Discovery of 3-Cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide: A Potent, Selective, and Orally Bioavailable Retinoic Acid Receptor-Related Orphan Receptor C2 Inverse Agonist.

The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.

Thyroid receptor ligands. Part 5: novel bicyclic agonist ligands selective for the thyroid hormone receptor beta.

Based on the examination of the crystal structure of rat TRbeta complexed with 3,5,3'-triiodo-l-thyronine (2) a novel TRbeta-selective indole derivative 6b was prepared and tested in vitro. This compound was found to be 14 times selective for TRbeta over TRalpha in binding and its beta-selectivity could be rationalized through the comparison of the X-ray crystallographic structures of 6b complexed with TRalpha and TRbeta.

Thyroid receptor ligands. Part 4: 4'-amido bioisosteric ligands selective for the thyroid hormone receptor beta.

Based on the examination of the X-ray crystallographic structures of the LBD of TRalpha and TRbeta in complex with KB-141 (2), a number of novel 4'-hydroxy bioisosteric thyromimetics were prepared. Optimal affinity and beta-selectivity (33 times), was found with a medium-sized alkyl-substituted amido group; iso-butyl (12c). It can be concluded that bioisosteric replacements of the 4'-hydroxy position represent a new promising class of TRbeta-selective synthetic thyromimetics.

Detection of phosphorylated peptides in proteomic analyses using microfluidic compact disk technology.

A compact disk (CD)-based microfluidic method for selective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinity chromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin and processed on the CD using nanoliter scale IMAC with and without subsequent in situ alkaline phosphatase treatment. This is followed by on-CD matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Dephosphorylation of the IMAC-enriched peptides allows selective phosphopeptide detection based on the differential mass maps generated (mass shifts of 80 Da or multiples of 80 Da). The CD contains 96 microstructures, each with a 16 nL IMAC microfluidic column. Movement of liquid is controlled by differential spinning of the disk. Up to 48 samples are distributed onto the CD in two equal sets. One set is for phosphopeptide enrichment only, the other for identical phosphopeptide enrichment but combined with in situ dephosphorylation. Peptides are eluted from the columns directly into MALDI target areas, still on the CD, using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into a MALDI mass spectrometer for analysis down to the femtomole level. The average success rate in phosphopeptide detection is over 90%. Applied to noncharacterized samples, the method identified two novel phosphorylation sites, Thr 735 and Ser 737, in the ligand-binding domain of the human mineralocorticoid receptor.

Thyroid receptor ligands. Part 2: Thyromimetics with improved selectivity for the thyroid hormone receptor beta.

A set of thyromimetics having improved selectivity for TR-beta1 were prepared by replacing the 3'-isopropyl group of 2 and 3 with substituents having increased steric bulk. From this limited SAR study, the most potent and selective compounds identified were derived from 2 and contained a 3'-phenyl moiety bearing small hydrophobic groups meta to the biphenyl link. X-ray crystal data of 15c complexed with TR-beta1 LBD shows methionine 442 to be displaced by the bulky R3' phenyl ethyl amide side chain. Movement of this amino acid side chain provides an expanded pocket for the bulky side chain while the ligand-receptor complex retains full agonist activity.

Thyroid receptor ligands. 1. Agonist ligands selective for the thyroid receptor beta1.

Endogenous thyroid receptor hormones 3,5,3',5'-tetraiodo-l-thyronine (T(4), 1) and 3,5,3'-triiodo-l-thyronine (T(3), 2) exert a significant effects on growth, development, and homeostasis in mammals. They regulate important genes in intestinal, skeletal, and cardiac muscles, the liver, and the central nervous system, influence overall metabolic rate, cholesterol and triglyceride levels, and heart rate, and affect mood and overall sense of well being. The literature suggests many or most effects of thyroid hormones on the heart, in particular on the heart rate and rhythm, are mediated through the TRalpha(1) isoform, while most actions of the hormones on the liver and other tissues are mediated more through the TRbeta(1) isoform of the receptor. Some effects of thyroid hormones may be therapeutically useful in nonthyroid disorders if adverse effects can be minimized or eliminated. These potentially useful features include weight reduction for the treatment of obesity, cholesterol lowering for treating hyperlipidemia, amelioration of depression, and stimulation of bone formation in osteoporosis. Prior attempts to utilize thyroid hormones pharmacologically to treat these disorders have been limited by manifestations of hyperthyroidism and, in particular, cardiovascular toxicity. Consequently, development of thyroid hormone receptor agonists that are selective for the beta-isoform could lead to safe therapies for these common disorders while avoiding cardiotoxicity. We describe here the synthesis and evaluation of a series of novel TR ligands, which are selective for TRbeta(1) over TRalpha(1). These ligands could potentially be useful for treatment of various disorders as outlined above. From a series of homologous R(1)-substituted carboxylic acid derivatives, increasing chain length was found to have a profound effect on affinity and selectivity in a radioreceptor binding assay for the human thyroid hormone receptors alpha(1) and beta(1) (TRalpha(1) and TRbeta(2)) as well as a reporter cell assay employing CHOK1-cells (Chinese hamster ovary cells) stably transfected with hTRalpha(1) or hTRbeta(1) and an alkaline phosphatase reporter-gene downstream thyroid response element (TRAFalpha(1) and TRAFbeta(1)). Affinity increases in the order formic, acetic, and propionic acid, while beta-selectivity is highest when the R(1) position is substituted with acetic acid. Within this series 3,5-dibromo-4-[(4-hydroxy-3-isopropylphenoxy)phenyl]acetic acid (11a) and 3,5-dichloro-4-[(4-hydroxy-3-isopropylphenoxy)phenyl]acetic acid (15) were found to reveal the most promising in vitro data based on isoform selectivity and were selected for further in vivo studies. The effect of 2, 11a, and 15 in a cholesterol-fed rat model was monitored including potencies for heart rate (ED(15)), cholesterol (ED(50)), and TSH (ED(50)). Potency for tachycardia was significantly reduced for the TRbeta selective compounds 11a and 15 compared with 2, while both 11a and 15 retained the cholesterol-lowering potency of 2. This left an approximately 10-fold therapeutic window between heart rate and cholesterol, which is consistent with the action of ligands that are approximately 10-fold more selective for TRbeta(1). We also report the X-ray crystallographic structures of the ligand binding domains of TRalpha and TRbeta in complex with 15. These structures reveal that the single amino acid difference in the ligand binding pocket (Ser277 in TRalpha or Asn331 in TRbeta) results in a slightly different hydrogen bonding pattern that may explain the increased beta-selectivity of 15.