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Adenine phosphoribosyltransferase plays a role in purine salvage by catalyzing the direct conversion of adenine to adenosine monophosphate. The involvement of the purine salvage pathway in tumor proliferation and angiogenesis makes adenine phosphoribosyltransferase a potential target for oncology drug discovery. We have expressed and characterized recombinant, N-terminally His-tagged human adenine phosphoribosyltransferase. Two assay formats were assessed for use in a high throughput screen: a spectrophotometric-based enzyme-coupled assay system and a radiometric ionic capture scintillation proximity bead assay format. Ultimately, the scintillation proximity assay format was chosen because of automated screening compatibility limitations of the coupled assay. We describe here the biochemical characterization of adenine phosphoribosyltransferase and the development of a robust, homogeneous, 384-well assay suitable for high throughput screening.

NAD synthetase is responsible for the conversion of nicotinic acid adenine dinucleotide to nicotinamide adenine dinucleotide. This reaction provides a biosynthetic route of the coenzyme and, thus, a source of cellular reducing equivalents. Alterations in the oxidative reductive potential of the cell have been implicated as a contributing factor in many disease states. Thus, this enzyme represents a new class of potential drug targets, and, hence, our efforts were focused upon developing a robust assay for utilization in a high throughput screen. Toward that end, we describe a coupled enzyme assay format for the measurement of recombinant human NAD synthetase by employing lactate dehydrogenase in a cycling/amplification reaction linked ultimately to the fluorescence generation of resorufin from resazurin via diaphorase. We present kinetics of the reaction of NAD synthetase in the coupled assay format, optimization conditions, and inhibition of the reaction by gossypol {1,1',6,6',7,7'-hexahydroxy-3,3'-dimethyl-5,5'-bis(1-methylethyl)-[2,2'- binaphthalene]-8,8'-dicarboxaldehyde}and illustrate the robustness of the assay by demonstrating 384-well microtiter plate uniformity statistics. Collectively, our results show that the assay method is both robust and well suited for this class of enzymes involved in the NAD+biosynthetic pathway.

Fatty acyl coenzyme A (CoA) synthetases are a group of enzymes responsible for the activation of fatty acids through ligated high-energy CoA thioester bonds. Ultimately these fatty acyl-CoA conjugates are routed toward either anabolic or catabolic pathways. Long-chain-fatty-acid-CoA ligase 5 (LACS 5) utilizes a wide range of saturated fatty acids with a substrate preference for C16–C18 unsaturated fatty acids. This enzyme represents a new class of potential drug targets, and, hence, our efforts were focused upon developing a robust assay for utilization in a high throughput screen. Toward that end, we describe a radiometric homogeneous measurement of the enzymatic reaction by employing ionic capture of the reaction product onto YSi scintillation proximity assay (SPA) beads. We present kinetic and inhibition data for LACS 5 using this SPA format. Our results show that the assay method is both robust and well suited for this class of lipid-metabolizing enzymes.

We have characterized a recombinantly expressed N-terminally tagged GST fusion of the tyrosine kinase domain of human EphB3. The EphB3 kinase domain was shown to phosphorylate a group of synthetic tyrosine-containing peptides derived from a proprietary biotinylated kinase-biased peptide substrate library. In addition, the enzyme activity was stimulated by the divalent cation, manganese, and inhibited by addition of magnesium. The most active tyrosine-containing peptide, a biotinylated 49-mer, displayed saturation kinetics with an apparent Kmof ~0.4 μM. The apparent Kmfor ATP was determined to be ~3 μM. The kinetics of the reaction was linear from concentrations of enzyme of 0.5 to 2 nM, and at or below the Kmconcentrations of the two substrates for at least 2 h at room temperature. Moreover, the tryrosine kinase inhibitor, PP2, produced an IC50of roughly 0.8 μM. In addition, the enzyme tolerated the solvent DMSO and was stable to multiple freeze/thaw cycles. Stability of the enzyme at 4°C storage was seen out to 6 h with an ~50% reduction of activity by 24 h. Formatting the assay in a 384-well microtiter plate produced good uniformity of signal at 100% inhibition, 50% inhibition, and no inhibition. The coefficient of variance was at or below 10% with a signal-to-background ratio of ~24 and a z value of 0.72. Collectively, these results showed the ability to configure a robust HTS for a truncated recombinantly expressed family member of the Ephrin tyrosine kinases.

Inositol-specific PLCs comprise a family of enzymes that utilize phosphoinositide substrates, e.g., PIP2, to generate intracellular second messengers for the regulation of cellular responses. In the past, monitoring this reaction has been difficult due to the need for radiolabeled substrates, separation of the reaction products by organic-phase extraction, and finally radiometric measurements of the segregated products. In this report, we have studied the enzymatic characteristics of two novel PLCs that were derived from functional genomic analyses using a phospholipid-modified solid scintillating support. This method allows for the hydrophobic capture of the [3H]phosphoinositide substrate on a well defined scintillation surface and the homogenous measurement of the enzymatic hydrolysis of the substrate by proximity effects. Our results show that the assay format is robust and well suited for this class of lipid-metabolizing enzymes.

Inositol-specific phospholipase Cs(PLCs) are a group of enzymes involved in the signal transduction pathway of many plasma membrane receptor mediated events. We developed a modified solid surface to capture [3H] PIP2onto the Basic FlashPlate® in order to monitor PLC activity. Our results clearly demonstrate the utility of [3H] PIP2-Coated Phospholipid FlashPlate® microtiter plates for assessing PLC activity for HTS of receptor-coupled functional assays. The results show that PLC activity can be measured easily from a variety of sources including purified recombinant enzyme preparations, crude HL60 cell lysates and permeabilized A431 human carcinoma cells. Moreover, this format provides a surface comparable to that used for classical solution based radiolabeled mixed phospholipid micelle studies and illustrates the feasibility of this assay for measuring PLC activation in a variety of different drug screening assays.

Inositol-specific phospholipase Cs(PLCs) are a group of enzymes involved in the signal transduction pathway of many plasma membrane receptor mediated events. We developed a modified solid surface to capture [3H] PIP2onto the Basic FlashPlate® in order to monitor PLC activity. Our results clearly demonstrate the utility of [3H] PIP2-Coated Phospholipid FlashPlate® microtiter plates for assessing PLC activity for HTS of receptor-coupled functional assays. The results show that PLC activity can be measured easily from a variety of sources including purified recombinant enzyme preparations, crude HL60 cell lysates and permeabilized A431 human carcinoma cells. Moreover, this format provides a surface comparable to that used for classical solution based radiolabeled mixed phospholipid micelle studies and illustrates the feasibility of this assay for measuring PLC activation in a variety of different drug screening assays.

When the terminal amino group in the side chain of primaquine was blocked with an ethoxyacetyl group shown in 2, or eliminated by oxidative deamination to carboxylic acid 3, the antimalarial effect was markedly reduced in a screening assay which measures tissue schizonticidal activity. The optical isomers 1Aand 1Bof primaquine had similar antimalarial potency to the racemic mixture but 1Bappeared less toxic. The 5-phenoxy-substituted analogue 4, belonging to a new class of antimalarials, showed similar potency in the assays to either 1Aor 1Bbut seemed less cytotoxic than (±)-primaquine. Compounds 1Aand 1Bwere found to be competitive inhibitors of human monoamine oxidase (MAO) A and B ( Kirange 103–225 μM), but 4showed 10–30-fold greater competitive inhibition of MAO A ( Ki= 6.8 μM) and 40–90-fold greater non-competitive inhibition of MAO B ( Ki= 2.3 μM).

Bacterially expressed synapse-specific clathrin assembly protein, AP-3 (F1-20/AP180/NP185/pp155), bound with high affinity both inositol hexakisphosphate (InsP6) (Kd= 239 nM) and diphosphoinositol pentakisphosphate (PP-InsP5) (Kd= 22 nM). The specificity of this ligand binding was demonstrated by competitive displacement of bound [3H]InsP6. IC50values were as follows: PP-InsP5= 50 nM, InsP6= 240 nM, inositol-1,2,4,5,6-pentakisphosphate (Ins(1,2,4,5,6)P5) = 2.2 μM, inositol-1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) = 5 μM, inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) > 10 μM, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) > 10 μM. Moreover, 10 μM inositol hexasulfate (InsS6) displaced only 15% of [3H]InsP6. The physiological significance of this binding is the ligand-specific inhibition of clathrin assembly (PP-InsP5> InsP6> Ins(1,2,4,5,6)P5); Ins(1,3,4,5,6)P5and InsS6did not inhibit clathrin assembly. We also observed high affinity binding of InsP6to purified bovine brain AP-3. We separately expressed the 33-kDa amino terminus and the 58-kDa carboxyl terminus, and it was the former that contained the high affinity inositol polyphosphate binding site. These studies suggest that specific inositol polyphosphates may play a role in the regulation of synaptic function by interacting with the synapse-specific clathrin assembly protein AP-3.

When the terminal amino group in the side chain of primaquine was blocked with an ethoxyacetyl group shown in 2, or eliminated by oxidative deamination to carboxylic acid 3, the antimalarial effect was markedly reduced in a screening assay which measures tissue schizonticidal activity. The optical isomers 1A and 1B of primaquine had similar antimalarial potency to the racemic mixture but 1B appeared less toxic. The 5‐phenoxy‐substituted analogue 4, belonging to a new class of antimalarials, showed similar potency in the assays to either 1A or 1B but seemed less cytotoxic than (±)‐primaquine. Compounds 1A and 1B were found to be competitive inhibitors of human monoamine oxidase (MAO) A and B (Kirange 103–225 μM), but 4 showed 10–30‐fold greater competitive inhibition of MAO A (Ki= 6.8 μM) and 40–90‐fold greater non‐competitive inhibition of MAO B (Ki= 2.3 μM).

The neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and its deuterated analogues were oxidized to their corresponding dihydropyridinium species (MPDP+) by preparations of pure human liver MAO B : monoclonal antibody complex to investigate the mechanism of MPTP activation. Lineweaver‐Burk plots of initial reaction rates revealed that the Km,appvalues for the various deuterated MPTP analogues were similar to those of MPTP. In contrast, Vmax,appvalues were substantially decreased by substitution of deuterium for hydrogen on the tetrahydropyridinium ring, especially at C‐6. Deuterium substitution on the N‐methyl group alone did not significantly reduce Vmax,app. These studies support the interpretation that oxidation of MPTP at the C‐6 position on the tetrahydropyridine ring is a major rate‐determining step in its biotransformation by MAO B.

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its deuterated analogues were oxidized to their corresponding dihydropyridinium species (MPDP +) by preparations of pure human liver MAO B : monoclonal antibody complex to investigate the mechanism of MPTP activation. Lineweaver-Burk plots of initial reaction rates revealed that the Km,appvalues for the various deuterated MPTP analogues were similar to those of MPTP. In contrast, Vmax,appvalues were substantially decreased by substitution of deuterium for hydrogen on the tetrahydropyridinium ring, especially at C-6. Deuterium substitution on the N-methyl group alone did not significantly reduce Vmax,app. These studies support the interpretation that oxidation of MPTP at the C-6 position on the tetrahydropyridine ring is a major rate-determining step in its biotransformation by MAO B.