A Novel Multi-step Virtual Screening for the Identification of Human and Mouse mPGES-1 Inhibitors.

We present here the development of a novel virtual screening protocol combining Structure-based and Ligand-based drug design approaches for the identification of mouse mPGES-1 inhibitors. We used the existing 3D structural data of the murine enzyme to hypothesize the inhibitors binding mode, which was the starting point for docking simulations, shape screening, and pharmacophore hypothesis screening. The protocol allowed the identification of 16 mouse mPGES-1 inhibitors with low micromolar activity, which, notably, also inhibit the human enzyme in the same concentration range. The inhibitors predicted binding mode is expected to be the base for the rational drug design of new potent dual species inhibitors of human and murine mPGES-1.

Development of an enzyme-linked immunosorbent assay, using a monoclonal antibody against alpha2-macroglobulin, for the diagnosis of systemic lupus erythematosus.

OBJECTIVES: To develop an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (mab) directed against abnormally glycosylated serum alpha2-macroglobulin (alpha2-M) from patients with systemic lupus erythematosus (SLE). DESIGN AND METHODS: Serum alpha2-M purified by HPLC from patients with SLE was injected in a Balb/c, CB6 F1 female mouse and hybrid cell lines were screened using alpha2-M Glu-C fragments derived from SLE and normal donors (NHS). A mab was selected and used to develop an ELISA by which sera from NHS (n = 14), SLE (n = 34), rheumatoid arthritis (n = 15), Sjögren's syndrome (n = 11), mixed connective tissue diseases (n = 12), and liver diseases (n = 11) were analyzed. RESULTS: The affinity of the mab for alpha2-M from SLE, but not from the other diseases, was higher compared to NHS, as demonstrated by immunoblotting and ELISA. CONCLUSIONS: The ELISA was capable of recognizing changes of glycosylation of alpha2-M in SLE and may be useful for its differential diagnosis.

Effect of modifications of the alkylpiperazine moiety of trazodone on 5HT2A and alpha1 receptor binding affinity.

A series of triazolopyridine derivatives (compounds 2a-l) were synthesized in order to explore the effect of modifications of the alkylpiperazine moiety of trazodone (fragment A) on binding affinity for 5HT2A and alpha1 receptors. All of the synthesized compounds show a decrease of affinity for both 5HT2A and alpha1 receptors, as compared to trazodone, with the exception of compounds 2b,c which bear a methyl group in an alpha position to the aliphatic nitrogen atom N1. These compounds showed a decrease of affinity only for the alpha1 receptor. The stereochemical influence of the piperazine moiety of compound 2c was also evaluated. Enantiomer (S)-2c showed the most significant differences between 5HT2A and alpha1 receptor affinity (IC50 values) and among the corresponding functional properties (pA2 values). Since (S)-2c cannot generate the metabolite 4-(3-chlorophenyl)piperazine this product was selected for further pharmacological studies.

Radical scavenger activity of bendazac, an anticataract non-steroidal anti-inflammatory agent.

Oxidative damage to lens components is associated with cataract formation and reactive oxygen species (ROS) overproduction at inflammation sites is thought to lead to the development of inflammatory disorders. Bendazac is a non-steroidal anti-inflammatory drug able to delay the cataractogenic process. Aim of the present study is to characterize, both chemically and biologically, the activity of this anticataract agent as a radical scavenger. Bendazac has been shown to be a strong reacting substrate in a chemical oxidizing system, which mimics a physiological pathway of hydroxy radical generation. In the Fenton-Cier reaction the drug rapidly forms a mixture of hydroxylated derivatives, among which 5-hydroxybendazac, bendazac's main metabolite, being a hydroxy radical scavenger itself. Moreover, by means of a rapid and sensitive flow cytometric method able to determine reactive oxygen intermediate production, bendazac and its 5-hydroxy derivative were shown to inhibit oxidative burst activation in polymorphonuclear neutrophil leukocytes (PMNLs).

Response of alpha 2-macroglobulin messenger ribonucleic acid expression to acute inflammation in the testis is different from the response in the liver and brain.

Recent studies from this laboratory have shown that Sertoli cells derived from 20-day-old rats and cultured in vitro synthesize and secrete a nonspecific protease inhibitor that is structurally and immunologically similar to serum alpha 2-macroglobulin (alpha 2-MG). In contrast to its serum homologue, the testicular alpha 2-MG is not an acute-phase protein in the rat since its protein concentration in the rete-testis fluid does not increase in response to inflammation. In the present study we examined the expression of alpha 2-MG mRNA in the rat testis in comparison to that in the brain and liver following induced inflammation. alpha 2-MG mRNA in the testis did not respond to induced inflammation, whereas its protein concentration in serum and its mRNA level in the brain and liver increased significantly in 20-day-old inflamed rats. In 8-day-old rat testis, where the blood-testis barrier is not yet formed, alpha 2-MG mRNA expression also did not respond to induced inflammation. The mRNA expression of clusterin, another authentic Sertoli cell protein whose secretion appears to be closely related to cell-cell interactions in the seminiferous epithelium, was shown to be unaffected by induced inflammation in the testis, brain, and liver. In view of the unexpected differential expression of alpha 2-MG mRNA to induced inflammation in the testis and liver, we sought to examine whether Sertoli cell alpha 2-MG would respond to FSH and testosterone (T), the major regulators of testicular function. Interestingly, expression of alpha 2-MG and clusterin mRNA in the Sertoli cell was not regulated by FSH, T, or a combination of FSH and T. Since there is an intimate morphological relationship between Sertoli cells and germ cells, we next examined the effect of germ cell-conditioned medium (GCCM) on Sertoli cell alpha 2-MG and clusterin mRNA expression. It was noted that GCCM caused a dose-dependent stimulation of alpha 2-MG and inhibition of clusterin mRNA expression in Sertoli cells, respectively. Therefore, our studies have shown that the regulatory mechanism that modulates the expression of alpha 2-MG mRNA in the rat testis is different from its counterpart in the brain and liver.