Abstract
Hyaluronidases are enzymes that degrade hyaluronan, an important component of the extracellular matrix. The mammalian hyaluronidases are considered to be involved in many (patho)physiological processes like fertilization, tumor growth, and metastasis. Bacterial hyaluronidases, also termed hyaluronate lyases, contribute to the spreading of microorganisms in tissues. Such roles for hyaluronidases suggest that inhibitors could be useful pharmacological tools. Potent and selective inhibitors are not known to date, although L-ascorbic acid has been reported to be a weak inhibitor of Streptococcus pneumoniae hyaluronate lyase (SpnHL). The x-ray structure of SpnHL complexed with L-ascorbic acid has been elucidated suggesting that additional hydrophobic interactions might increase inhibitory activity. Here we show that L-ascorbic acid 6-hexadecanoate (Vcpal) is a potent inhibitor of both streptococcal and bovine testicular hyaluronidase (BTH). Vcpal showed strong inhibition of Streptococcus agalactiae hyaluronate lyase with an IC(50) of 4 microM and weaker inhibition of SpnHL and BTH with IC(50) values of 100 and 56 microM, respectively. To date, Vcpal has proved to be one of the most potent inhibitors of hyaluronidase. We also determined the x-ray structure of the SpnHL-Vcpal complex and confirmed the hypothesis that additional hydrophobic interactions with Phe-343, His-399, and Thr-400 in the active site led to increased inhibition. A homology structural model of BTH was also generated to suggest binding modes of Vcpal to this hyaluronidase. The long alkyl chain seemed to interact with an extended, hydrophobic channel formed by mostly conserved amino acids Ala-84, Leu-91, Tyr-93, Tyr-220, and Leu-344 in BTH.
Highlights
Hyaluronidases are enzymes that degrade hyaluronan, an important component of the extracellular matrix
In contrast to bovine testicular hyaluronidase (BTH), Vc inhibits the Streptococcus pneumoniae hyaluronate lyase (SpnHL) with an IC50 value of around 6 mM under the experimental conditions used [25]
In our normalized assays with equiactive concentrations the bacterial enzymes were weakly inhibited by Vc with IC50 values of 6 mM for SagHL and 32 mM for SpnHL, respectively, whereas the bovine enzyme was not affected up to 100 mM
Summary
Materials—Hyaluronan (hyaluronic acid) from Streptococcus zooepidemicus was purchased from Aqua Biochem (Dessau, Germany). Determination of Inhibitory Activity on Hyaluronidases—The inhibitory effect of Vc and Vcpal on the hyaluronate lyases from S. pneumoniae, S. agalactiae strain 4755, and hyaluronidase from bovine testis was measured using a turbidimetric assay [33]. After incubation of the assay mixture for 30 min at 37 °C, 720 l of a 2.5% (w/v) cetyltrimethylammonium bromide solution (2.5 g of cetyltrimethylammonium bromide dissolved in 100 ml of 0.5 M sodium hydroxide solution, pH 12.5) were added to precipitate the residual high molecular weight substrate and to stop the enzyme reaction This mixture was again incubated at 25 °C for 20 min, and the turbidity of each sample was determined at 600 nm with a Uvikon 930 UV spectrophotometer (Kontron, Eching, Germany). The ligand Vcpal was modeled into the catalytic site according to the strong, immediately evident differ-
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