The observation that levels of the mitogenic peptide endothelin-1 are elevated in the human coronary sinus after percutaneous transluminal coronary angioplasty (PTCA) has implicated endothelin-1 in the etiology of vascular restenosis. The present study examined this hypothesis in both an in vitro and an in vivo rat model of neointimal formation by using the novel nonpeptide endothelin receptor antagonist SB 209670. In vitro, endothelin-1 (1 nmol/L) induced a ninefold increase in rat aortic vascular smooth muscle [3Hlthymidine incorporation. This endothelin A receptor-mediated effect was completely inhibited by SB 209670 (IC50, 6.2±2.2 nmol/L). In vivo, acute intra-arterial administration of exogenous endothelin-1 (5 to 500 pmol/kg over a 30-minute period immediately after angioplasty) dose-dependently augmented the degree of neointimal formation (by up to 150% when assessed 14 days after surgery). This response was evident as early as 7 days after angioplasty. Hemodynamic studies indicated that this action was unrelated to a systemic pressor action of the peptide. Administration of SB 209670 (2.5 mg/kg IP, twice a day for 3 days before and for 2 weeks after surgery) reduced neointimal formation by '50% relative to control animals. Thus, the data indicate for the first time that (1) endothelin-1 promotes neointimal formation in vivo and (2) tensin II and norepinephrine, endothelin-1 is now recognized as playing an intrinsic role in the endogenous control of systemic hemodynamics.3 Since levels of endothelin-1 are elevated in the human coronary sinus after PTCA, it has been suggested that endothelin-1 is involved in the pathogenesis of vascular restenosis.4In addition to its actions on vascular smooth muscle tonus, endothelin-1 is also a potent mitogen in several cultured cell lines of both cardiovascular and noncarReceived February 1, 1994; accepted March 21, 1994.

A novel series of tertiary amines as retinoid-related orphan receptor gamma-t (RORγt) inverse agonists was discovered through agonist/inverse agonist conversion. The level of RORγt inhibition can be enhanced by modulating the conformational disruption of H12 in RORγt LBD. Linker exploration and rational design led to the discovery of more potent indole-based RORγt inverse agonists.KEYWORDS: RORγt, agonists, inverse agonists, Th17 cell differentiation, cocrystal structure, structure-based design R etinoid-related orphan receptor gamma-t (RORγt) is a member of the nuclear receptor superfamily. RORγt is a key regulator of the development and functions of T-helper 17 (Th17) cells which are implicated in the pathology of a variety of human inflammatory and autoimmune disorders. 1,2 The RORγt inhibitors have potential utility in controlling the activity of Th17 cells and can be developed as therapeutic agents for treatment of Th17-related autoimmune diseases. A few small molecule inhibitors of RORγt have been reported in the literature. 3−10 In this paper, we report the discovery of tertiary amines and indoles as potent RORγt inverse agonists using structure-and knowledge-based compound design.A high-throughput screen (HTS) of the GSK in-house compound collection using a RORγ fluorescence resonance energy transfer (FRET) assay 11 resulted in identification of thiazole amide 1 as a RORγt inverse agonist with IC 50 of 1.0 μM. The binding of 1 to the RORγt ligand binding domain (LBD) was confirmed with a thermal shift of 7.1°C in a thermal shift assay. 11 SAR exploration on the left-hand side (LHS) of 1 led to the identification of tertiary amine 2 as a potent RORγt agonist with a EC 50 of 0.02 μM in dual FRET assay (Scheme 1). 12 Dual FRET assay, using the same technology as the FRET assay but without adding a surrogate agonist, only relies on the basal level of RORγt activity and is able to measure both agonists and inverse agonists. Peptide profiling study using dual FRET assay showed that coactivator peptide (e.g., steroid receptor coactivator 1 (SRC1)) was recruited upon binding of 2 to RORγt LBD whereas corepressor peptide (e.g., nuclear receptor corepressor 2 (NCOR2)) was not. 12 Given the structure similarity of 1 and 2, we assume that they adopt a similar binding mode within RORγt LBD despite their difference as agonist and inverse agonist. To understand the binding mode of the chemical series, an in-silico docking study for 2 based on a reported RORγt crystal structure 13 was conducted.A RORγt LBD crystal structure (PDB accession code: 3KYT) was selected and processed for the docking study. A total of 40 poses with the best scores were obtained and visually inspected after docking with Surflex-Dock v2.3 14−16 in Sybyl 8.1, 17 among which the top 10 poses were found to be representative and thus further ranked using MM/GBSA 18−20 affinity scores based on the VSGB2.0 solvent model. 21,22 As a

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Selection and validation of novel molecular targets have become of paramount importance in light of the plethora of new potential therapeutic drug targets that have emerged from human gene sequencing. In response to this revolution within the pharmaceutical industry, the development of high-throughput methods in both biology and chemistry has been necessitated. This review addresses these technological advances as well as several new areas that have been created by necessity to deal with this new paradigm, such as bioinformatics, cheminformatics, and functional genomics. With many of these key components of future drug discovery now in place, it is possible to map out a critical path for this process that will be used into the new millennium.

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