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.

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.

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.

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.

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.

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.

Activation of the liver X receptor protects against hepatic injury in endotoxemia by suppressing Kupffer cell activation.

Recent reports have demonstrated that liver X receptors (LXRs) of the nuclear receptor family have anti-inflammatory effects on macrophages. Here we examine whether activation of LXR by the synthetic agonist GW3965 can ameliorate the liver injury/dysfunction caused by endotoxins in the rat. Male Wistar rats received GW3965 (0.3 mg/kg) or vehicle (50% dimethyl sulfoxide) 30 min before coadministration of lipopolysaccharide (LPS, 5 mg/kg i.v.) and peptidoglycan (1 mg/kg i.v.). Treatment with GW3965 attenuated the increase in the plasma levels of alanine aminotransferase and bilirubin (markers of liver injury/dysfunction) as well as the focal hepatocyte necrosis (histology) caused by coadministration of LPS and peptidoglycan. This protective effect of GW3965 treatment was associated with reduced infiltration of mast cells in the liver (histopathology) and reduced gene expression of the chemokines eotaxins 1 and 2, whereas MIP-2 mRNA levels were not affected. Plasma levels of tumor necrosis factor alpha and prostaglandin E2 were significantly attenuated by GW3965, whereas plasma interleukins 6 and 10 were not altered. High expression of LXRalpha mRNA was observed in Kupffer cell cultures, suggesting that Kupffer cells are targets of GW3965. Subsequent in vitro studies in Kupffer cells demonstrated that exposure to GW3965 attenuated the LPS-induced release of tumor necrosis factor alpha and prostaglandin E2 in a dose-dependent manner. In conclusion, this study demonstrates that activation of LXR by GW3965 protects against liver injury and dysfunction in a rat model of endotoxemia, in part by exerting an anti-inflammatory effect on Kupffer cells.

Structure-guided synthesis of tamoxifen analogs with improved selectivity for the orphan ERRgamma.

The design and synthesis of 4-hydroxytamoxifen (4-OHT) derivatives are described. The binding affinities of these compounds toward the orphan estrogen-related receptor gamma and the classical estrogen receptor alpha demonstrate that analogs bearing hydroxyalkyl groups display improved binding selectivity profiles compared with that of 4-OHT. An X-ray crystal structure of one of the designed compounds bound to ERRgamma LBD confirms the molecular basis of the selectivity.

Identification and structure-activity relationship of phenolic acyl hydrazones as selective agonists for the estrogen-related orphan nuclear receptors ERRbeta and ERRgamma.

The first small molecule agonists of the estrogen-related receptors have been identified. GSK4716 (3) and GSK9089 (4) show binding to ERRgamma with remarkable selectivity over the classical estrogen receptors. Notably, in cell-based reporter assays, 3 mimics the protein ligand PGC-1alpha in activation of human ERRbeta and ERRgamma.

Liver X receptor activators display anti-inflammatory activity in irritant and allergic contact dermatitis models: liver-X-receptor-specific inhibition of inflammation and primary cytokine production.

Activators of liver X receptors (LXR) stimulate epidermal differentiation and development, but inhibit keratinocyte proliferation. In this study, the anti-inflammatory effects of two oxysterols, 22(R)-hydroxy-cholesterol (22ROH) and 25-hydroxycholesterol (25OH), and a nonsterol activator of LXR, GW3965, were examined utilizing models of irritant and allergic contact dermatitis. Irritant dermatitis was induced by applying phorbol 12-myristate-13-acetate (TPA) to the surface of the ears of CD1 mice, followed by treatment with 22ROH, 25OH, GW3965, or vehicle alone. Whereas TPA treatment alone induced an approximately 2-fold increase in ear weight and thickness, 22ROH, 25OH, or GW3965 markedly suppressed the increase (greater than 50% decrease), and to an extent comparable to that observed with 0.05% clobetasol treatment. Histology also revealed a marked decrease in TPA-induced cutaneous inflammation in oxysterol-treated animals. As topical treatment with cholesterol did not reduce the TPA-induced inflammation, and the nonsterol LXR activator (GW3965) inhibited inflammation, the anti-inflammatory effects of oxysterols cannot be ascribed to a nonspecific sterol effect. In addition, 22ROH did not reduce inflammation in LXRbeta-/- or LXRalphabeta-/- animals, indicating that LXRbeta is required for this anti-inflammatory effect. 22ROH also caused a partial reduction in ear thickness in LXRalpha-/- animals, however (approximately 50% of that observed in wild-type mice), suggesting that this receptor also mediates the anti-inflammatory effects of oxysterols. Both ear thickness and weight increased (approximately 1.5-fold) in the oxazolone-induced allergic dermatitis model, and 22ROH and GW3965 reduced inflammation by approximately 50% and approximately 30%, respectively. Finally, immunohistochemistry demonstrated an inhibition in the production of the pro-inflammatory cytokines interleukin-1alpha and tumor necrosis factor alpha in the oxysterol-treated sites from both TPA- and oxazolone-treated animals. These studies demonstrate that activators of LXR display potent anti-inflammatory activity in both irritant and allergic contact models of dermatitis, requiring the participation of both LXRalpha and LXRbeta. LXR activators could provide a new class of therapeutic agents for the treatment of cutaneous inflammatory disorders.

Identification of bile acid precursors as endogenous ligands for the nuclear xenobiotic pregnane X receptor.

Sterol 27-hydroxylase (CYP27A1) is required for bile acid synthesis by both the classical and alternate pathways. Cyp27a1(-/-) mice exhibit a dramatic increase in the activity of cytochrome P450 3A (CYP3A), which catalyzes side-chain hydroxylations of bile acid intermediates, thereby facilitating their excretion in the bile and urine. We examine the role of the nuclear xenobiotic receptor PXR (pregnane X receptor) in this process. We demonstrate that expression of Cyp3a11 and other established PXR target genes is increased in the Cyp27a1(-/-) mice. WhenCyp27a1(-/-) mice are fed a diet containing either cholic acid or chenodeoxycholic acid, expression of CYP7A1, which catalyzes the rate-limiting step in bile acid biosynthesis, is strongly suppressed. In parallel, the induction of Cyp3a11 observed in these mice is reversed, suggesting that bile acid intermediates serve as PXR activators. In support of this hypothesis, three potentially toxic sterols (7alpha-hydroxy-4-cholesten-3-one, 5beta-cholestan-3alpha,7alpha,12alpha-triol, and 4-cholesten-3-one), including two that are known to accumulate in Cyp27a1(-/-) mice, are efficacious activators of mouse PXR. All three compounds are more potent activators of mouse PXR than of human PXR, which may explain in part why humans who lack functional CYP27A1 do not display a corresponding increase in CYP3A activity and are stricken with the disease cerebrotendinous xanthomatosis. Taken together, these results reveal the existence of a feedforward regulatory loop by which potentially toxic bile acid intermediates activate PXR and induce their own metabolism. In addition, this study demonstrates that animal models with alterations in gene expression can be used to identify endogenous ligands for orphan nuclear receptors.

Identification of a nonsteroidal liver X receptor agonist through parallel array synthesis of tertiary amines.

A potent, selective, orally active LXR agonist was identified from focused libraries of tertiary amines. GW3965 (12) recruits the steroid receptor coactivator 1 to human LXRalpha in a cell-free ligand-sensing assay with an EC(50) of 125 nM and profiles as a full agonist on hLXRalpha and hLXRbeta in cell-based reporter gene assays with EC(50)'s of 190 and 30 nM, respectively. After oral dosing at 10 mg/kg to C57BL/6 mice, 12 increased expression of the reverse cholesterol transporter ABCA1 in the small intestine and peripheral macrophages and increased the plasma concentrations of HDL cholesterol by 30%. 12 will be a valuable chemical tool to investigate the role of LXR in the regulation of reverse cholesterol transport and lipid metabolism.

Functional consequences of cysteine modification in the ligand binding sites of peroxisome proliferator activated receptors by GW9662.

In the course of a high throughput screen to search for ligands of peroxisome proliferator activated receptor-gamma (PPARgamma), we identified GW9662 using a competition binding assay against the human ligand binding domain. GW9662 had nanomolar IC(50) versus PPARgamma and was 10- and 600-fold less potent in binding experiments using PPARalpha and PPARdelta, respectively. Pretreatment of all three PPARs with GW9662 resulted in the irreversible loss of ligand binding as assessed by scintillation proximity assay. Incubation of PPAR with GW9662 resulted in a change in the absorbance spectra of the receptors consistent with covalent modification. Mass spectrometric analysis of the PPARgamma ligand binding domain treated with GW9662 established Cys(285) as the site of covalent modification. This cysteine is conserved among all three PPARs. In cell-based reporter assays, GW9662 was a potent and selective antagonist of full-length PPARgamma. The functional activity of GW9662 as an antagonist of PPARgamma was confirmed in an assay of adipocyte differentiation. GW9662 showed essentially no effect on transcription when tested using both full-length PPARdelta and PPARalpha. Time-resolved fluorescence assays of ligand-modulated receptor heterodimerization, coactivator binding, and corepressor binding were consistent with the effects observed in the reporter gene assays. Control activators increased PPAR:RXR heterodimer formation and coactivator binding to both PPARgamma and PPARdelta. Corepressor binding was decreased. In the case of PPARalpha, GW9662 treatment did not significantly increase heterodimerization and coactivator binding or decrease corepressor binding. The experimental data indicate that GW9662 modification of each of the three PPARs results in different functional consequences. The selective and irreversible nature of GW9662 treatment, and the observation that activity is maintained in cell culture experiments, suggests that this compound may be a useful tool for elucidation of the role of PPARgamma in biological processes.

A novel method for the generation of nitrile oxides on solid phase: application to the synthesis of substituted benzopyranoisoxazoles.

A solid-phase synthesis of substituted benzopyranoisoxazoles is described. The six-step synthesis features a novel method of generating nitrile oxides on a polymer support followed by an intramolecular 1,3-dipolar cycloaddition with a tethered alkyne for assembly of the benzopyranoisoxazole scaffold. Furthermore, the utilization of single-bead attenuated total reflectance Fourier transform infrared (ATR-IR) microspectroscopy as an essential analytical tool for reaction optimization is highlighted.