Motomi Matsukawa

A Screening System of Prodrugs Selective for MAO-A or MAO-B

We synthesized several prodrugs of glycine and gamma-aminobutyric acid. In order to establish a screening system from the prodrugs of selective activity to MAO-A or MAO-B, we examined purification conditions such as solubilization with Triton X-100, precipitation with ammonium sulfate, gel filtration and anion exchange chromatography. MAO-B was purified from various tissues such as guinea pig brain, kidney and spleen. MAO-A from human placenta without MAO-B was unstable in above purifications and used as crude. At each purification step, we checked sensitivity of the enzyme to specific inhibitors by developing a convenient fluorescence assay, in which hydrogen peroxide produced by the enzyme was reacted with p-hydroxyphenylpropionic acid. A fluorescence microplate reader measured a fluorescence of the fluorescent product from p-hydroxyphenylpropionic acid with horseradish peroxidase. In comparison with milacemide, N,N-bis(carbamoylmethyl)-N-pentylamine was the best and exclusive substrate for MAO-B. 2-N-(phenylethylamino)-acetoamide was the good substrate for MAO-A and MAO-B same as milacemide. 4-N-(n-pentylamino)-butyric acid and 4-(N-phenylethylamino)-butyric acid were the moderate substrates for both enzymes, which should release gamma-aminobutyric acid. These drugs will be new leading compounds.

Development and Application of a Microplate Assay Method for the Mass Screening of MMP Inhibitors

The matrix metalloproteinases (MMPs) are membrane-bond zinc endopeptidases and are thought to be essential for the diverse invasive processes of angiogenesis and tumor metastasis. Thus, inhibition of MMPs has generated considerable interest as a therapeutic strategy. Two approaches have been applied in a search for useful MMP inhibitors: substrate-based design of pseudopeptide derivatives, and random screening of compound libraries and natural products. A number of MMP inhibitors (MMPIs), which are mainly synthetic, have entered into Phase III clinical trials. To find novel MMPIs that are oral, active, and substrate specific, a simple and convenient assay method is necessary. For this purpose, we developed a microplate assay method, a modification of the flow injection analysis (FIA) method. 1 Two recombinant MMPs (r-MMP-2 and r-MT1-MMP) were expressed as latent forms in E. coli in the manner previously used for expression of r-MMP-7. 2 After activation, MMPs were incubated with a fluorogenic substrate peptide (MOCAc-Pro-Leu-GlyLeu-A 2 pr(DNP)-Ala-Arg-NH 2 ). The fluorescent intensity of a proteolytic peptide (MOCAc-PropLeu-Gly) was measured every 15 min up to 2 hr with a microplate reader by monitoring at 340 nm for excitation and at 400 nm for emission. The IC 50 values were calculated from the data obtained after 2 hr of incubation. The IC 50 values of a PRCGVPD-containing peptide (BS-10; MQKPRCGVPD) and of its analogue peptides against r-MMP-7 were similar to the IC 50 values obtained by FIA. To confirm the activities of r-MMP-2 and MT1-MMP, the IC 50 values of the same peptides were measured, and a broad spectrum of inhibitory activities was observed. 3 By using this method, inhibitory activities of more than 80 test samples can be analyzed in only 4 min. For application to the development of novel MMP inhibitors, Chinese drugs and crude drugs were studied by this method. The inhibitory activities of 35 crude drugs are summarized in F IGURE 1. The results of two assays of the components of a green tea are illustrated in F IGURE 2. Only one day was required to mea-