Design and synthesis of AX7574: a microcystin-derived, fluorescent probe for serine/threonine phosphatases.

The design and synthesis of AX7574, a microcystin-derived probe for serine/threonine phosphatases, is described. A key step in the synthesis was the conjugation under basic conditions of a tetramethylrhodamine 1,3-diketone derivative to the arginine side chain present in microcystin-LR. The resulting conjugate specifically labeled the active site of protein phosphatases 1 (PP-1) with a 1:1 stoichiometry and IC50 of 4.0 nM. AX7574 was used to isolate and identify PP-1, PP-2A, PP-4, and PP-6 in Jurkat cells. Finally, AX7574 was able to record changes in the phosphatase activity levels of calyculin A treated Jurkat cells versus untreated control cells.

Cytochrome P450 inhibition using recombinant proteins and mass spectrometry/multiple reaction monitoring technology in a cassette incubation.

Detailed cytochrome P450 (P450) inhibition profiles are now required for the registration of novel molecular entities. This method uses combined substrates (phenacetin, diclofenac, S-mephenytoin, bufuralol, and midazolam) with combined recombinant P450 enzymes (CYP1A2, 2C9, 2C19, 2D6, and 3A4) in an attempt to limit interactions with other more minor P450s and associated reductases. Kinetic analysis of single substrate with single P450 (sP450) yielded apparent Km values of 25, 2, 20, 9, and 3 microM, for CYP1A2, 2C9, 2C19, 2D6, and 3A4, respectively. Combined substrates with combined P450s (cP450) yielded apparent Km values of 65, 4, 19, 7, and 2 microM. Selectivity of the substrates for each P450 isoform was checked. Phenacetin proved to be the least selective substrate. However, the ratio of the various P450s was modified in the final assay such that metabolism of phenacetin by other enzymes was approximately 20% of the metabolism by CYP1A2. IC50 determinations with alpha-naphthoflavone (0.04 microM), sulfaphenazole (0.26 microM), tranylcypromine (9 microM), quinidine (0.02 microM), and ketoconazole (0.01 microM) were similar for sP450 and cP450 enzymes. The assay was further evaluated with 11 literature compounds and 52 in-house new chemical entities, and the data compared with radiometric/fluorescent values. The overall protein level of the assay was reduced from the original starting point, as this led to some artificially high IC50 measurements when compared with existing lower protein assays (radiometric/fluorometric). This method offers high throughput P450 inhibition profiling with potential advantages over current radiometric or fluorometric methods.

Synthesis of N-acetylglucosamine derivatives as probes for specificity of chicken hepatic lectin.

Syntheses of the following compounds are described: 6-(Trifluoroacetylamino)hexyl 2-acetamido-2,6-dideoxy-beta-D-glucopyranoside and 2-acetamido-2-deoxy-alpha-D-xylopyranoside, two allyl 2-acetamido-2-deoxy-alpha-D-glucopyranosiduronic acid derivatives, and several allyl 2-acylamido-2-deoxy-beta-D-glucopyranosides having different acyl groups. These and other compounds were used as inhibitors in the binding assay for the chicken hepatic lectin specific for N-acetylglucosamine. We found that: 1) The inhibitory potency of N-acylglucosamine derivatives decreased progressively with increase in the size of acyl group, 2) absence of either 3- or 4-OH group of N-acetylglucosamine lowered the binding affinity more than 100-fold, and 3) the presence of a negatively charged group (carboxylic acid) at the C-6 position did not lower the affinity. The first two items are similar to the mammalian hepatic galactose/N-acetylgalactosamine lectins, but the last item is in a strong contrast to the mammalian lectins.