Martin Missbach

Pineal gland hormone melatonin binds and activates an orphan of the nuclear receptor superfamily.

Nuclear orpan receptors are members of the superfamily of structurally related, ligand-inducible transcription factors for which no ligand has yet been identified. Over the past few years many nuclear orphan receptors have been cloned, but only for the retinoid X receptor (RXR) has a natural ligand (9-cis-retinoic acid) been found. Here we report the identification of melatonin as a ligand for the recently cloned orphan receptor retinoid Z receptor beta (RZR beta). We found RZR beta expression in the rat brain nearly coincident with binding sites for the pineal gland hormone melatonin (5-methoxy-N-acetyltryptamine). We show here binding and activation of RZR beta by melatonin with Kd and EC50 values in the low nanomolar range. A nuclear signaling pathway for melatonin may contribute to some of the diverse and profound effects of this hormone, for example, in the context of circadian rhythmicity.

A novel inhibitor of the tyrosine kinase Src suppresses phosphorylation of its major cellular substrates and reduces bone resorption in vitro and in rodent models in vivo

The tyrosine kinase Src has been implicated in the process of osteoclast-mediated bone resorption. Here, we describe a novel class of Src inhibitors, substituted 5,7-diphenyl-pyrrolo[2,3-d]pyrimidines, and characterize one of them, CGP77675, in vitro and in models of bone resorption in vivo. In vitro, CGP77675 inhibited phosphorylation of peptide substrates and autophosphorylation of purified Src (concentration producing half-maximal inhibition [IC50] values 5-20 and 40 nmol/L, respectively). The compound was selective toward other protein kinases: the Src IC50 value was lower than those for Cdc2 (>500-fold), epidermal growth factor (EGF) receptor (7.5-fold), and vascular endothelial growth factor receptor (>50-fold), and for v-Abl (15-fold) and focal adhesion kinase (Fak) (>25-fold). The Src kinase family members Lck and Yes were inhibited with IC50 values 20-fold higher than or equal to Src. To measure the inhibition of cellular Src activity, we identified the major tyrosine-phosphorylated proteins in an Src-overexpressing cell line IC8.1 as Src, Fak, and paxillin. CGP77675 potently inhibited tyrosine phosphorylation of the Src substrates Fak and paxillin, but had much less effect on Src (IC50 values 0.3, 0.5, and 5.7 micromol/L). The phosphorylation of Src in IC8.1 cells reflected phosphorylation of the negative regulatory tyrosine 527 (Y527); thus, the inhibitor was selective against the Y527 C-terminal Src kinase Csk. In osteoblastic MC3T3-E1 cells, CGP77675 inhibited signaling induced by PDGF at the receptor level, but not signaling by EGF, basic fibroblast growth factor, insulin-like growth factor-1, and phorbol 12-myristate 13-acetate. The effect of CGP77675 on bone resorption was evaluated in vitro and in vivo. The parathyroid hormone-induced bone resorption in rat fetal long bone cultures was inhibited with an IC50 of 0.8 micromol/L. CGP77675 dose-dependently reduced the hypercalcemia induced in mice by interleukin-1beta and partly prevented bone loss and microarchitectural changes in young ovariectomized rats, showing that the protective effect on bone was exerted via the inhibition of bone resorption. Thus, specific Src family kinase inhibitors may be useful for the treatment of diseases associated with elevated bone loss.

Thiazolidine Diones, Specific Ligands of the Nuclear Receptor Retinoid Z Receptor/Retinoid Acid Receptor-related Orphan Receptor α with Potent Antiarthritic Activity

Rat adjuvant arthritis is a chronic T cell-dependent autoimmune disease with many similarities to rheumatoid arthritis. We have identified a class of thiazolidine diones with high potency in suppressing chronic inflammation and joint destruction in this experimental model. The lead compound CGP 52608 (1-(3-allyl-4-oxothiazolidine-2-ylidene)-4-methylthiosemicarbazone) exhibits antiarthritic activity at daily oral doses between 0.01 and 1 mg/kg and was shown to specifically activate the retinoid Z receptor/retinoid acid receptor-related orphan receptor α (RZR/RORα) in low nanomolar concentrations. This receptor is a novel member of the superfamily of ligand-inducible transcription factors, and we have recently identified the pineal gland hormone melatonin as a natural ligand. Structure-activity relationship studies with 13 closely related analogues of CGP 52608 revealed a striking correlation between RZR/RORα activation and antiarthritic activity. We therefore suggest that nuclear signaling via RZR/RORα is a key mechanism in mediating the antiarthritic effects of these thiazolidine diones and may open a novel therapeutic approach for the treatment of rheumatoid arthritis and other autoimmune diseases. The existence of a nuclear melatonin receptor may lead to a better understanding of the immunomodulatory actions of melatonin.

7-Alkyl- and 7-cycloalkyl-5-aryl-pyrrolo[2,3-d]pyrimidines--potent inhibitors of the tyrosine kinase c-Src.

7-Substituted-5-aryl-pyrrolo[2,3-d]pyrimidines have been prepared starting from alpha-bromoacetophenones. These compounds represent a novel class of potent inhibitors of the tyrosine kinase pp60(c-Src) with good specificity towards other tyrosine kinases (EGF-R, v-Abl).

7-Pyrrolidinyl- and 7-piperidinyl-5-aryl-pyrrolo[2,3-d]pyrimidines--potent inhibitors of the tyrosine kinase c-Src.

7-Heterocyclyl-5-aryl-pyrrolo[2,3-d]pyrimidines represent a new class of highly potent and selective inhibitors of the tyrosine kinase pp60(c-Src).

Substituted 5,7-diphenyl-pyrrolo[2,3d]pyrimidines: potent inhibitors of the tyrosine kinase c-Src.

5,7-Diphenyl-pyrrolo[2,3d]pyrimidines represent a new class of highly potent inhibitors of the tyrosine kinase c-Src (IC50 < 50 nM) with specificity against a panel of different tyrosine kinases. The substitution pattern on the two phenyl rings determines potency and specificity and provides a means to modulate cellular activity.

1-Alkyl-4-phenyl-6-alkoxy-1H-quinazolin-2-ones: a novel series of potent calcium-sensing receptor antagonists.

Parathyroid hormone (PTH) is an effective bone anabolic agent. However, only when administered by daily sc injections exposure of short duration is achieved, a prerequisite for an anabolic response. Instead of applying exogenous PTH, mobilization of endogenous stores of the hormone can be envisaged. The secretion of PTH stored in the parathyroid glands is mediated by a calcium sensing receptor (CaSR) a GPCR localized at the cell surface. Antagonists of CaSR (calcilytics) mimic a state of hypocalcaemia and stimulate PTH release to the bloodstream. Screening of the internal compound collection for inhibition of CaSR signaling function afforded 2a. In vitro potency could be improved >1000 fold by optimization of its chemical structure. The binding mode of our compounds was predicted based on molecular modeling and confirmed by testing with mutated receptors. While the compounds readily induced PTH release after iv application a special formulation was needed for oral activity. The required profile was achieved by using microemulsions. Excellent PK/PD correlation was found in rats and dogs. High levels of PTH were reached in plasma within minutes which reverted to baseline in about 1-2 h in both species.

N7-Substituted-5-aryl-pyrrolo[2,3-d]pyrimidines Represent a Versatile Class of Potent Inhibitors of the Tyrosine Kinase c-Src

5-Aryl-pyrrolo[2,3-d]pyrimidines incorporating different N(7)-substituents have been prepared and evaluated for their inhibitory potency towards the tyrosine kinase c-Src. Optimization of these compounds resulted in highly potent c-Src inhibitors, some (e.g. 4g, 6g, 7h, 8l) with excellent specificity towards other receptor and nonreceptor tyrosine kinases. In addition compounds 4g, 5b and 5c are characterized by a good pharmacokinetic profile.

Phenothiazines are potent inhibitors of osteoclastic bone resorption

1. We have previously shown that promethazine inhibits osteoclastic bone resorption in the in vitro bone slice assay (IC50 = 0.8 microM), but the mechanism(s) involved are unclear. 2. We have now tested the effects on osteoclast activity of five other structurally related compounds. Phenothiazine, chlorpromazine, amitriptyline, phenazine, and phenoxazine had IC50 values of 0.8, 0.9, 6, 7, and > 10 microM, respectively, in the bone slice assay, indicating that the basic phenothiazine structural element itself is important for osteoclast inhibitory activity. 3. The results are discussed in terms of the known effects of phenothiazines on plasma membrane fluidity, blocking of ion channels, and inhibition of calmodulin.

Penta-substituted benzimidazoles as potent antagonists of the calcium-sensing receptor (CaSR-antagonists).

A series of novel benzimidazole derivatives has been designed via a scaffold morphing approach based on known calcilytics chemotypes. Subsequent lead optimisation led to the discovery of penta-substituted benzimidazoles that exhibit attractive in vitro and in vivo calcium-sensing receptor (CaSR) inhibitory profiles. In addition, synthesis and structure-activity relationship data are provided.