ML067 : TSHR (Thyroid Stimulating Hormone Receptor) Agonist
ML067
Target Name
Thyroid Stimulating Hormone Receptor
Target Alias
TSHR
Target Class
G-protein Coupled Receptor
Mechanism of Action
Agonist of TSHR
Biological / Disease Relevance
Thyroid homeostasis, TSHR biology, G-protein Coupled Receptor (GPCR)
In vitro activity
TSHR cAMP assay (EC50)In vitro activity
TSHR TR-FRET assay (EC50)Target Information
Thyroid Stimulating Hormone (TSH) is an α/β heterodimeric glycoprotein hormone secreted from the anterior pituitary gland which belongs to the glycoprotein hormone family, including Chorionic Gonadotropin (CG), Luteinizing Hormone (LH), and Follicle Stimulating Hormone (FSH). TSH binds to the TSH receptor (TSHR), which couples preferentially to the G-alpha (s) (Gs) protein, resulting in activation of adenylate cyclase and increase in cyclic adenosine 3’, 5’ monophosphate (cAMP). THSR is mainly expressed in thyroid follicular cells and regulates their growth and function. Recombinant TSH is currently used for the diagnosis in patients with thyroid cancer receiving thyroid hormone suppression therapy, and for screening of residual tumor after surgery, but it is expensive and must be administered intramuscularly. An orally active small molecule TSHR agonist would serve as a valuable research tool for studying TSHR pharmacology and physiology, and would have multiple advantages for therapeutic applications. However, no selective small molecule agonist of the TSHR exists, and no small molecule screen for TSHR agonists has ever been reported. This cell-based assay utilized a cyclic nucleotide gated ion channel (CNG) as a biosensor for measurement of increase in intracellular cAMP level stimulated by TSHR agonists. The HEK293 cell line expressing human TSHR and a modified CNG was used as the primary screen assay. The membrane potential dye was used to detect membrane depolarization upon the activation of CNG by increased cAMP level. This assay was optimized in a homogenous 1536-well plate format.
Properties
ML067
MLS000098157
| Physical & chemical properties | ||||
|---|---|---|---|---|
| Molecular Weight | 317.4 g/mol | |||
| Molecular Formula | C18H23NO4 | |||
| cLogP | 2.8 | |||
| PSA | 68.5 Ų | |||
| Storage | ||||
| Solubility | 10 mM stock in DMSO | |||
| CAS Number | 380487-05-8 | |||
SMILES:
C1C2CC3CC1CC(C2)(C3)C(=O)OCC(=O)NCC4=CC=CO4
InChI:
InChI=1/C18H23NO4/c20-16(19-10-15-2-1-3-22-15)11-23-17(21)18-7-12-4-13 (8-18)6-14(5-12)9-18/h1-3,12-14H,4-11H2,(H,19,20)/f/h19H
InChIKey:
MOSOSUPDUHNNDQ-UHFFFAOYSA-N
Activity
Summary activity statement /
ML067 (MLS000098157, SID 26752280, CID 2998659) is found to activate the TSHR TR-FRET based cAMP assay but not the FSHR and LHR, indicating selectivity.
In vitro activity - Selectivity Assay
| TSHR MP (AC50) | FSHR (AC50) | LHR (AC50) | |
|---|---|---|---|
|
ML067 |
2.0 uM | > 100 uM | > 100 uM |
Summary /
ML067 (MLS000098157, SID 26752280, CID 2998659) is observed to selectively activate TSHR expressing cell line (membrane potential, MP assay) in a dose response manner with a low micromolar EC50 (Figure 1). No activation was observed in the counterscreen parental cell line. Moreover, this probe is inactive against two other closely related GPCR family members Follicle Stimulating Hormone Receptor (FSHR) and Luteinizing Hormone Receptor (LHR) TR-FRET based assays. Analogs for the probe were also purchased and tested (Figure 2). Compounds NCGC00161862, NCGC00161863, NCGC00161864 were inactive in all three cell lines. Compounds NCGC00161866 and NCGC00161867 activated TSHR with even higher efficacies than ML069 (MLS000098157) (61%, 65%, versus 40% respectively) but similar potencies (15.8, 10.0, and 8.5 uM).
Fig 1. A representative positive compound, MLS000098157, in the TSHR cell line (red squares) and parental cells (black triangles). The EC50 value in the TSHR cell line was 0.79 uM. It was not active in the parental cell line. The sample was reordered and confirmed EC50 value of 2.0 uM.
Fig. 2. Concentration-response curves of representative compounds in TR-FRET based cAMP assays in the cell lines with TSHR (red triangles), FSHR (black squares), and LHR (open circles). MLS000098157 showed activity specific to the TSH cell line and had an EC50 value of 12.6 uM. NCGC00161867-01 had an EC 50 value of 10.0 uM in the TSH cell line and was inactive in FSH and LH. NCGC00161866-01 had an EC50 value of 14.1 uM in the TSH cell line and was inactive in FSH and LH. NCGC00161862-01 and NCGC00161863-01 were inactive in all 3 cell lines.
References
- Quantitative High-Throughput Screen for Agonists of Thyroid Stimulating Hormone Receptor: Summary
- Titus S, Huang W, Marugan J, et al. Identification of Potent and Selective Thyroid Stimulating Hormone Receptor Agonists. 2009 Sep 1 [Updated 2011 Mar 25]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK56234/