Kenneth H. Pearce

Rev-erbα, a Heme Sensor That Coordinates Metabolic and Circadian Pathways

The circadian clock temporally coordinates metabolic homeostasis in mammals. Central to this is heme, an iron-containing porphyrin that serves as prosthetic group for enzymes involved in oxidative metabolism as well as transcription factors that regulate circadian rhythmicity. The circadian factor that integrates this dual function of heme is not known. We show that heme binds reversibly to the orphan nuclear receptor Rev-erbα, a critical negative component of the circadian core clock, and regulates its interaction with a nuclear receptor corepressor complex. Furthermore, heme suppresses hepatic gluconeogenic gene expression and glucose output through Rev-erbα–mediated gene repression. Thus, Rev-erbα serves as a heme sensor that coordinates the cellular clock, glucose homeostasis, and energy metabolism.

DNA methylation status predicts cell type-specific enhancer activity

Cell‐selective glucocorticoid receptor (GR) binding to distal regulatory elements is associated with cell type‐specific regions of locally accessible chromatin. These regions can either pre‐exist in chromatin (pre‐programmed) or be induced by the receptor (de novo). Mechanisms that create and maintain these sites are not well understood. We observe a global enrichment of CpG density for pre‐programmed elements, and implicate their demethylated state in the maintenance of open chromatin in a tissue‐specific manner. In contrast, sites that are actively opened by GR (de novo) are characterized by low CpG density, and form a unique class of enhancers devoid of suppressive effect of agglomerated methyl‐cytosines. Furthermore, treatment with glucocorticoids induces rapid changes in methylation levels at selected CpGs within de novo sites. Finally, we identify GR‐binding elements with CpGs at critical positions, and show that methylation can affect GR–DNA interactions in vitro. The findings present a unique link between tissue‐specific chromatin accessibility, DNA methylation and transcription factor binding and show that DNA methylation can be an integral component of gene regulation by nuclear receptors.

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.

Identification of peptides that inhibit the DNA binding, trans-activator, and DNA replication functions of the human papillomavirus type 11 E2 protein.

ABSTRACT Peptide antagonists of the human papillomavirus type 11 (HPV-11) E2-DNA association were identified using a filamentous bacteriophage random peptide library. Synthetic peptides antagonized the E2-DNA interaction, effectively blocked E2-mediated transcriptional activation of a reporter gene in cell culture, and inhibited E1-E2-mediated HPV-11 DNA replication in vitro. These peptides may prove to be useful tools for characterizing E2 function and for exploring the effectiveness of E2-inhibitor-based treatments for HPV-associated diseases.

A High-Throughput Screening-Compatible Strategy for the Identification of Inositol Pyrophosphate Kinase Inhibitors

Pharmacological tools—‘chemical probes’—that intervene in cell signaling cascades are important for complementing genetically-based experimental approaches. Probe development frequently begins with a high-throughput screen (HTS) of a chemical library. Herein, we describe the design, validation, and implementation of the first HTS-compatible strategy against any inositol phosphate kinase. Our target enzyme, PPIP5K, synthesizes ‘high-energy’ inositol pyrophosphates (PP-InsPs), which regulate cell function at the interface between cellular energy metabolism and signal transduction. We optimized a time-resolved, fluorescence resonance energy transfer ADP-assay to record PPIP5K-catalyzed, ATP-driven phosphorylation of 5-InsP7 to 1,5-InsP8 in 384-well format (Z’ = 0.82 ± 0.06). We screened a library of 4745 compounds, all anticipated to be membrane-permeant, which are known—or conjectured based on their structures—to target the nucleotide binding site of protein kinases. At a screening concentration of 13 μM, fifteen compounds inhibited PPIP5K >50%. The potency of nine of these hits was confirmed by dose-response analyses. Three of these molecules were selected from different structural clusters for analysis of binding to PPIP5K, using isothermal calorimetry. Acceptable thermograms were obtained for two compounds, UNC10112646 (Kd = 7.30 ± 0.03 μM) and UNC10225498 (Kd = 1.37 ± 0.03 μM). These Kd values lie within the 1–10 μM range generally recognized as suitable for further probe development. In silico docking data rationalizes the difference in affinities. HPLC analysis confirmed that UNC10225498 and UNC10112646 directly inhibit PPIP5K-catalyzed phosphorylation of 5-InsP7 to 1,5-InsP8; kinetic experiments showed inhibition to be competitive with ATP. No other biological activity has previously been ascribed to either UNC10225498 or UNC10112646; moreover, at 10 μM, neither compound inhibits IP6K2, a structurally-unrelated PP-InsP kinase. Our screening strategy may be generally applicable to inhibitor discovery campaigns for other inositol phosphate kinases.

Activation of the Human Growth Hormone Receptor

The hematopoietic family of cytokines and receptors, to which human growth hormone (hGH) and its receptor (hGHR) belong, are a set of hormone-receptor pairs that are classified on the basis of having similar three-dimensional topologies, as well as similar functional characteristics (1–3). The complex between hGH and the hGHR is one of the best understood among the ligand-receptor pairs of the hematopoietic cytokine family (for other reviews see refs. 4 and 5). A number of the ligands in this family (e.g, hGH, prolactin, erythropoietin, thrombopoietin) transduce signals through their receptors via a sequential dimerization mechanism. In this process, a single hormone molecule can bind to the extracellular domain of two receptors. Here, the manner by which hGH recognizes and binds to its receptor and the molecular aspects whereby hGH transduces its cell proliferative signal through the membrane are reviewed in more detail.

Retraction: A High-Throughput Screening-Compatible Strategy for the Identification of Inositol Pyrophosphate Kinase Inhibitors (PLoS ONE (2016) 11, 10 (e0164378) DOI: 10.1371/journal.pone.0164378)

The authors of the above paper retract this article due to concerns about the integrity of the data and the validity of the conclusions. The first author, Brandi M. Baughman, has admitted to the co-authors and the Office of Research Integrity at NIH that she falsified and/or fabricated data and text concerning Figs 2, 3, 4, 5, 6, 8, S1, S2, S3, S4, and S5, for which she takes sole responsibility. Subsequent to the paper being published, further experiments by co-authors Wang, Stashko, and Pearce have verified that a major conclusion of this article is invalid: UNC10112646, UNC10225354, and UNC10225498 are not inhibitors of PPIP5K, contrary to the claims in the published paper. In light of these concerns, all of the authors have agreed to retract this article.