Recruitment of FBXO22 for targeted degradation of NSD2.

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain-of-function mutation p.E1099K, resulting in growth suppression, apoptosis and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 to recruit the SCFFBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22. Overall, we present a potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a new FBXO22-recruitment strategy for TPD.

Ube3a unsilencer for the potential treatment of Angelman syndrome.

Deletion of the maternal UBE3A allele causes Angelman syndrome (AS); because paternal UBE3A is epigenetically silenced by a long non-coding antisense (UBE3A-ATS) in neurons, this nearly eliminates UBE3A protein in the brain. Reactivating paternal UBE3A holds promise for treating AS. We previously showed topoisomerase inhibitors can reactivate paternal UBE3A, but their therapeutic challenges prompted our search for small molecule unsilencers with a different mechanism of action. Here, we found that (S)-PHA533533 acts through a novel mechanism to significantly increase paternal Ube3a mRNA and UBE3A protein levels while downregulating Ube3a-ATS in primary neurons derived from AS model mice. Furthermore, peripheral delivery of (S)-PHA533533 in AS model mice induces widespread neuronal UBE3A expression. Finally, we show that (S)-PHA533533 unsilences paternal UBE3A in AS patient-derived neurons, highlighting its translational potential. Our findings provide a lead for developing a small molecule treatment for AS that could be safe, non-invasively delivered, and capable of brain-wide unsilencing of paternal UBE3A.

Recruitment of FBXO22 for Targeted Degradation of NSD2.

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here, we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain of function mutation p.E1099K, resulting in growth suppression, apoptosis, and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 in a covalent and reversible manner to recruit the SCF FBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCF FBXO22 . Overall, we present a highly potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a novel FBXO22-dependent TPD strategy.

Exploration of phenylpropanoic acids as agonists of the free fatty acid receptor 4 (FFA4): Identification of an orally efficacious FFA4 agonist.

The long chain free fatty acid receptor 4 (FFA4/GPR120) has recently been recognized as lipid sensor playing important roles in nutrient sensing and inflammation and thus holds potential as a therapeutic target for type 2 diabetes and metabolic syndrome. To explore the effects of stimulating this receptor in animal models of metabolic disease, we initiated work to identify agonists with appropriate pharmacokinetic properties to support progression into in vivo studies. Extensive SAR studies of a series of phenylpropanoic acids led to the identification of compound 29, a FFA4 agonist which lowers plasma glucose in two preclinical models of type 2 diabetes.

Identification of diarylsulfonamides as agonists of the free fatty acid receptor 4 (FFA4/GPR120).

The exploration of a diarylsulfonamide series of free fatty acid receptor 4 (FFA4/GPR120) agonists is described. This work led to the identification of selective FFA4 agonist 8 (GSK137647A) and selective FFA4 antagonist 39. The in vitro profile of compounds 8 and 39 is presented herein.

Enzymatic- and iridium-catalyzed asymmetric synthesis of a benzothiazepinylphosphonate bile acid transporter inhibitor.

A synthesis of the benzothiazepine phosphonic acid 3, employing both enzymatic and transition metal catalysis, is described. The quaternary chiral center of 3 was obtained by resolution of ethyl (2-ethyl)norleucinate (4) with porcine liver esterase (PLE) immobilized on Sepabeads. The resulting (R)-amino acid (5) was converted in two steps to aminosulfate 7, which was used for construction of the benzothiazepine ring. Benzophenone 15, prepared in four steps from trimethylhydroquinone 11, enabled sequential incorporation of phosphorus (Arbuzov chemistry) and sulfur (Pd(0)-catalyzed thiol coupling) leading to mercaptan intermediate 18. S-Alkylation of 18 with aminosulfate 7 followed by cyclodehydration afforded dihydrobenzothiazepine 20. Iridium-catalyzed asymmetric hydrogenation of 20 with the complex of [Ir(COD)2BArF] (26) and Taniaphos ligand P afforded the (3R,5R)-tetrahydrobenzothiazepine 30 following flash chromatography. Oxidation of 30 to sulfone 31 and phosphonate hydrolysis completed the synthesis of 3 in 12 steps and 13% overall yield.

Discovery of a highly potent, nonabsorbable apical sodium-dependent bile acid transporter inhibitor (GSK2330672) for treatment of type 2 diabetes.

The apical sodium-dependent bile acid transporter (ASBT) transports bile salts from the lumen of the gastrointestinal (GI) tract to the liver via the portal vein. Multiple pharmaceutical companies have exploited the physiological link between ASBT and hepatic cholesterol metabolism, which led to the clinical investigation of ASBT inhibitors as lipid-lowering agents. While modest lipid effects were demonstrated, the potential utility of ASBT inhibitors for treatment of type 2 diabetes has been relatively unexplored. We initiated a lead optimization effort that focused on the identification of a potent, nonabsorbable ASBT inhibitor starting from the first-generation inhibitor 264W94 (1). Extensive SAR studies culminated in the discovery of GSK2330672 (56) as a highly potent, nonabsorbable ASBT inhibitor which lowers glucose in an animal model of type 2 diabetes and shows excellent developability properties for evaluating the potential therapeutic utility of a nonabsorbable ASBT inhibitor for treatment of patients with type 2 diabetes.

Liver X receptor protects against liver injury in sepsis caused by rodent cecal ligation and puncture.

BACKGROUND: Liver X receptor (LXR) is a transcription factor of the nuclear receptor family, regulating genes involved in metabolism, inflammation, and apoptosis. In the present investigation, we examined the role of LXR in organ injury and systemic inflammation in rodent models of polymicrobial peritonitis caused by cecal ligation and puncture (CLP). METHODS: Rats were subjected to CLP sepsis or a sham operation. Some were treated with the synthetic LXR agonist GW3965 0.3 mg/kg 30 min prior to the CLP procedure, and organs and plasma were harvested at 3, 10, 18, or 24 h. Organs were analyzed for RNA expression by quantitative polymerase chain reaction or for morphologic differences by histologic review. Organ injury and inflammatory markers were measured in plasma. RESULTS: Expression of the LXRα gene was decreased in the livers of CLP rats compared with sham-operated rats. Administration of a synthetic agonist of LXR (GW3965) reduced biochemical indices of liver injury in the blood of CLP rats. We also demonstrated that liver injury associated with CLP is aggravated in LXRα- and LXRαß-deficient mice compared with wild-type and LXRß-deficient mice, indicating a role for LXRα in protecting the liver. The enhanced liver injury in LXR-deficient mice was associated with elevated plasma concentrations of high mobility group box 1, a late mediator of inflammation and a known factor in the pathology of this model. CONCLUSIONS: Collectively, these results argue in favor of a role for LXRα in protection against liver injury in experimental sepsis induced by CLP.

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.

Discovery of tertiary sulfonamides as potent liver X receptor antagonists.

Tertiary sulfonamides were identified in a HTS as dual liver X receptor (LXR, NR1H2, and NR1H3) ligands, and the binding affinity of the series was increased through iterative analogue synthesis. A ligand-bound cocrystal structure was determined which elucidated key interactions for high binding affinity. Further characterization of the tertiary sulfonamide series led to the identification of high affinity LXR antagonists. GSK2033 (17) is the first potent cell-active LXR antagonist described to date. 17 may be a useful chemical probe to explore the cell biology of this orphan nuclear receptor.

Liver X receptor agonist GW3965 dose-dependently regulates lps-mediated liver injury and modulates posttranscriptional TNF-alpha production and p38 mitogen-activated protein kinase activation in liver macrophages.

Modulation of the host inflammatory response to infection may be a key approach to improve the outcome of patients with sepsis and organ injury. We previously reported that pretreatment of rats with the liver X receptor (LXR) agonist GW3965 reduced the liver injury associated with endotoxemia and attenuated the production of TNF-alpha by rat Kupffer cells. Here, we examine the dose-dependent effect of GW3965 on liver injury and cytokine production in a rat model of endotoxemia and explore the mechanisms underlying TNF-alpha attenuation in Kupffer cells. Low doses of GW3965 (0.1 or 0.3 mg/kg) administered 30 min before infusion of LPS and peptidoglycan significantly attenuated the increase in plasma levels of the liver injury markers alanine aminotransferase and bilirubin (6 h) as well as the inflammatory mediators TNF-alpha (1 h) and prostaglandin E2 (6 h) associated with endotoxemia. In contrast, pretreatment with a higher dose of GW3965 (1.0 mg/kg) had no such effect. Studies in primary cultures of rat Kupffer cells demonstrated that LXR agonist treatment attenuated both the secreted and cell-associated levels of TNF-alpha, whereas TNF-alpha mRNA levels were not altered. Phosphorylated p38 mitogen-activated protein kinase, which plays a major role in production of TNF-alpha at the posttranscriptional level, was attenuated by GW3965 treatment in Kupffer cells. Experiments in murine LXR-deficient Kupffer cells demonstrated enhanced production of TNF-alpha in Kupffer cells from LXR-alpha(-/-) mice when challenged with LPS compared with LXR-beta(-/-) and wild-type Kupffer cells. Taken together, these results argue in favor of a novel mechanism for LXR-mediated attenuation of liver injury by interfering with posttranscriptional regulation of TNF-alpha in Kupffer cells.

Structure-guided design of N-phenyl tertiary amines as transrepression-selective liver X receptor modulators with anti-inflammatory activity.

A cocrystal structure of T1317 (3) bound to hLXRbeta was utilized in the design of a series of substituted N-phenyl tertiary amines. Profiling in binding and functional assays led to the identification of LXR modulator GSK9772 ( 20) as a high-affinity LXRbeta ligand (IC 50 = 30 nM) that shows separation of anti-inflammatory and lipogenic activities in human macrophage and liver cell lines, respectively. A cocrystal structure of the structurally related analog 19 bound to LXRbeta reveals regions within the receptor that can affect receptor modulation through ligand modification. Mechanistic studies demonstrate that 20 is greater than 10-fold selective for LXR-mediated transrepression of proinflammatory gene expression versus transactivation of lipogenic signaling pathways, thus providing an opportunity for the identification of LXR modulators with improved therapeutic indexes.

Liver X receptor agonists increase airway reactivity in a model of asthma via increasing airway smooth muscle growth.

The liver X receptors (LXRalpha/beta) are orphan nuclear receptors that are expressed in a large number of cell types and have been shown to have anti-inflammatory properties. Nuclear receptors have previously proved to be amenable targets for small molecular mass pharmacological agents in asthma, and so the effect of an LXR ligand was assessed in models of allergic airway inflammation. LXR agonist, GW 3965, was profiled in rat and mouse models of allergic asthma. In the Brown Norway rats, GW 3965 (3-30 mg/kg) was unable to reduce the bronchoalveolar lavage eosinophilia associated with this model and had no impact on inflammatory biomarkers (eotaxin and IL-1beta). The compound did significantly stimulate ABCA-1 (ATP-binding cassette A1) mRNA expression, indicating that there was adequate exposure/LXR activation. In the mouse model, the LXR ligand surprisingly increased airway reactivity, an effect that was apparent in both the Ag and nonchallenged groups. This increase was not associated with a change in lung tissue inflammation or number of mucus-containing cells. There was, however, a marked increase in airway smooth muscle thickness in both treated groups. We demonstrated an increase in contractile response to exogenous methacholine in isolated airways taken from LXR agonist-treated animals compared with the relevant control tissue. We corroborated these findings in a human system by demonstrating increased proliferation of cultured airway smooth muscle. This phenomenon, if evidenced in man, would indicate that LXR ligands may directly increase airway reactivity, which could be detrimental, especially in patients with existing respiratory disease and with already compromised lung function.

Selective partial agonism of liver X receptor alpha is related to differential corepressor recruitment.

Classically, activated transcription by nuclear receptors (NRs) is due to a ligand-induced switch from corepressor- to coactivator-bound states. However, coactivators and corepressors recognize overlapping surfaces of liganded and unliganded NRs, respectively. Here we show that, at sufficiently high concentration, the NR corepressor (NCoR) influences the activity of the liver X receptor (LXR) even in the presence of a potent full agonist that destabilizes NCoR binding. Partial agonist ligands that less effectively dissociate NCoR from LXR are even more sensitive to NCoR levels, in a target gene-selective manner. Thus, differential recruitment of NCoR is a major determinant of partial agonism and selective LXR modulation of target genes.

Recent advances in liver X receptor biology and chemistry.

The liver X receptors LXR alpha and LXR beta are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. Seminal studies with genetic and chemical tools were instrumental in the elucidation of cholesterol metabolism, gluconeogenesis, inflammation, and lipogenesis as signaling pathways that are controlled by the LXRs. First generation non-steroidal LXR agonists show beneficial effects in multiple animals models of human disease yet have not progressed in the clinic due to deleterious side effects in the liver. Numerous reports have appeared in the the recent literature that disclose new LXR signaling pathways and the identification of novel LXR chemotypes that may show improved therapeutic indices. This review will provide a brief historical perspective but will primarily focus on recent advances in LXR biology and chemistry.

ApoE promotes the proteolytic degradation of Abeta.

Apolipoprotein E is associated with age-related risk for Alzheimer's disease and plays critical roles in Abeta homeostasis. We report that ApoE plays a role in facilitating the proteolytic clearance of soluble Abeta from the brain. The endolytic degradation of Abeta peptides within microglia by neprilysin and related enzymes is dramatically enhanced by ApoE. Similarly, Abeta degradation extracellularly by insulin-degrading enzyme is facilitated by ApoE. The capacity of ApoE to promote Abeta degradation is dependent upon the ApoE isoform and its lipidation status. The enhanced expression of lipidated ApoE, through the activation of liver X receptors, stimulates Abeta degradation. Indeed, aged Tg2576 mice treated with the LXR agonist GW3965 exhibited a dramatic reduction in brain Abeta load. GW3965 treatment also reversed contextual memory deficits. These data demonstrate a mechanism through which ApoE facilitates the clearance of Abeta from the brain and suggest that LXR agonists may represent a novel therapy for AD.

The phytoestrogen coumestrol is a naturally occurring antagonist of the human pregnane X receptor.

Antagonizing the action of the human nuclear xenobiotic receptor pregnane X receptor (PXR) may have important clinical implications in preventing drug-drug interactions and improving therapeutic efficacy. We provide evidence that a naturally occurring phytoestrogen, coumestrol, is an antagonist of the nuclear receptor PXR (NR1I2). In transient transfection assays, coumestrol was able to suppress the agonist effects of SR12813 on human PXR activity. PXR activity was assessed and correlated with effects on the metabolism of the anesthetic tribromoethanol and on gene expression in primary human hepatocytes. We found that coumestrol was able to suppress the effects of PXR agonists on the expression of the known PXR target genes, CYP3A4 and CYP2B6, in primary human hepatocytes as well as inhibit metabolism of tribromoethanol in humanized PXR mice. Coumestrol at concentrations above 1.0 microm competed in scintillation proximity assays with a labeled PXR agonist for binding to the ligand-binding cavity. However, mammalian two-hybrid assays and transient transcription data using ligand-binding-cavity mutant forms of PXR show that coumestrol also antagonizes coregulator recruitment. This effect is likely by binding to a surface outside the ligand-binding pocket. Taken together, these data imply that there are antagonist binding site(s) for coumestrol on the surface of PXR. These studies provide the basis for development of novel small molecule inhibitors of PXR with the ultimate goal of clinical applications toward preventing drug-drug interactions.

Novel role for the liver X nuclear receptor in the suppression of lung inflammatory responses.

The liver X receptors (LXRalpha/beta) are part of the nuclear receptor family and are believed to regulate cholesterol and lipid homeostasis. It has also been suggested that LXR agonists possess anti-inflammatory properties. The aim of this work was to determine the effect of LXR agonists on the innate immune response in human primary lung macrophages and a pre-clinical rodent model of lung inflammation. Before profiling the impact of the agonist, we established that both the human macrophages and the rodent lungs expressed LXRalpha/beta. We then used two structurally distinct LXR agonists to demonstrate that activation of this transcription factor reduces cytokine production in THP-1 cells and lung macrophages. Then, using the expression profile of ATP binding cassettes A1 (ABCA-1; a gene directly linked to LXR activation) as a biomarker for lung exposure of the compound, we demonstrated an LXR-dependent reduction in lung neutrophilia rodents in vivo. This inhibition was not associated with a suppression of c-Fos/c-Jun mRNA expression or NF-kappaB/AP-1 DNA binding, suggesting that any anti-inflammatory activity of LXR agonists is not via inhibition of NF-kappaB/AP-1 transcriptional activity. These data do not completely rule out an impact of these agonists on these two prominent transcription factors. In summary, this study is the first to demonstrate anti-inflammatory actions of LXRs in the lung. Chronic innate inflammatory responses observed in some airway diseases is thought to be central to disease pathogenesis. Therefore, data suggest that LXR ligands have utility in the treatment of lung diseases that involves chronic inflammation mediated by macrophages and neutrophils.

Ligand activation of LXR beta reverses atherosclerosis and cellular cholesterol overload in mice lacking LXR alpha and apoE.

Liver X receptors (LXRs) alpha and beta are transcriptional regulators of cholesterol homeostasis and potential targets for the development of antiatherosclerosis drugs. However, the specific roles of individual LXR isotypes in atherosclerosis and the pharmacological effects of synthetic agonists remain unclear. Previous work has shown that mice lacking LXRalpha accumulate cholesterol in the liver but not in peripheral tissues. In striking contrast, we demonstrate here that LXRalpha(-/-)apoE(-/-) mice exhibit extreme cholesterol accumulation in peripheral tissues, a dramatic increase in whole-body cholesterol burden, and accelerated atherosclerosis. The phenotype of these mice suggests that the level of LXR pathway activation in macrophages achieved by LXRbeta and endogenous ligand is unable to maintain homeostasis in the setting of hypercholesterolemia. Surprisingly, however, a highly efficacious synthetic agonist was able to compensate for the loss of LXRalpha. Treatment of LXRalpha(-/-)apoE(-/-) mice with synthetic LXR ligand ameliorates the cholesterol overload phenotype and reduces atherosclerosis. These observations indicate that LXRalpha has an essential role in maintaining peripheral cholesterol homeostasis in the context of hypercholesterolemia and provide in vivo support for drug development strategies targeting LXRbeta.

The relative transcription index: a gene expression based metric for prioritization of drug candidates.

Efficient compound selection remains a key challenge in drug discovery today. The goal is to identify developable drug candidates early in the screening process while simultaneously flagging compounds with off-target effects indicative of liabilities or alternate indications. This goal overlaps but is distinct from the goal of toxicogenomics which is focused primarily on identifying toxicity signatures of lead candidates in key tissues. We propose a framework where global changes in gene expression levels in response to compounds can be used as an objective metric for early compound prioritization. We call this metric the Relative Transcription Index (RTI). RTI is a measure of the relative activity of compounds as ascertained by their effects on transcription at a genome-wide level. Compounds with a low RTI affect the expression of only a few genes whereas compounds with a high RTI affect the expression of a large number of genes. This information is useful for differentiating compounds that, based on phenotypic assays alone, may appear to be equally efficacious. Since compounds with high RTI are more likely to display off-target effects, the RTI metric, if implemented early in the screening process, can become a valuable tool for compound selection. The utility of the RTI metric is demonstrated by its application to two different gene expression datasets--one involving modulators of the liver X receptor (LXR) and the other concerning antibacterial compounds belonging to diverse mechanistic classes.