Elizabeth A. Nichols

Design and synthesis of potent, orally bioavailable dihydroquinazolinone inhibitors of p38 MAP kinase.

The development of potent, orally bioavailable (in rat) and selective dihydroquinazolinone inhibitors of p38alpha MAP kinase is described. These analogues are hybrids of a pyridinylimidazole p38alpha inhibitor reported by Merck Research Laboratories and VX-745. Optimization of the C-5 phenyl and the C-7 piperidinyl substituents led to the identification of 15i which gave excellent suppression of TNF-alpha production in LPS-stimulated whole blood (IC(50)=10nM) and good oral exposure in rats (F=68%, AUCn PO=0.58 microM h).

Sphingosine 1‐phosphate receptor agonist FTY720‐phosphate causes marginal zone B cell displacement

FTY720 is an immunosuppressive agent that modulates lymphocyte trafficking. It is phosphorylated in vivo to FTY720‐phosphate (FTY‐P) and binds to a family of G protein‐coupled receptors recognizing sphingosine 1‐phosphate (S1P) as the natural ligand. It has previously been reported that FTY‐P blocks egress of lymphocytes from the thymus and lymph nodes, resulting in peripheral blood lymphopenia. We now report that FTY‐P also causes displacement of marginal zone (MZ) B cells to the splenic follicles, an effect that is similar to that observed after in vivo administration of lipopolysaccharide. This effect is specific to B cells in the MZ, as treatment with FTY‐P does not cause redistribution of the resident macrophage population. A small but statistically significant decrease in the expression of β1 integrin on MZ B cells was observed with FTY‐P treatment. The redistribution of MZ B cells from the MZ sinuses does not abolish the ability of these cells to respond to the T‐independent antigen, trinitrophenol‐Ficoll. It has been proposed that the displacement of MZ B cells to the follicles is an indication of cell activation. Consistent with this, FTY‐P caused an increase in percentage of MZ B cells expressing activation markers CD9, CD1d, and CD24. These results suggest that S1P receptors on MZ B cells are responsible for their mobilization to follicles.

Large, activated B cells are the primary B-cell target of 8-bromoguanosine and 8-mercaptoguanosine

Previous studies have documented the ability of 8-bromoguanosine (8-BrGuo) and 8-mercaptoguanosine (8-MGuo) to induce polyclonal proliferation and differentiation of B cells from a variety of mouse strains. In the present study, we have defined the cellular target of this mitogenic activity. Using B cells fractionated according to size, we have found that large B cells are responsive to 8-BrGuo- and 8-MGuo-induced proliferation and differentiation whereas small, resting B cells are relatively unresponsive to these compounds. Addition of splenic adherent cells to the small B-cell fraction partially restored the proliferative but not the differentiative responses to 8-BrGuo and 8-MGuo. Although small B cells alone did not proliferate or differentiate in response to 8-BrGuo and 8-MGuo, cell surface expression of Ia antigens increased following incubation with these compounds. Thus, the biological activity of 8-BrGuo and 8-MGuo appears to be dictated by the cell type upon which it is acting. Small B cells are activated as evidenced by increased levels of surface Ia whereas large B cells are not only activated but are also induced to proliferate and differentiate.

High level expression of the plasma cell antigen PC.1 on the T-cell subset expanding in MRL/MpJ-lpr/lpr mice: detection with a xenogeneic monoclonal antibody and alloantisera.

MRL/MpJ-lpr/lpr (MRL-lpr/lpr) mice spontaneously develop a systemic lupus erythematosus-like syndrome associated with the expansion of a T-cell subset exerting helper activity for autoantibody production. Several studies have demonstrated that these T cells have unusual phenotypic characteristics including the expression of the B220 B-cell marker. To further characterize the antigenic profiles of these T cells, we have generated monoclonal antibodies (MAb) by immunizing rats with MRL-lpr/lpr T cells. Using flow cytofluorometry analysis, one of these MAb (4G6), described here, was found to react strongly with T cells of MRL-lpr/lpr mice but weakly with T cells of congenic mice lacking the lpr mutation (MRL/MpJ-+/+ mice). This MAb also stained brightly T cells from C3H/Hej-lpr/lpr mice and dimly those from normal C3H/Hej mice. However, it failed to react with T cells from C57Bl/6-lpr/lpr mice or normal C57Bl/6 (B6) mice. Analysis of 4G6 reactivity (weak vs negative) of T cells in a series of inbred strains demonstrated a correlation with the Pca-1a genotype known to result in expression of the PC.1 antigen on plasma cells. Immunoprecipitation studies revealed that the 4G6 antigen has a mean apparent molecular weight of 115,000, under reducing conditions, similar to that of PC.1. Moreover, high expression of 4G6 was found on plasmacytoma lines and B blasts but not on T blasts. Identity of the 4G6 antigen with PC.1 was confirmed by the finding that conventional anti-PC.1 alloantisera could block the cell surface binding of the 4G6 MAb. Therefore, T cells from MRL-lpr/lpr (and C3H-lpr/lpr) mice aberrantly carry high levels of a plasma cell antigen, detected by the 4G6 MAb, which substantiates further that these T cells represent a unique subset with some surface properties of the B-cell lineage.

5-Halo-6-phenyl pyrimidinones and 8-substituted guanosines: Biological response modifiers with similar effects on B cells

5-Halo-6-phenyl pyrimidinones, represented by 2-amino-5-bromo-6-phenyl-4(3H)-pyrimidinone (ABPP) and 2-amino-5-iodo-6-phenyl-4(3H)-pyrimidinone (AIPP), and 8-substituted guanosines, represented by 8-bromoguanosine (8-BrGuo) and 8-mercaptoguanosine (8-MGuo), are well-documented biological response modifiers. We have found that these substituted pyrimidinones and guanosines are very similar in their abilities to activate B cells. ABPP, AIPP, 8-BrGuo, and 8-MGuo induced murine B cells to polyclonally proliferate and differentiate in vitro. The maximal B-cell response levels and the kinetics of the responses elicited with both classes of compounds were comparable; however, ABPP and AIPP were approximately 10-fold more potent than 8-BrGuo and 8-MGuo. An additional similarity observed between the two classes was that polyclonal activation of B cells by ABPP, AIPP, 8-BrGuo, and 8-MGuo was limited to large B cells which had probably been activated previously in vivo. This is in contrast to lipopolysaccharide which is capable of inducing both large, activated B cells and small, resting B cells to proliferate and differentiate. Although substituted pyrimidinones and guanosines were not able to induce new DNA synthesis or antibody production in small B cells, both classes of compounds increased the expression of Ia antigens on the surface of both small and large B cells. These data, together with the recent observations that 8-BrGuo, like ABPP and AIPP, can stimulate NK and cytotoxic macrophage activity via the induction of interferon, strongly suggest that 5-halo-6-phenyl pyrimidinones and 8-substituted guanosines belong to the same structural class of biological response modifiers. Thus, the residues held in common by these two classes of stimulators may interact with the same cellular constituent in the target cells.

SAR for MHC class II binding tetrapeptides: Correlation with potential binding site

Abstract Comparison of SAR for a tetrapeptide inhibitor of MHC class II with existing information endorses a binding mode consistent with naturally loaded full length peptides.

Tetrapeptide derived inhibitors of complexation of a class II MHC: the peptide backbone is not inviolate.

Major histocompatabilty (MHC) proteins rely heavily on peptide backbone recognition for ligation. Nonetheless, modifications to the polyamide backbone of a tetrapeptide ligand can be made without abrogating binding.