Background: Novel small molecular ligands (SMLs) to the thyrotropin receptor (TSHR) have potential as improved molecular probes and as therapeutic agents for the treatment of thyroid dysfunction and thyroid cancer. Methods: To identify novel SMLs to the TSHR, we developed a transcription-based luciferase-cAMP highthroughput screening system and we screened 48,224 compounds from a 100K library in duplicate. Results: We obtained 62 hits using the cut-off criteria of the mean -three standard deviations above the baseline. Twenty molecules with the greatest activity were rescreened against the parent CHO-luciferase cell for nonspecific activation, and we selected two molecules (MS437 and MS438) with the highest potency for further study. These lead molecules demonstrated no detectible cross-reactivity with homologous receptors when tested against luteinizing hormone (LH)/human chorionic gonadotropin receptor and follicle stimulating hormone receptor-expressing cells. Molecule MS437 had a TSHR-stimulating potency with an EC 50 of 13 · 10 -8 M, and molecule MS438 had an EC 50 of 5.3 · 10 -8 M. The ability of these small molecule agonists to bind to the transmembrane domain of the receptor and initiate signal transduction was suggested by their activation of a chimeric receptor consisting of an LHR ectodomain and a TSHR transmembrane. Molecular modeling demonstrated that these molecules bound to residues S505 and E506 for MS438 and T501 for MS437 in the intrahelical region of transmembrane helix 3. We also examined the G protein activating ability of these molecules using CHO cells co-expressing TSHRs transfected with luciferase reporter vectors in order to measure G sa , G bc , G aq , and G a12 activation quantitatively. The MS437 and MS438 molecules showed potent activation of G sa , G aq , and G a12 similar to TSH, but neither the small molecule agonists nor TSH showed activation of the G bc pathway. The small molecules MS437 and MS438 also showed upregulation of thyroglobulin (Tg), sodium iodine symporter (NIS), and TSHR gene expression. Conclusions: Pharmacokinetic analysis of MS437 and MS438 indicated their pharmacotherapeutic potential, and their intraperitoneal administration to normal female mice resulted in significantly increased serum thyroxine levels, which could be maintained by repeated treatments. These molecules can therefore serve as lead molecules for further development of powerful TSH agonists.

Chronic myeloid leukemia (CML) is characterized in 90% of patients by the presence of the reciprocal translocation t(9;22)(q34;q11) leading to the fusion of the BCR and ABL genes. Most patients with Philadelphia chromosome positive CML express either the e13a2 (b2a2) or e14a2 (b3a2) MBCR-ABL mRNA. Some variant cases have been reported expressing the fusion e1a2 (mBCR-ABL) or e19a2 (microBCR-ABL). Very rare atypical transcripts such as e13a3, e14a3 or e6a2 have been described. We report here a sixth case of a Ph positive patient with an e6a2 BCR-ABL fusion transcript and describe for the first time a chimeric molecule alternatively spliced for exon 5 of the BCR gene.

SR31747A is a new sigma ligand exhibiting immunosuppressive properties and antiproliferative activity on lymphocyte cells. Only two subtypes of sigma receptor, namely the sigma 1 receptor and emopamilbinding protein, have been characterised molecularly. Only the σ 1 receptor has been shown to bind (Z)Ncyclohexyl-N-ethyl-3-(3-chloro4-cyclohexylphenyl)propen-2-ylamine hydrochloride (SR31747A) with high affinity. It was demonstrated that the SR31747A effect on the inhibition of T-cell proliferation was consistent with a sigma 1 receptor-mediated event. In this report, binding experiments and sterol isomerase assays, using recombinant yeast strains, indicate that the recently cloned emopamil-binding protein is a new SR31747A-binding protein whose activity is inhibited by SR31747A. Sterol analyses reveal the accumulation of a ∆8-cholesterol isomer at the expense of cholesterol in SR31747A-treated cells, suggesting that cholesterol biosynthesis is inhibited by SR31747A at the ∆8-∆7 sterol isomerase step in animal cells. This observation is consistent with a sterol isomerase role of the emopamil-binding protein in the cholesterol biosynthetic pathway in animal cells. In contrast, there is no evidence for such a role of the sigma 1 receptor, in spite of the structural similarity shared by this protein and yeast sterol isomerase. We have found that SR31747A also exerts anti-proliferative effects at nanomolar concentrations on various established cell lines. The antiproliferative activity of SR31747A is reversed by cholesterol. Sterol-isomerase overproduction enhances resistance of CHO cells. This last observation strongly suggests that sterol isomerase is implicated in the antiproliferative effect of the drug in established cell lines.