| Summary: | Degradation of high molecular weight hyaluronic acid (HA) in humans is mainly catalyzed by hyaluronidase Hyal1. This enzyme is involved in many pathophysiological processes and therefore appears an interesting target for drug discovery. Until now, only a few inhibitors of human Hyal1 are known due to obstacles in obtaining active enzymes for inhibitor screening. The aim of the present work was to provide a convenient enzyme activity assay and show its feasibility by the identification of new inhibitors. By autodisplay, <i>Escherichia</i><i> </i><i>coli</i> F470 can present active Hyal1 on its surface. In this study, the inducible expression of Hyal1 on the cell surface of <i>E.</i><i> </i><i>coli</i> under the control of a rhamnose-dependent promoter (P<sub>rha</sub>) was performed and optimized. Enzyme activity per single cell was increased by a factor of 100 compared to the constitutive Hyal1 surface display, as described before. An activity of 6.8 × 10<sup>-4</sup> mU per single cell was obtained under optimal reaction conditions. By this modified activity assay, two new inhibitors of human Hyal1 were identified. Chicoric acid, a natural compound belonging to the phenylpropanoids, showed an IC<sub>50</sub> value of 171 µM. The steroid derivative testosterone propionate showed and IC<sub>50</sub> value of 124 ± 1.1 µM. Both values were in the same order of magnitude as the IC<sub>50</sub> value of glycyrrhizic acid (177 µM), one of the best known inhibitors of human Hyal1 known so far. In conclusion, we established a new enzyme activity assay for human Hyal1 and identified new inhibitors with this new assay method.
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