The pharmacophore of the human C5a anaphylatoxin.

We have determined which amino acids contribute to the pharmacophore of human C5a, a potent inflammatory mediator. A systematic mutational analysis of this 74-amino acid protein was performed and the effects on the potency of receptor binding and of C5a-induced intracellular calcium ion mobilization were measured. This analysis included the construction of hybrids between C5a and the homologous but unreactive C3a protein and site-directed mutagenesis. Ten noncontiguous amino acids from the structurally well-defined 4-helix core domain (amino acids 1-63) and the C-terminal arginine-containing tripeptide were found to contribute to the pharmacophore of human C5a. The 10 mostly charged amino acids from the core domain generally made small incremental contributions toward binding affinity, some of which were independent. Substitutions of the C-terminal amino acid Arg 74 produced the largest single effect. We also found the connection between these 2 important regions to be unconstrained.

Identification of a high-affinity anti-phosphoserine antibody for the development of a homogeneous fluorescence polarization assay of protein kinase C.

In the last few years, fluorescence polarization (FP) has been applied to the development of robust, homogeneous, high throughput assays in molecular recognition research, such as ligand-protein interactions. Recently, this technology has been applied to the development of homogeneous tyrosine kinase assays, since there are high-affinity anti-phosphotyrosine antibodies available. Unlike tyrosine kinases, application of FP to assay development for serine/threonine kinases has been impeded because of lack of high-affinity anti-phosphoserine/threonine antibodies. In the present study, we report the discovery of a high-affinity, monoclonal anti-phosphoserine antibody, 2B9, with a Kd of 250 +/- 34 pM for a phosphoserine-containing peptide tracer, fluorescein-RFARKGS(PO(4))LRQKNV. Our data suggest that 2B9 is selective for fluorescein-RFARKGS(PO(4))LRQKNV. The antibody and tracer have been used for the development of a competitive FP assay for protein kinase C (PKC) in 384-well plates. Phosphatidylserine, which enhances the kinase activity of PKC in a Ca(2+)-dependent manner and has a structure similar to that of phosphoserine, did not interfere with binding of the peptide tracer to the antibody in the FP assay. The data indicate that the FP assay is more sensitive and robust than the scintillation proximity assay for PKC. The FP assay developed here can be used for rapid screening of hundreds of thousands of compounds for discovery of therapeutic leads for PKC-related diseases.

Measurement of cdk4 kinase activity using an affinity peptide-tagging technology.

Cyclin-dependent kinases such as Cdk4 are involved in the control of cell cycle progression, and misregulation of Cdk4 has been implicated in many types of cancers. In the present study, we report the development of a novel homogeneous assay using an affinity peptide-tagging technology for rapidly discovering Cdk4 inhibitors. The DNA sequence encoding a streptavidin recognition motif, or StrepTag (AWRHPQFGG), was cloned and expressed at the C-terminus of a fusion protein of a 152-amino acid hyperphosphorylation domain (Rb152) of the retinoblastoma protein (Rb) linked to GST at the N-terminus. This affinity peptide-tagged protein (GST-Rb152-StrepTag), which contains the two known phosphorylation sites of Rb, specifically phosphorylated by Cdk4 in vivo, was used as a substrate in the current in vitro kinase assay. After phosphorylation, scintillation proximity assay (SPA) scintillant beads coated with streptavidin were added. Radiolabeled GST-Rb152-StrepTag was brought in close proximity to the SPA scintillant beads through the interaction between StrepTag and streptavidin, resulting in the emission of light from beads. By applying the affinity peptide-tagging technology, we have eliminated the separation and wash steps which are normally required in a radioactive filtration assay. Therefore, this homogeneous method is simple, robust, and highly amenable to high-throughput screening of Cdk4-specific inhibitors. Furthermore, the affinity peptide tagging technique reported here is a simple, generic method that can be applied to many recombinant proteins for the development of kinase and protein-protein interaction assays.

Measurement of the protein tyrosine kinase activity of c-src using time-resolved fluorometry of europium chelates.

A nonradioactive, sensitive assay method to evaluate the activity of protein tyrosine kinases is described. This method utilizes europium chelate-labeled anti-phosphotyrosine antibodies to detect phosphate transfer to a polymeric substrate coated onto microtiter plate wells. The amount of phosphorylation is then detected using time-resolved, dissociation-enhanced fluorescence. Recombinant c-src was used to demonstrate that substrate phosphorylation was dependent on incubation time, enzyme concentration, and the amount of substrate used to coat the microtiter plate wells. A series of proprietary c-src inhibitors was evaluated in competition assays, and demonstrated a rank order of potency which was identical to that determined by other assay methods. Substrate phosphorylation was also demonstrated to be dependent on the concentration of ATP present during the kinase reaction. Because the kinase assay can be performed with different ATP concentrations (unlike with assays utilizing radioactive ATP analogs), the assay described can be used to distinguish compounds that compete for the ATP or substrate binding sites of the kinase.

Microxine, a new cdc2 kinase inhibitor from the Australian marine sponge Microxina species.

A new purine derivative microxine (1) was isolated from the Australian marine sponge Microxina sp. The compound was isolated via reversed-phase chromatography and its structure determined spectroscopically. Microxine was found to weakly inhibit cdc2 kinase activity with an IC(50) of 13 microM.

A solid-phase assay for the determination of protein tyrosine kinase activity of c-src using scintillating microtitration plates.

A solid-phase assay for the determination of protein tyrosine kinase (PTK) activity has been developed. The transfer of 33PO4 from ATP to the synthetic substrate poly(Glu, Tyr) 4:1 attached to the bioactive surface of scintillating microtiter plates served as the basis to evaluate enzyme activity. The procedure eliminates detection with phosphotyrosine antibodies, tedious separation of phosphorylated peptides with phosphocellulose membranes, and extensive washing steps. For these reasons, the traditionally time-consuming procedure can be performed with a simple three-step protocol. The method is highly accurate, rapid, and robotics friendly. The advantages over existing assays make this procedure especially suited for high throughput applications.

Novel C5a receptor antagonists regulate neutrophil functions in vitro and in vivo.

Novel recombinant human C5a receptor antagonists were discovered through modification of the C terminus of C5a. The C5a1-71T1M,C27S,Q71C monomer, (C5aRAM; CGS 27913), was a pure and potent functional antagonist. The importance of a C-terminal cysteine at position 71 to antagonist properties of C5aRAM was confirmed by studying C5a1-71 derivatives with replacements of Q71, C5a derivatives of various lengths (70-74) with C-terminal cysteines, and C5a derivatives of various lengths (71-74) with Q71C replacements. The majority of C5a1-71Q71 derivatives were agonists (C5a-like) in the human neutrophil C5a-induced intracellular calcium mobilization assay. The C5a1-71Q71C derivative was an antagonist. C5a derivatives of lengths 73 and 74 with C-terminal cysteines were agonists, while lengths 70 to 72 were antagonists. C5a derivatives of lengths 72, 73, and 74 with Q71C replacements were agonists, while, again, C5a1-71Q71C was an antagonist. C5aRAM and its adducts, including its dimer, C5aRAD (CGS 32359), were pure antagonists. Additionally, CSaRAM and CSaRAD inhibited binding of 125I-labeled recombinant human C5a to neutrophil membranes (Ki = 79 and 2 pM, respectively), C5a-stimulated neutrophil intracellular calcium mobilization (8 and 13 nM), CD11b integrin up-regulation (10 and 1 nM), superoxide generation (182 and 282 nM), lysozyme release (1 and 2 microM), and chemotaxis (11 and 7 microM). In vivo, intradermal injection of C5aRAM inhibited C5a-induced dermal edema in rabbits. Furthermore, a 5-mg/kg i.v. bolus of C5aRAD significantly inhibited C5a-induced neutropenia in micropigs when challenged with C5a 30 min after C5aRAD administration. C5aRAM and C5aRAD are novel, potent C5a receptor antagonists devoid of agonist or proinflammatory activity with demonstrated efficacy in vitro and in vivo.