Lisa A. Orband-Miller

HIV protease inhibitors block adipogenesis and increase lipolysis in vitro

AIDS therapies employing HIV protease inhibitors (PIs) are associated with changes in fat metabolism. However, the cellular mechanisms affected by PIs are not clear. Thus, the affects of PIs on adipocyte differentiation were examined in vitro using C3H10T1/2 stem cells. In these cells the PIs, nelfinavir, saquinavir, and ritonavir, reduced triglyceride accumulation, lipogenesis, and expression of the adipose markers, aP2 and LPL. Histological analysis revealed nelfinavir, saquinavir and ritonavir treatment decreased oil red O-staining of cytoplasmic fat droplets. Inhibition occurred in the presence of the RXR agonist LGD1069, indicating the inhibitory effects were not due to an absence of RXR ligand. Moreover, these three PIs increased acute lipolysis in adipocytes. In contrast, two HIV PIs, amprenavir and indinavir, had little effect on lipolysis, lipogenesis, or expression of aP2 and LPL. Although, saquinavir, inhibited ligand-binding to PPARgamma with an IC(50) of 12.7+/-3.2 microM, none of the other PIs bound to the nuclear receptors RXRalpha or PPARgamma, (IC(50)s>20 microM), suggesting that inhibition of adipogenesis is not due to antagonism of ligand binding to RXRalpha or PPARgamma. Taken together, the results suggest that some, but not all, PIs block adipogenesis and stimulate fat catabolism in vitro and this may contribute to the effects of PIs on metabolism in the clinic.

Identification of peroxisome proliferator-activated receptor ligands from a biased chemical library

BACKGROUND The peroxisome proliferator-activated receptors (PPARs) were cloned as orphan members of the nuclear receptor superfamily of transcription factors. The identification of subtype-selective ligands for PPARalpha and PPARgamma has led to the discovery of their roles in the regulation of lipid metabolism and glucose homeostasis. No subtype-selective PPARdelta ligands are available and the function of this subtype is currently unknown. RESULTS A three-component library was designed in which one of the monomers was biased towards the PPARs and the other two monomers were chosen to add chemical diversity. Synthesis and screening of the library resulted in the identification of pools with activity on each of the PPAR subtypes. Deconvolution of the pools with the highest activity on PPARdelta led to the identification of GW 2433 as the first high-affinity PPARdelta ligand. [3H]GW 2433 is an effective radioligand for use in PPARdelta competition-binding assays. CONCLUSIONS The synthesis of biased chemical libraries is an efficient approach to the identification of lead molecules for members of sequence-related receptor families. This approach is well suited to the discovery of small-molecule ligands for orphan receptors.

X-ray crystal structures of the estrogen-related receptor-gamma ligand binding domain in three functional states reveal the molecular basis of small molecule regulation.

X-ray crystal structures of the ligand binding domain (LBD) of the estrogen-related receptor-γ (ERRγ) were determined that describe this receptor in three distinct states: unliganded, inverse agonist bound, and agonist bound. Two structures were solved for the unliganded state, the ERRγ LBD alone, and in complex with a coregulator peptide representing a portion of receptor interacting protein 140 (RIP140). No significant differences were seen between these structures that both exhibited the conformation of ERRγ seen in studies with other coactivators. Two structures were obtained describing the inverse agonist-bound state, the ERRγ LBD with 4-hydroxytamoxifen (4-OHT), and the ERRγ LBD with 4-OHT and a peptide representing a portion of the silencing mediator of retinoid and thyroid hormone action protein (SMRT). The 4-OHT structure was similar to other reported inverse agonist bound structures, showing reorientation of phenylalanine 435 and a displacement of the AF-2 helix relative to the unliganded structures with little other rearrangement occurring. No significant changes to the LBD appear to be induced by peptide binding with the addition of the SMRT peptide to the ERRγ plus 4-OHT complex. The observed agonist-bound state contains the ERRγ LBD, a ligand (GSK4716), and the RIP140 peptide and reveals an unexpected rearrangement of the phenol-binding residues. Thermal stability studies show that agonist binding leads to global stabilization of the ligand binding domain. In contrast to the conventional mechanism of nuclear receptor ligand activation, activation of ERRγ by GSK4716 does not appear to involve a major rearrangement or significant stabilization of the C-terminal helix.

Pharmacologic Inhibition of RORγt Regulates Th17 Signature Gene Expression and Suppresses Cutaneous Inflammation In Vivo

IL-17–producing CD4+Th17 cells, CD8+Tc17 cells, and γδ T cells play critical roles in the pathogenesis of autoimmune psoriasis. RORγt is required for the differentiation of Th17 cells and expression of IL-17. In this article, we describe a novel, potent, and selective RORγt inverse agonist (TMP778), and its inactive diastereomer (TMP776). This chemistry, for the first time to our knowledge, provides a unique and powerful set of tools to probe RORγt-dependent functions. TMP778, but not TMP776, blocked human Th17 and Tc17 cell differentiation and also acutely modulated IL-17A production and inflammatory Th17-signature gene expression (Il17a, Il17f, Il22, Il26, Ccr6, and Il23) in mature human Th17 effector/memory T cells. In addition, TMP778, but not TMP776, inhibited IL-17A production in both human and mouse γδ T cells. IL-23–induced IL-17A production was also blocked by TMP778 treatment. In vivo targeting of RORγt in mice via TMP778 administration reduced imiquimod-induced psoriasis-like cutaneous inflammation. Further, TMP778 selectively regulated Th17-signature gene expression in mononuclear cells isolated from both the blood and affected skin of psoriasis patients. In summary, to our knowledge, we are the first to demonstrate that RORγt inverse agonists: 1) inhibit Tc17 cell differentiation, as well as IL-17 production by γδ T cells and CD8+ Tc17 cells; 2) block imiquimod-induced cutaneous inflammation; 3) inhibit Th17 signature gene expression by cells isolated from psoriatic patient samples; and 4) block IL-23–induced IL-17A expression. Thus, RORγt is a tractable drug target for the treatment of cutaneous inflammatory disorders, which may afford additional therapeutic benefit over existing modalities that target only IL-17A.

GSK4112, a Small Molecule Chemical Probe for the Cell Biology of the Nuclear Heme Receptor Rev-erbα

The identification of nonporphyrin ligands for the orphan nuclear receptor Rev-erbα will enable studies of its role as a heme sensor and regulator of metabolic and circadian signaling. We describe the development of a biochemical assay measuring the interaction between Rev-erbα and a peptide from the nuclear receptor corepressor-1 (NCoR). The assay was utilized to identify a small molecule ligand for Rev-erbα, GSK4112 (1), that was competitive with heme. In cells, 1 profiled as a Rev-erbα agonist in cells to inhibit expression of the circadian target gene bmal1. In addition, 1 repressed the expression of gluconeogenic genes in liver cells and reduced glucose output in primary hepatocytes. Therefore, 1 is useful as a chemical tool to probe the function of Rev-erbα in transcriptional repression, regulation of circadian biology, and metabolic pathways. Additionally, 1 may serve as a starting point for design of Rev-erbα chemical probes with in vivo pharmacological activity.

Quinoline 3-sulfonamides inhibit lactate dehydrogenase A and reverse aerobic glycolysis in cancer cells

BackgroundMost normal cells in the presence of oxygen utilize glucose for mitochondrial oxidative phosphorylation. In contrast, many cancer cells rapidly convert glucose to lactate in the cytosol, a process termed aerobic glycolysis. This glycolytic phenotype is enabled by lactate dehydrogenase (LDH), which catalyzes the inter-conversion of pyruvate and lactate. The purpose of this study was to identify and characterize potent and selective inhibitors of LDHA.MethodsHigh throughput screening and lead optimization were used to generate inhibitors of LDHA enzymatic activity. Effects of these inhibitors on metabolism were evaluated using cell-based lactate production, oxygen consumption, and 13C NMR spectroscopy assays. Changes in comprehensive metabolic profile, cell proliferation, and apoptosis were assessed upon compound treatment.Results3-((3-carbamoyl-7-(3,5-dimethylisoxazol-4-yl)-6-methoxyquinolin-4-yl) amino) benzoic acid was identified as an NADH-competitive LDHA inhibitor. Lead optimization yielded molecules with LDHA inhibitory potencies as low as 2 nM and 10 to 80-fold selectivity over LDHB. Molecules in this family rapidly and profoundly inhibited lactate production rates in multiple cancer cell lines including hepatocellular and breast carcinomas. Consistent with selective inhibition of LDHA, the most sensitive breast cancer cell lines to lactate inhibition in hypoxic conditions were cells with low expression of LDHB. Our inhibitors increased rates of oxygen consumption in hepatocellular carcinoma cells at doses up to 3 microM, while higher concentrations directly inhibited mitochondrial function. Analysis of more than 500 metabolites upon LDHA inhibition in Snu398 cells revealed that intracellular concentrations of glycolysis and citric acid cycle intermediates were increased, consistent with enhanced Krebs cycle activity and blockage of cytosolic glycolysis. Treatment with these compounds also potentiated PKM2 activity and promoted apoptosis in Snu398 cells.ConclusionsRapid chemical inhibition of LDHA by these quinoline 3-sulfonamids led to profound metabolic alterations and impaired cell survival in carcinoma cells making it a compelling strategy for treating solid tumors that rely on aerobic glycolysis for survival.

Small molecule agonists of the orphan nuclear receptors steroidogenic factor-1 (SF-1, NR5A1) and liver receptor homologue-1 (LRH-1, NR5A2).

The crystal structure of LRH-1 ligand binding domain bound to our previously reported agonist 3-(E-oct-4-en-4-yl)-1-phenylamino-2-phenyl-cis-bicyclo[3.3.0]oct-2-ene 5 is described. Two new classes of agonists in which the bridgehead anilino group from our first series was replaced with an alkoxy or 1-ethenyl group were designed, synthesized, and tested for activity in a peptide recruitment assay. Both new classes gave very active compounds, particularly against SF-1. Structure-activity studies led to excellent dual-LRH-1/SF-1 agonists (e.g., RJW100) as well as compounds selective for LRH-1 (RJW101) and SF-1 (RJW102 and RJW103). The series based on 1-ethenyl substitution was acid stable, overcoming a significant drawback of our original bridgehead anilino-substituted series. Initial studies on the regulation of gene expression in human cell lines showed excellent, reproducible activity at endogenous target genes.

Discovery of novel N-(5-(arylcarbonyl)thiazol-2-yl)amides and N-(5-(arylcarbonyl)thiophen-2-yl)amides as potent RORγt inhibitors

Novel series of N-(5-(arylcarbonyl)thiazol-2-yl)amides and N-(5-(arylcarbonyl)thiophen-2-yl)amides were discovered as potent retinoic acid receptor-related orphan receptor-gamma-t (RORγt) inhibitors. SAR studies of the RORγt HTS hit 6a led to identification of thiazole ketone amide 8h and thiophene ketone amide 9g with high binding affinity and inhibitory activity of Th17 cell differentiation. Compound 8h showed in vivo efficacy in both mouse experimental autoimmune encephalomyelitis (EAE) and collagen induced arthritis (CIA) models via oral administration.

Discovery of Tertiary Amine and Indole Derivatives as Potent RORγt Inverse Agonists

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.

Optimized chemical probes for REV-ERBα

REV-ERBα has emerged as an important target for regulation of circadian rhythm and its associated physiology. Herein, we report on the optimization of a series of REV-ERBα agonists based on GSK4112 (1) for potency, selectivity, and bioavailability. (1) Potent REV-ERBα agonists 4, 10, 16, and 23 are detailed for their ability to suppress BMAL and IL-6 expression from human cells while also demonstrating excellent selectivity over LXRα. Amine 4 demonstrated in vivo bioavailability after either iv or oral dosing.