High Throughput Screening for Ligands for Seven Transmembrane-spanning Receptors

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Seven transmembrane-spanning receptors (7TMRs or G protein-coupled receptors, GPCRs) represent the largest family of signal-transducing molecules known. 7TMRs convey signals for light and many extracellular regulatory molecules, such as, hormones, growth factors and neurotransmitters, that regulate every cell in the body. Dysregulation of 7TMRs has been found in a growing number of human diseases and 7TMRs have been estimated to be the targets of more than 30% of the drugs used in clinical medicine today. Thus, discovery of probes/drugs for 7TMRs is an important goal of biomedical research. We use high throughput screening (HTS) for small molecule ligands (SMLs) for 7TMRs with the receptors for thyroid-stimulating hormone (TSH-R) and thyrotropin-releasing hormone (TRH-R). During this year, we continued our development of these SMLs. 1) We previously reported on a SML agonist for TSH-R that could be used in an animal model to stimulate an increase in radioactive iodine uptake by the thyroid gland and in thyroid hormone secretion after oral administration. We had recognized that the SML was a racemic mixture of two enantiomers. We now have shown that the two enantiomers can be separated and that they exhibit different potencies in vitro and, more importantly, that one is far more effective than the other in vivo. We have now discovered a formulation that increases the bioavailability of the more active enantiomer. This new formulation is highly active in stimulating the thyroid glands of mice and rats after oral administration. This is a big step in the pre-clinical development of this drug. 2) We previously reported that a G protein-independent pathway, which uses beta-arrestin-1 as a signal transduction molecule, was important in the signaling by the TSH-R in bone precursor cells. We have now completed a high-throughput screen (in collaboration with NCATS) for SMLs that activate the TSH-R via a beta-arrestin-1 dependent, G protein-independent mechanism and found several lead compounds. These compounds not only have activity by themselves but act a positive allosteric modulators (PAMs) to increase the activity of TSH. We are currently working with chemists at NCATS to improve the pharmacological characteristics of these lead compounds. We think these compounds may be lead drugs for the treatment of osteoporosis in humans. 3) In preliminary studies, it was previously shown that TRH itself when administered orally to patients with cancer-related fatigue (CRF) relieved some of their fatigue symptoms. However, TRH is not a good drug as it is rapidly degraded in the circulation and does not cross the blood brain barrier efficiently. We were intrigued by this observation and with a goal eventually to develop new TRH-R agonists, we decided to develop a mouse model of fatigue to test the activities of new compounds on fatigue. We have now developed a modified treadmill test for fatigue and showed that it reported quantitative readouts of fatigue in mice. We are now in the process of testing new TRH-R ligands in fatigue induced by cancer burden, chemotherapeutic agents and radiation.