Discovery of the TLR7/8 Antagonist MHV370 for Treatment of Systemic Autoimmune Diseases.

Toll-like receptor (TLR) 7 and TLR8 are endosomal sensors of the innate immune system that are activated by GU-rich single stranded RNA (ssRNA). Multiple genetic and functional lines of evidence link chronic activation of TLR7/8 to the pathogenesis of systemic autoimmune diseases (sAID) such as Sjögren's syndrome (SjS) and systemic lupus erythematosus (SLE). This makes targeting TLR7/8-induced inflammation with small-molecule inhibitors an attractive approach for the treatment of patients suffering from systemic autoimmune diseases.

Discovery of potent, orally bioavailable in vivo efficacious antagonists of the TLR7/8 pathway.

Antagonism of the Toll-like receptors (TLRs) 7 and TLR8 has been hypothesized to be beneficial to patients suffering from autoimmune conditions. A phenotypic screen for small molecule antagonists of TLR7/8 was carried out in a murine P4H1 cell line. Compound 1 was identified as a hit that showed antagonistic activity on TLR7 and TLR8 but not TLR9, as shown on human peripheral blood mononuclear cells (hPBMCs).

Tetrahydroquinolin-3-yl carbamate glucocorticoid receptor agonists with reduced PEPCK activation.

Continuing studies on tetrahydroquinoline glucocorticoid receptor anti-inflammatory agents lead to the identification of several tetrahydroquinolin-3-yl carbamates that exhibited steroid-like activity in in vitro transrepression assays with reduced transactivation of phosphoenol pyruvate carboxykinase (PEPCK), a key enzyme in the gluconeogenesis pathway.

Nonsteroidal 2,3-dihydroquinoline glucocorticoid receptor agonists with reduced PEPCK activation.

Continuing studies based on dihydroquinoline glucocorticoid receptor agonists lead to the discovery of a series of C4-oxime analogs. Representative compounds exhibited potent transrepression activity with minimal transactivation of phosphoenolpyruvate caboxykinase (PEPCK), a key protein in the gluconeogenesis pathway. These compounds represent promising leads in identifying GR agonists with high anti-inflammatory activity and attenuated potential for glucose elevation.

Discovery of orally available tetrahydroquinoline-based glucocorticoid receptor agonists.

A series of tetrahydroquinoline derivatives were synthesized and profiled for their ability to act as glucocorticoid receptor selective modulators. Structure-activity relationships of the tetrahydroquinoline B-ring lead to the discovery of orally available GR-selective agonists with high in vivo activity.

Tetrahydroquinoline glucocorticoid receptor agonists: discovery of a 3-hydroxyl for improving receptor selectivity.

We have previously disclosed a series of glucocorticoid receptor (GR) ligands derived from 6-indole-1,2,3,4-tetrahydroquinolines through structure-activity relationship (SAR) of the pendent C6-indole ring. In parallel with this effort, we now report SAR of the tetrahydroquinoline A-ring that identified the importance of a C3 hydroxyl in improving GR selectivity within a series of non-steroidal GR agonists.

Activation of the orphan nuclear receptor steroidogenic factor 1 by oxysterols.

Steroidogenic factor 1 (SF-1), an orphan member of the intracellular receptor superfamily, plays an essential role in the development and function of multiple endocrine organs. It is expressed in all steroidogenic tissues where it regulates the P450 steroidogenic genes to generate physiologically active steroids. Although many of the functions of SF-1 in vivo have been defined, an unresolved question is whether a ligand modulates its transcriptional activity.

Identification and characterization of a versatile retinoid response element (retinoic acid receptor response element-retinoid X receptor response element) in the mouse tissue transglutaminase gene promoter.

Tissue transglutaminase (transglutaminase type II) is an intracellular protein cross-linking enzyme that accumulates in connective tissue and in cells undergoing apoptosis. Retinoids regulate the transcription of the mouse tissue transglutaminase gene via activation of regulatory elements contained within 4 kilobases of the 5'-end of the gene. Co-transfection studies with retinoid receptor expression vectors in CV-1 cells demonstrated that the mouse tissue transglutaminase promoter is activated by ligand activation of either retinoic acid receptor-retinoid X receptor (RAR.

Vector PCR.

A strategy employing PCR technology to facilitate the amplification of DNA segments inserted in plasmid vectors is described. Nine oligonucleotide primers specific for vector sequences bracketing cloning sites in seven commonly used vectors were designed. We used these primers for the amplification of 25 different inserts ranging in size from 0.

Microvillin: a 200-kilodalton protein in microvilli of rat mammary cells detected by a monoclonal antibody.

We have isolated a monoclonal antibody that was raised against rat mammary tumor cells. The antibody stains the lumenal edge of mammary duct epithelial cells. It immunoprecipitates a 200-kilodalton nonglycosylated, nonphosphorylated protein, which, by immunofluorescence and electron microscopy, can be localized in microvilli.

Developmental regulation of cytokeratins in cells of the rat mammary gland studied with monoclonal antibodies.

We have isolated two monoclonal antibodies to cytokeratins and determined their cell specificities. They display interesting localization within the rat mammary gland. One (1A10) shows specificity for myoepithelial cells; the other (24B42) is specific for lumenal cells at various stages of development.

Epithelial cell types and their evolution in the rat mammary gland determined by immunological markers.

We have used immunological reagents for characterizing epithelial cell types in the adult rat mammary gland. Some were sera against purified proteins; others were monoclonal antibodies to mammary cells. On the basis of marker distribution we have identified 10 different cell types.