In the case of hyaluronidases the exact determination of Ki values of inhibitors is extremely difficult. On the one hand, the substrate hyaluronan is difficult to quantify as the used preparations are not homogeneous but consist of a mixture of different HA molecules with different molar weights. Additionally, HA is cleaved by the action of hyaluronidases and the resulting hyaluronan fragments are again substrates for another catalytic reaction which affects the substrate concentration. On the other hand, the competing transglycosylation reaction also influences the substrate concentration, a necessary parameter for the calculation of Ki values. Therefore, the determination of IC50 values is a viable approach to collect comparable inhibitor data for libraries of compounds with respect to the elucidation of structure-activity relationships. As IC50 values are strongly dependent on the method and the assay conditions (e.g. incubation conditions, enzymes, enzyme concentrations, substrate concentrations, pH value), huge discrepancies can occur, so that a direct comparison of data from different laboratories is impossible in most cases, For example, DSCG is designated as one of the most potent inhibitors of bovine testicular hyaluronidase
with an IC50 value of 29 µM17. When this compound was investigated upon BTH inhibition under the assay conditions applied in this work, the compound proved to be much less potent: no inhibition of the enzyme was detectable at concentrations below 2 mM at pH 5.0 in the turbidimetric assay, and an IC50 value as high as 1.24 mM was determined in the colorimetric Morgan Elson assay38 by Salmen39.
Therefore, several reference compounds described in the literature were investigated for inhibition of the hyaluronidases under standardized conditions in the turbidimetric activity assay based on the method of Di Ferrante40 (see chapter 3) to gain reliable and comparable data with respect to the classification of the synthesized inhibitors in terms of potency. The structures of the tested reference compounds are depicted in Figure 5.1, the obtained IC50 values are summarized in Table 5.1.
Chapter 5 Inhibitors of hyaluronidases: reference compounds
Figure 5.1. Structures of investigated small molecule hyaluronidase inhibitors.
Table 5.1. Inhibitory activities of reference compounds on four investigated hyaluronidases, Hyal-1 PH-20 BTH SagHyal4755
Compound
IC50 [µM]a
Glycyrrhizic acid 26 ± 1 17 ± 1 737 ± 30 46 ± 2 Glycyrrhetinic acid > 100 6.9 ± 0.7 > 100 54 ± 3 Carbenoxolone > 130 3.2 ± 0.5 134 ± 7 20 ± 1 Indomethacin 390 ± 30 82 ± 9 540b 350c
Diclofenac > 500 68 ± 7 > 1000 161 ± 11 DSCG 474 ± 75 > 900 > 2000 1208 ± 81 Phloretin 358 ± 49 200 ± 12 > 1400 213 ± 8 Kaempferol > 1000 288 ± 23 > 1000 237 ± 15
Sylibinin > 1000 111 ± 10 > 1000b 296 ± 18 Lauryl gallate > 100 20 ± 3 > 100 13 ± 1 L-Ascorbic acid > 60000 7564 ± 883 > 100000d 6100 ± 100d 1,3-Diacetylbenzimidazole-2-thione > 1000 > 1000 > 1000b 160c
3-Phenyl-1-(2-thioxobenzo[d]
oxazol-3(2H)-yl)propan-1-one > 100 > 100 > 100d 15 ± 1d Tetradecane sulfonic acid 21 ± 1 7.6 ± 0.3 93 ± 5 12 ± 1 Lauric acid > 100 25 ± 2 > 150 > 150 Tannic acid 3.6 ± 0.5 1.1 ± 0.1 > 160 8.9 ± 0.6
a inhibition of enzyme expressed as IC50 (µM), mean values ± SEM; IC50 values determined at pH 5.0 (PH-20, BTH, SagHyal4755) or 3.5 (Hyal-1) in the turbidimetric assay; bas determined by Salmen39 at pH 3.6; cas determined by Salmen39; das determined by Braun41.
It is obvious that broadly accepted inhibitors like kaemferol, DSCG or indomethacin only weakly inhibit the investigated mammalian hyaluronidases and the bacterial hyaluronate lyase from Streptococcus agalactiae strain 4755. Among the investigated inhibitors, strongest inhibition of Hyal-1 was found for the detergent tetradecane sulfonic acid and for glycyrrhizic acid with IC50 values of 21 µM and 26 µM, respectively. The related compound carbenoxolone together with tetradecane sulfonic acid were found to be the most potent inhibitors of recombinantly expressed PH-20. The weak inhibitor DSCG was found to be outstanding due to the fact that certain selectivity for Hyal-1 versus human PH-20 was obtained, whereas the opposite was found for all other reference compounds. The substances were only very weakly active or inactive on BTH, whereas the bacterial enzyme was inhibited by most compounds in the (sub)millimolar range. The most potent inhibitors of the bacterial enzyme were the antioxidant lauryl gallate and the detergent tetradecane sulfonic acid with IC50 values of 13 µM and 12 µM, respectively. 1,3-Diacetylbenzimidazole-2-thione and
3-phenyl-1-(2-thioxobenzo[d]oxazol-3(2H)-Chapter 5 Inhibitors of hyaluronidases: reference compounds
hyaluronate lyase in our workgroup39, 41 proved to be inactive when tested on the mammalian enzymes.
Tannic acid, which is commonly used for the immobilization of enzymes42, is composed of gallic acid moieties attached to a glucose core und must therefore be clearly separated from the other nonpolymeric low molecular weight inhibitors which are subject of this work. Other sulfated polymers like sodium-polystyrene-4-sulfonate (PSS) also possess inhibitory potency in the (sub)micromolar range as reported by Isoyama et al.15, but it should be stressed that the molecular weight of those derivatives is extremely high (e.g. 519,000 g/mol for PSS990000). Thus, IC50 values in the same range as those of low molecular weight inhibitors are obtained when calculated on a weight concentration basis. Although the mentioned polymers or detergents like tetradecane sulfonic acid are rather potent hyaluronidase inhibitors, these compounds are far from being druglike and their utility as pharmacological tools for in vivo investigations is limited. Thus, the search for more potent and selective low molecular weight inhibitors is necessary.
5.3 References
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Chapter 5 Inhibitors of hyaluronidases: reference compounds
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(39) Salmen, S. Inhibitors of bacterial and mammalian hyaluronidase. Synthesis and structure-activity relationships. Doctoral Thesis, Regensburg, 2003. http://www.opus-bayern.de/uni-regensburg/volltexte/2004/320/
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Chapter 6 Derivatives of the glucurono-6,3-lactone as hyaluronidase inhibitors