Induction of heme oxygenase I (HMOX1) by HPP-4382: a novel modulator of Bach1 activity.

Oxidative stress is generated by reactive oxygen species (ROS) produced in response to metabolic activity and environmental factors. Increased oxidative stress is associated with the pathophysiology of a broad spectrum of inflammatory diseases. Cellular response to excess ROS involves the induction of antioxidant response element (ARE) genes under control of the transcriptional activator Nrf2 and the transcriptional repressor Bach1. The development of synthetic small molecules that activate the protective anti-oxidant response network is of major therapeutic interest. Traditional small molecules targeting ARE-regulated gene activation (e.g., bardoxolone, dimethyl fumarate) function by alkylating numerous proteins including Keap1, the controlling protein of Nrf2. An alternative is to target the repressor Bach1. Bach1 has an endogenous ligand, heme, that inhibits Bach1 binding to ARE, thus allowing Nrf2-mediated gene expression including that of heme-oxygenase-1 (HMOX1), a well described target of Bach1 repression. In this report, normal human lung fibroblasts were used to screen a collection of synthetic small molecules for their ability to induce HMOX1. A class of HMOX1-inducing compounds, represented by HPP-4382, was discovered. These compounds are not reactive electrophiles, are not suppressed by N-acetyl cysteine, and do not perturb either ROS or cellular glutathione. Using RNAi, we further demonstrate that HPP-4382 induces HMOX1 in an Nrf2-dependent manner. Chromatin immunoprecipitation verified that HPP-4382 treatment of NHLF cells reciprocally coordinated a decrease in binding of Bach1 and an increase of Nrf2 binding to the HMOX1 E2 enhancer. Finally we show that HPP-4382 can inhibit Bach1 activity in a reporter assay that measures transcription driven by the human HMOX1 E2 enhancer. Our results suggest that HPP-4382 is a novel activator of the antioxidant response through the modulation of Bach1 binding to the ARE binding site of target genes.

Effects of AgRP inhibition on energy balance and metabolism in rodent models.

Activation of brain melanocortin-4 receptors (MC4-R) by α-melanocyte-stimulating hormone (MSH) or inhibition by agouti-related protein (AgRP) regulates food intake and energy expenditure and can modulate neuroendocrine responses to changes in energy balance. To examine the effects of AgRP inhibition on energy balance, a small molecule, non-peptide compound, TTP2515, developed by TransTech Pharma, Inc., was studied in vitro and in rodent models in vivo. TTP2515 prevented AgRP from antagonizing α-MSH-induced increases in cAMP in HEK 293 cells overexpressing the human MC4-R. When administered to rats by oral gavage TTP2515 blocked icv AgRP-induced increases in food intake, weight gain and adiposity and suppression of T4 levels. In both diet-induced obese (DIO) and leptin-deficient mice, TTP2515 decreased food intake, weight gain, adiposity and respiratory quotient. TTP2515 potently suppressed food intake and weight gain in lean mice immediately after initiation of a high fat diet (HFD) but had no effect on these parameters in lean chow-fed mice. However, when tested in AgRP KO mice, TTP2515 also suppressed food intake and weight gain during HFD feeding. In several studies TTP2515 increased T4 but not T3 levels, however this was also observed in AgRP KO mice. TTP2515 also attenuated refeeding and weight gain after fasting, an effect not evident in AgRP KO mice when administered at moderate doses. This study shows that TTP2515 exerts many effects consistent with AgRP inhibition however experiments in AgRP KO mice indicate some off-target effects of this drug. TTP2515 was particularly effective during fasting and in mice with leptin deficiency, conditions in which AgRP is elevated, as well as during acute and chronic HFD feeding. Thus the usefulness of this drug in treating obesity deserves further exploration, to define the AgRP dependent and independent mechanisms by which TTP2515 exerts its effects on energy balance.

Validation of a fluorescent imaging plate reader membrane potential assay for high-throughput screening of glycine transporter modulators.

A fluorescent imaging plate reader (FLIPR) membrane potential (V(m)) assay was evaluated for pharmacological characterization and high-throughput screening (HTS) of rat glycine transporter type 2 (rGlyT(2)) in a stable rGlyT(2)-HEK cell line. Data show that glycine activation of rGlyT(2) consistently results in a concentration-dependent V(m) response on the FLIPR that is blocked by the potent and selective GlyT(2) antagonist 4-benzyloxy-3,5-dimethoxy-N-[1-dimethylamino-cyclopentyl)methyl]-benz-amide (Org-25543). Agonist and antagonist pharmacologies match those reported using conventional [(3)H]glycine uptake assays and electrophysiology. The glycine response is dependent on buffer ionic composition consistent with GlyT(2) physiology. Assay signal-to-background and coefficient of variation meets sufficient statistical criteria to conduct HTS. The results of a screen of the chemical inventory demonstrate that the assay is able to successfully identify and confirm GlyT(2) inhibitors. The advantages of this assay are its homogeneity, compatibility with both 96- and 384-well formats, and lack of radioactivity usage. Thus, the authors conclude that a fluorescence-based V(m) assay on FLIPR is a viable approach for identification and pharmacological profiling of small molecule modulators of the electrogenic transporter rGlyT(2).

DiPOA ([8-(3,3-diphenyl-propyl)-4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]dec-3-yl]-acetic acid), a novel, systemically available, and peripherally restricted mu opioid agonist with antihyperalgesic activity: I. In vitro pharmacological characterization and pharmacokinetic properties.

Mu opioid receptors are present throughout the central and peripheral nervous systems. Peripheral inflammation causes an increase in mu receptor levels on peripheral terminals of primary afferent neurons. Recent studies indicate that activation of peripheral mu receptors produces antihyperalgesic effects in animals and humans. Here, we describe the in vitro pharmacological and in vivo pharmacokinetic properties of a novel, highly potent, and peripherally restricted mu opioid agonist, [8-(3,3-diphenyl-propyl)-4-oxo-1-phenyl-1,3,8-triaza-spiro[4.5]dec-3-yl]-acetic acid (DiPOA). In a radioligand binding assay, DiPOA inhibited [(3)H]-diprenorphine binding to recombinant human mu receptors with a K(i) value of approximately 0.8 nM. The rank order of affinity for DiPOA binding to recombinant human opioid receptors was mu > kappa approximately ORL-1 >> delta. DiPOA showed potent agonist effects in a human mu receptor guanosine 5'-O-(3-[(35)S]thio)triphosphate functional assay, with an EC(50) value of approximately 33 nM and efficacy of approximately 85% [normalized to the mu agonist, [d-Ala2,MePhe4,Gly(ol)5]enkephalin]. Low potency agonist activity was also seen at ORL-1 and kappa receptors. DiPOA bound competitively to the opioid binding site of human mu receptors as demonstrated by a parallel rightward shift in its concentration-response curve in the presence of increasing concentrations of naltrexone. High and sustained (> or =5 h) plasma levels for DiPOA were achieved following intraperitoneal administration at 3 and 10 mg/kg; central nervous system penetration, however, was < or =4% of the plasma concentration, even at levels exceeding 1500 ng/ml. As such, DiPOA represents a systemically available, peripherally restricted small molecule mu opioid agonist that will aid in understanding the role played by mu opioid receptors in the periphery.

4-(2-pyridyl)piperazine-1-carboxamides: potent vanilloid receptor 1 antagonists.

A series of 4-(2-pyridyl)piperazine-1-carboxamide analogues based on the lead compound 1 was synthesized and evaluated for VR1 antagonist activity in capsaicin-induced (CAP) and pH (5.5)-induced (pH) FLIPR assays in a rat VR1-expressing HEK293 cell line. Potent VR1 antagonists were identified through SAR studies. From these studies, 18 was found to be very potent in the in vitro assay [IC(50)=4.8 nM (pH) and 35 nM (CAP)] and orally available in rat (F%=15.1).