Src homology-2 domain binding assays by scintillation proximity and surface plasmon resonance.

Various SH2 competitive binding assays, based on different techniques, have been described in the literature to identify and characterize SH2 ligands. The consideration that most reported methods show experimental limitations associated with assay parameters has prompted us to base our Src-SH2 inhibitor discovery program on the use of two different assays. In this study, two conceptually different biochemical methods designed to discover Src-SH2 inhibitors, respectively scintillation proximity assay (SPA) and surface plasmon resonance (SPR), have been evaluated and compared. For its high sensitivity and adaptability to automation SPA was chosen for high capacity screening (primary screen), whereas SPR was used for hits confirmation (secondary screening). However with the drastic improvement of inhibitor affinities, the limit of sensitivity was rapidly reached for the SPR assay based on the canonical pYEEI ligand. The substitution of the natural, monophosphorylated peptide ligand with a triphosphorylated peptide has allowed us to remarkably increase its sensitivity, so that molecules with nanomolar affinities could be easily differentiated in terms of IC(50) ranking. Such a new, improved SPR assay can be of great interest for the study of high affinity ligands of different SH2-based drug targets.

Discovery of thioazepinone ligands for Src SH2: from non-specific to specific binding.

The structure-based design and synthesis of new thioazepinones as ligands for Src SH2 protein is presented. From benzothioazepinones, ligands with somewhat unspecific binding properties, simpler thioazepinones were designed, the best ones demonstrated nanomolar affinity for Src SH2. A few of these new ligands were crystallized with the protein and demonstrated a specific binding mode with the protein.

Bone-targeted Src SH2 inhibitors block Src cellular activity and osteoclast-mediated resorption.

Src, a nonreceptor tyrosine kinase, is an important regulator of osteoclast-mediated resorption. We have investigated whether compounds that bind to the Src SH2 domain inhibit Src activity in cells and decrease osteoclast-mediated resorption. Compounds were examined for binding to the Src SH2 domain in vitro using a fluorescence polarization binding assay. Experiments were carried out with compounds demonstrating in vitro binding activity (nmol/L range) to determine if they inhibit Src SH2 binding and Src function in cells, demonstrate blockade of Src signaling, and lack cellular toxicity. Cell-based assays included: (1) a mammalian two-hybrid assay; (2) morphological reversion and growth inhibition of cSrcY527F-transformed cells; and (3) inhibition of cortactin phosphorylation in csk-/- cells. The Src SH2 binding compounds inhibit Src activity in all three of these mechanism-based assays. The compounds described were synthesized to contain nonhydrolyzable phosphotyrosine mimics that bind to bone. These compounds were further tested and found to inhibit rabbit osteoclast-mediated resorption of dentine. These results indicate that compounds that bind to the Src SH2 domain can inhibit Src activity in cells and inhibit osteoclast-mediated resorption.

Inhibition of production of tumor necrosis factor by monoclonal antibodies to lipopolysaccharides.

Murine monoclonal antibodies to lipopolysaccharides (LPS) from rough J5 mutant of Escherichia coli O111:B4 protected D-galactosamine-treated mice against lethal effects of LPS and provided protection in an experimental infection model with live E. coli. Three previously prepared anti-LPS antibodies were evaluated for ability to inhibit LPS-induced tumor necrosis factor (TNF) secretion. In vivo, TNF production was induced in mice treated with D-galactosamine and challenged with LPS. Two antibodies (D6B3 and D6B4) decreased serum TNF levels and prevented lethal effects of LPS. Nonprotective anti-LPS antibody (D9A2) and an unrelated antibody to neomycin did not reduce circulating TNF levels after LPS challenge. Pretreatment of mice with D6B3 and D6B4 protected mice from infection with E. coli and prevented serum TNF increases induced by E. coli infection. In nonprotected animals, high levels of TNF were detected in serum 3-6 h after infection; animals died within 24 h. In vitro, addition of protective antibodies to macrophage cultures at initiation of LPS stimulation inhibited TNF production. Nonprotective antibody D9A2 failed to block LPS-induced TNF production.

Toxicity and activity of purified trichosanthin.

Trichosanthin was purified from fresh Chinese root tubers of Trichosanthes kirilowii and evaluated for anti-HIV activity. Trichosanthin inhibited syncytium formation between infected H9 cells and uninfected Sup-T1 cells from 0.5 to 4 micrograms/ml. Trichosanthin also inhibited HIV replication in H9 and CEM-SS cells at 1 microgram/ml, but was toxic for MT-4 cells (HTLV-I-positive), at doses greater than 0.25 microgram/ml. This new purification procedure confirms the anti-HIV activity of trichosanthin on some cell lines in different biological assays.

Murine monoclonal antibodies to Klebsiella pneumoniae protect against lethal endotoxemia and experimental infection with capsulated K. pneumoniae.

To prepare monoclonal antibodies (MAbs) directed against the core-lipid A fractions of smooth lipopoly-saccharide (LPS) from Klebsiella pneumoniae O1:K2, we immunized BALB/c mice with the LPS-associated proteins plus LPS. This preparation exposed the core-lipid A moiety, which is normally hidden in the micellar structure of classical LPS preparations. Among 10 MAbs selected for their reactivity with LPS-associated proteins plus LPS from K. pneumoniae O1:K2, 6 (3A3, 3C2, 3C4, 7D2, 11C3, and 12B6) were directed against the core fraction and 2 (6C5 and 10A5) were directed against the lipid A fraction. Only one (2A4) recognized the O antigen, and one (6D5) had an undefined specificity. When injected before challenge with K. pneumoniae O1:K2 LPS in galactosamine-sensitized mice, five of the MAbs (3C4, 6D5, 7D2, 11C3, and 12B6) provided protection in this model of lethal endotoxemia. MAb 7D2 was also protective in an experimental infection with capsulated K. pneumoniae O1:K2.

Protective effects of murine monoclonal antibodies in experimental septicemia: E. coli antibodies protect against different serotypes of E. coli.

Murine monoclonal antibodies that bind outer membrane antigens of the J5 mutant of Escherichia coli O111:B4 were derived from spleen cells of BALB/c mice immunized with killed whole cells and boosted with lipopolysaccharide (LPS) and LPS-associated proteins. Seven hybridomas were selected for their reactivity against the J5 LPS; they cross-reacted with O111, O55, O127, and O128 E. coli LPS. One (B7B3) also reacted with the Serratia marcescens LPS and Klebsiella pneumoniae lipid A. A protective effect was obtained with D6B4 antibody in a lethal endotoxemia model induced by LPS from O111, O127, and O128 E. coli serotypes in D-galactosamine-sensitized mice. D6B4 and D6B3 antibodies protected mice infected with E. coli O111:B4, when administered before infection. The D6B4 antibody was also protective when administered after infection. The antibodies D6B3 and D4B5 were protective in heterologous infection induced by E. coli O2:K1.