leading to biosynthesis and secretion of thyroid hormones. Because TSHR is involved in several thyroid pathologies, there is a strong rationale for the design of small molecule "drug-like" ligands. Recombinant human TSH (rhTSH, Thyrogen(®)) has been used in the follow-up of patients with thyroid cancer to increase the sensitivity for detection of recurrence or metastasis. rhTSH is difficult to produce and must be administered by injection. A small molecule TSHR agonist could produce the same beneficial effects as rhTSH but with greater ease of oral administration. We developed a small molecule ligand that is a full agonist at TSHR. Importantly for its clinical potential, this agonist elevated serum thyroxine and stimulated thyroidal radioiodide uptake in mice after its absorption from the gastrointestinal tract following oral administration. Graves' disease (GD) is caused by persistent, unregulated stimulation of thyrocytes by thyroid-stimulating antibodies (TSAbs) that activate TSHR. We identified the first small molecule TSHR antagonists that inhibited TSH- and TSAb-stimulated signalling in primary cultures of human thyrocytes. Our results provide proof-of-principle for effectiveness of small molecule agonists and antagonists for TSHR. We suggest that these small molecule ligands are lead compounds for the development of higher potency ligands that can be used as probes of TSHR biology with therapeutic potential.

C-peptide of preproinsulin-like peptide 7: localization in the rat brain and activity in vitro.

With the use of a rabbit polyclonal antiserum against a conserved region (54-118) of C-peptide of human preproinsulin-like peptide 7, referred to herein as C-INSL7, neurons expressing C-INSL7-immunoreactivity (irC-INSL7) were detected in the pontine nucleus incertus, the lateral or ventrolateral periaqueductal gray, dorsal raphe nuclei and dorsal substantia nigra. Immunoreactive fibers were present in numerous forebrain areas, with a high density in the septum, hypothalamus and thalamus. Pre-absorption of C-INSL7 antiserum with the peptide C-INSL7 (1 microg/ml), but not the insulin-like peptide 7 (INSL7; 1 microg/ml), also known as relaxin 3, abolished the immunoreactivity. Optical imaging with a voltage-sensitive dye bis-[1,3-dibutylbarbituric acid] trimethineoxonol (DiSBAC4(3)) showed that C-INSL7 (100 nM) depolarized or hyperpolarized a small population of cultured rat hypothalamic neurons studied. Ratiometric imaging studies with calcium-sensitive dye fura-2 showed that C-INSL7 (10-1000 nM) produced a dose-dependent increase in cytosolic calcium concentrations [Ca2+]i in cultured hypothalamic neurons with two distinct patterns: (1) a sustained elevation lasting for minutes; and (2) a fast, transitory rise followed by oscillations. In a Ca2+-free Hanks' solution, C-INSL7 again elicited two types of calcium transients: (1) a fast, transitory increase not followed by a plateau phase, and (2) a transitory rise followed by oscillations. INSL7 (100 nM) elicited a depolarization or hyperpolarization in a small population of hypothalamic neurons, and an increase of [Ca2+]i with two patterns that were dissimilar from that of C-INSL7. [125I]C-INSL7 bindings to rat brain membranes were inhibited by C-INSL7 in a dose-dependent manner; the Kd and Bmax. values were 17.7 +/- 8.2 nM and 45.4 +/- 20.5 fmol/mg protein. INSL7 did not inhibit [125I]C-INSL7 binding to rat brain membranes, indicating that C-INSL7 and INSL7 bind to distinct binding sites. Collectively, our result raises the possibility that C-INSL7 acts as a signaling molecule independent from INSL7 in the rat CNS.

Tourette's syndrome.

Tourette's syndrome (TS) is a chronic disorder characterized by motor and vocal tics and a variety of associated behaviour disorders. Because current therapy is often unsatisfactory, there is expanding interest in new therapeutic strategies that are more effective, cause less side effects and ameliorate not only tics but also behavioural problems. From anecdotal reports and preliminary controlled studies it is suggested that - at least in a subgroup of patients - cannabinoids are effective in the treatment of TS. While most patients report beneficial effects when smoking marijuana (Cannabis sativa L.), available clinical trials have been performed using oral Δ9-tetrahydrocannabinol (THC). In otherwise treatment-resistant TS patients, therefore, therapy with THC should not be left unattempted. To date, it is unknown whether other drugs that interact with the endocannabinoid receptor system might be more effective in the treatment of TS than smoked marijuana or pure THC. Since it has been suggested that abnormalities within the endocannabinoid receptor system might underlie TS pathophysiology, it would be of interest to investigate the effect of substances that for example bind more selectively to the central cannabinoid receptor or inhibit the uptake or the degradation of different endocannabinoids.

Agonist-specific coupling of growth hormone secretagogue receptor type 1a to different intracellular signaling systems. Role of adenosine.

The growth hormone secretagogue receptor subtype 1a (GHSR-1a) is involved in biological actions of ghrelin by triggering intracellular second messengers coupled to heterotrimeric G-protein complex involving Galpha(q/11). Adenosine is a partial agonist of the GHSR-1a, binding to a binding pocket distinct from the one described for ghrelin. This suggests a variety of functions for the poorly understood GHSR1a receptor. In this work, a sequential analysis of the pathways involved in the regulation of GHSR-1a signaling was undertaken to characterize the intracellular calcium mobilization that is observed following adenosine binding. The results showed that adenosine induced, in a dose-dependent manner, a calcium mobilization from IP(3)-sensitive intracellular stores since the IP(3) receptor blocker 2-APB was able to suppress the calcium response. However, adenosine did not show any effect in the formation of inositol phosphates. The calcium-mobilizing activity was blocked after preincubation of cells with CTX, the inhibitor of adenylate cyclase MDL-12,330A and the protein kinase A blocker H-89. Furthermore, the administration of adenosine stimulated cAMP production. Based on the experimental data, a signaling pathway is proposed involving adenylate cyclase and protein kinase A, which causes phosphorylation of the IP(3) receptor, with a cross-talk between the signaling pathways activated by ghrelin and adenosine. The data described in this report suggest that GHSR-1a is able to activate different intracellular second-messenger systems depending on the agonist that activates it. The regulation of the ghrelin-activated earliest signaling pathways by adenosine may have unexpected implications in the GHSR-1a actions.