Ceramide kinase regulates acute wound healing by suppressing 5-oxo-ETE biosynthesis and signaling via its receptor OXER1.

The sphingolipid, ceramide-1-phosphate (C1P), has been shown to promote the inflammatory phase and inhibit the proliferation and remodeling stages of wound repair via direct interaction with group IVA cytosolic phospholipase A2, a regulator of eicosanoid biosynthesis that fine-tunes the behaviors of various cell types during wound healing. However, the anabolic enzyme responsible for the production of C1P that suppresses wound healing as well as bioactive eicosanoids and target receptors that drive enhanced wound remodeling have not been characterized. Herein, we determined that decreasing C1P activity via inhibitors or genetic ablation of the anabolic enzyme ceramide kinase (CERK) significantly enhanced wound healing phenotypes. Importantly, postwounding inhibition of CERK enhanced the closure rate of acute wounds, improved the quality of healing, and increased fibroblast migration via a "class switch" in the eicosanoid profile. This switch reduced pro-inflammatory prostaglandins (e.g., prostaglandin E2) and increased levels of 5-hydroxyeicosatetraenoic acid and the downstream metabolite 5-oxo-eicosatetraenoic acid (5-oxo-ETE). Moreover, dermal fibroblasts from mice with genetically ablated CERK showed enhanced wound healing markers, while blockage of the murine 5-oxo-ETE receptor (oxoeicosanoid receptor 1) inhibited the enhanced migration phenotype of these cell models. Together, these studies reinforce the vital roles eicosanoids play in the wound healing process and demonstrate a novel role for CERK-derived C1P as a negative regulator of 5-oxo-ETE biosynthesis and the activation of oxoeicosanoid receptor 1 in wound healing. These findings provide foundational preclinical results for the use of CERK inhibitors to shift the balance from inflammation to resolution and increase the wound healing rate.

Using Target Engagement Biomarkers to Predict Clinical Efficacy of MetAP2 Inhibitors.

Target-engagement pharmacodynamic (PD) biomarkers are valuable tools in the prioritization of drug candidates, especially for novel, first-in-class mechanisms whose robustness to alter disease outcome is unknown. Methionine aminopeptidase 2 (MetAP2) is a cytosolic metalloenzyme that cleaves the N-terminal methionine from nascent proteins. Inhibition of MetAP2 leads to weight loss in obese rodents, dogs and humans. However, there is a need to develop efficacious compounds that specifically inhibit MetAP2 with an improved safety profile. The objective of this study was to identify a PD biomarker for selecting potent, efficacious compounds and for predicting clinical efficacy that would result from inhibition of MetAP2. Here we report the use of NMet14-3-3γ for this purpose. Treatment of primary human cells with MetAP2 inhibitors resulted in an approx. 10-fold increase in NMet14-3-3γ levels. Furthermore, treatment of diet-induced obese mice with these compounds reduced body weight (approx. 20%) and increased NMet14-3-3γ (approx. 15-fold) in adipose tissues. The effects on target engagement and body weight increased over time and were dependent on dose and administration frequency of compound. The relationship between compound concentration in plasma, NMet14-3-3γ in tissue, and reduction of body weight in obese mice was used to generate a pharmacokinetic-pharmacodynamic-efficacy model for predicting efficacy of MetAP2 inhibitors in mice. We also developed a model for predicting weight loss in humans using a target engagement PD assay that measures inhibitor-bound MetAP2 in blood. In summary, MetAP2 target engagement biomarkers can be used to select efficacious compounds and predict weight loss in humans. SIGNIFICANCE STATEMENT: The application of target engagement pharmacodynamic biomarkers during drug development provides a means to determine the dose required to fully engage the intended target and an approach to connect the drug target to physiological effects. This work exemplifies the process of using target engagement biomarkers during preclinical research to select new drug candidates and predict clinical efficacy. We determine concentration of MetAP2 antiobesity compounds needed to produce pharmacological activity in primary human cells and in target tissues from an appropriate animal model and establish key relationships between pharmacokinetics, pharmacodynamics, and efficacy, including the duration of effects after drug administration. The biomarkers described here can aid decision-making in early clinical trials of MetAP2 inhibitors for the treatment of obesity.

MetAP2 inhibition increases energy expenditure through direct action on brown adipocytes.

Inhibitors of methionine aminopeptidase 2 (MetAP2) have been shown to reduce body weight in obese mice and humans. The target tissue and cellular mechanism of MetAP2 inhibitors, however, have not been extensively examined. Using compounds with diverse chemical scaffolds, we showed that MetAP2 inhibition decreases body weight and fat mass and increases lean mass in the obese mice but not in the lean mice. Obesity is associated with catecholamine resistance and blunted ß-adrenergic receptor signaling activities, which could dampen lipolysis and energy expenditure resulting in weight gain. In the current study, we examined effect of MetAP2 inhibition on brown adipose tissue and brown adipocytes. Norepinephrine increases energy expenditure in brown adipose tissue by providing fatty acid substrate through lipolysis and by increasing expression of uncoupled protein-1 (UCP1). Metabolomic analysis shows that in response to MetAP2 inhibitor treatment, fatty acid metabolites in brown adipose tissue increase transiently and subsequently decrease to basal or below basal levels, suggesting an effect on fatty acid metabolism in this tissue. Treatment of brown adipocytes with MetAP2 inhibitors enhances norepinephrine-induced lipolysis and energy expenditure, and prolongs the activity of norepinephrine to increase ucp1 gene expression and energy expenditure in norepinephrine-desensitized brown adipocytes. In summary, we showed that the anti-obesity activity of MetAP2 inhibitors can be mediated, at least in part, through direct action on brown adipocytes by enhancing ß-adrenergic-signaling-stimulated activities.

Regulation of metabolism by nuclear hormone receptors.

The worldwide epidemic of metabolic disease indicates that a better understanding of the pathways contributing to the pathogenesis of this constellation of diseases need to be determined. Nuclear hormone receptors comprise a superfamily of ligand-activated transcription factors that control development, differentiation, and metabolism. Over the last 15 years a growing number of nuclear receptors have been identified that coordinate genetic networks regulating lipid metabolism and energy utilization. Several of these receptors directly sample the levels of metabolic intermediates and use this information to regulate the synthesis, transport, and breakdown of the metabolite of interest. In contrast, other family members sense metabolic activity via the presence or absence of interacting proteins. The ability of these nuclear receptors to impact metabolism and inflammation will be discussed and the potential of each receptor subfamily to serve as drug targets for metabolic disease will be highlighted.

The nature of FSH induction by GnRH.

Follicle-stimulating hormone (FSH), a major regulator of mammalian gonadal function, is induced by gonadotropin-releasing hormone (GnRH), but it is unclear how much induction is direct or indirect and what relevance each has in vivo. Two advances now make it possible to address these issues, which are central to understanding FSH regulation. The first is the use of transformed L beta T2 gonadotropes to define key promoter sequences of FSHB (the gene encoding the FSH-beta subunit) that are needed for induction by GnRH and/or other factors; and the second is the ability to express FSHB promoter-reporter constructs in transgenic mouse gonadotropes to test the physiological relevance of promoter elements identified by using L beta T2 cells. Here, we summarize past studies on GnRH induction of FSH, and propose questions and approaches for the future.

Identification of a negative regulatory surface within estrogen receptor alpha provides evidence in support of a role for corepressors in regulating cellular responses to agonists and antagonists.

Several lines of evidence have indicated that the estrogen receptor (ER) can recruit the corepressors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid receptors (SMRT), to target genes in the presence of tamoxifen, suggesting a possible role for NCoR/SMRT in regulating ER pharmacology. However, a tamoxifen-dependent, direct interaction between NCoR/SMRT and ER in vitro has not been demonstrated. To investigate the possible involvement of different corepressors in the actions of antiestrogen-bound ER, we have constructed a phage display library that expresses 23-amino acid peptides containing the canonical CoRNR box motif in an otherwise random background. Screening of the CoRNR box library with apo-ER or ER treated with tamoxifen or ICI 182,780 led to the isolation of peptides whose ability to interact with ER was influenced by the nature of the bound ligand. Using a series of ERalpha mutants, we found that helix 12 was not required for the binding of CoRNR box peptides, whereas disruption of helixes 3 and 5 had a marked effect on peptide binding. One mutant, ER-L372R, lost the ability to interact with CoRNR box-containing peptides without affecting its binding to LXXLL motif-containing peptides. The estradiol- and tamoxifen-mediated transcriptional activity of ER-L372R was dramatically increased by 11- and 3-fold, respectively, compared with that of wild-type ERalpha. The ICI 182,780-mediated repressional activity of this mutant was also reduced by 4-fold compared with that of wild-type ERalpha. These results suggest that leucine 372 may be an important part of the interaction surface on ER that is responsible for corepressor binding. In addition, our data suggest that corepressors, other than NCoR/SMRT, may be involved in ER signaling.