Therapeutically important enzymes with polar substrates or products: characterization by capillary electrophoresis and identification of inhibitors

Abstract

Enzymes are important drug targets, since they participate in many disease processes and can be inhibited by small drug molecules. In our research projects, a group of therapeutically important enzymes with polar substrates or products has been investigated by means of capillary electrophoresis (CE). <br /> The first enzyme we investigated was ecto-5’-nucleotidase (eN), which can catalyze the hydrolysis of the phosphoric acid ester bond of AMP yielding the corresponding nucleoside adenosine and inorganic phosphate. Treatment with eN inhibitors may be a promising novel strategy for cancer therapy, because eN has been found to be overexpressed on many cancer cells, and eN-generated adenosine prevents tumor destruction by inhibiting antitumor immunity. We investigated nucleotide mimetics which consisted of a nucleoside scaffold substituted in the 5’-position with a dipeptide moiety. The compounds were investigated at rat eN using a capillary electrophoresis (CE)-based assay, and potent inhibitors were further investigated at human eN. The test results showed that the inhibitory potency of the compounds appeared to be pH-dependent: when the buffer pH was decreased, the potency of the compounds increased. Since tumor tissues typically show low extracellular pH values, the new inhibitors might act as tumor-selective eN inhibitors without affecting physiologically important functions of eN, such as the production of adenosine in blood vessels, which shows vasodilatory effects.<br /> The second enzyme that we investigated was cerebroside sulfotransferase (CST), which catalyzes the transfer of a sulfate group from the co-substrate 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to cerebroside yielding cerebroside sulphate and adenosine-3′,5′-diphosphate (PAP). CST is a promising new therapeutic target for the treatment of metachromatic leukodystrophy (MLD), a devastating genetic disease. In the absence of an effective therapy, MLD leads to early death of the young patients. In the present study we developed a CE-based assay for monitoring the catalytic activity of CST. A low nanomolar limit of detection (LOD = 66.6 nM) was achieved for the enzymatic product PAP by using dynamic pH junction stacking. Our CE method was sensitive, robust and suitable for CST inhibitor screening, Ki value determination, and enzyme kinetic studies. Several reference compounds were tested including cerebrosides, psychosine and Congo Red in order to validate our method. We investigated analogues of the CST substrate cerebroside with this newly developed CE method, in order to study structure-activity relationship (SAR) and to identify and develop novel CST inhibitors. A substrate analogue α-galactosylceramide was identified to be a novel inhibitor, which will be used as a lead structure for developing more potent competitive inhibitors, which are urgently needed for the treatment of MLD. Moreover, SAR information for the CST cerebroside binding site would be highly useful especially since a crystal structure of the CST cerebroside binding site is not available yet. <br /> A further group of investigated enzymes were capsule biosynthesis enzymes from Staphylococcus aureus. We characterized the enzymes CapD and CapE of the Staphylococcus aureus serotype 5 biosynthesis cluster, which catalyze the first steps in the synthesis of the soluble capsule precursors UDP-D-FucNAc and UDP-L-FucNAc, respectively. A capillary CE-based method applying micellar electrokinetic chromatography (MEKC) was developed for the functional characterization of the enzymes using dynamic coating of the capillary with polybrene at pH 12.4. The limits of detection for the CapD and CapE products UDP-2-acetamido-2,6-dideoxy-α-D-xylo-hex-4-ulose and UDP-2-acetamido-2,6-dideoxy-β-L-arabino-hex-4-ulose, respectively, were below 1 µM. Using this new, robust and sensitive method we performed kinetic studies for CapD and CapE, and screened a compound library in search for enzyme inhibitors. Several active compounds were identified and characterized. Our studies contribute to a profound understanding of the capsule biosynthesis in pathogenic bacteria. This approach may lead to the identification of novel anti-virulence and antibiotic drugs.