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.

No abstract

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-dimethylaminocyclopentyl)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 . (Journal of Biomolecular Screening 2005:365-373)

In the search for more efficacious drugs to treat neuropathic pain states, a series of phenoxyphenyl pyridines was designed based on 4-(4-flurophenoxy)benzaldehyde semicarbazone. Through variation of the substituents on the pyridine ring, several potent state-dependent sodium channel inhibitors were identified. From these compounds, 23 dose dependently reversed tactile allodynia in the Chung model of neuropathic pain. Administered orally at 10 mg/kg the level of reversal was ca. 50%, comparable to the effect of carbamazepine administered orally at 100 mg/kg.

No abstract