Morgan-Elson assay

The test compounds, dissolved in DMSO (18 µl), were incubated at 37 °C in an incubation mixture containing 200 µl of buffer (if not otherwise indicated: McIlvaine’s buffer: solution A: 0.2 M Na2HPO4, 0.1 M NaCl, solution B: 0.1 M citric acid, 0.1 M NaCl; solution A and B were mixed in appropriate portions to adjust the required pH), 50 µl of BSA solution (0.2 mg/ml in water), 50 µl of HA solution (5 mg/ml in water), 82 µl of water and 50 µl of an enzyme solution. The final DMSO concentration was 3.9

% (v/v). After incubation at 37 °C, the enzyme reaction was stopped by addition of 110 µl of alkaline borate solution and subsequent heating for 4.5 min at 100 °C. The alkaline borate solution was prepared immediately before use from a borate solution (17.3 g H3BO4 and 7.8 g KOH in 100 ml water) and a potassium carbonate solution (8.0 g K2CO3 in 10 ml water). After cooling on ice for 2 min 1250 µl of N,N-dimethylaminobenzaldehyde (20.0 g N,N-N,N-dimethylaminobenzaldehyde dissolved in 25 ml of concentrated HCl and 75 ml of glacial acetic acid; the solution was diluted with 4 volumes of glacial acetic acid immediately before use) was added and the mixture was incubated at 37 °C for 20 min. The samples were transferred into acryl cuvettes and the absorbance of the colored product was measured photometrically at a wavelength of 586 nm using an Uvikon 930 UV spectrophotometer (Kontron, Eching, Germany). As reference for 100 % enzyme activity the formation of the red colored product of a sample without inhibitor (18 µl of DMSO was used instead) was quantified. In absence of both enzyme and inhibitor (50 µl of BSA solution and 18 µl

be measured. In the cases where only quantification of the enzyme activity without inhibitor was necessary, the inhibitor solution was replaced by water (18 µl).

The enzyme activity was calculated from the formation of the red colored product measured at 586 nm. The effect of the test compounds on the enzymatic activity was calculated according to the equation:

(Asample – Aenzyme)

relative activity [in %] = ––––––––––––––––––––– · 100 % (Ainhibitor – Aboth)

where Aenzyme is the absorbance of the reference sample without enzyme (50 µl of BSA (0.2 mg/ml) solution was used instead), Ainhibitor is the absorbance of the reference sample without inhibitor (7 µl of DMSO was used instead) and Aboth is the absorbance without both, enzyme and inhibitor.

3.5.3 Turbidimetric assay

The turbidimetric hyaluronidase activity assay described by Di Ferrante¹² was modified to allow for the performance in 96-well plates as recently described²²:

Incubation mixtures contained the following compounds: 31 µL incubation buffer, 8 µL BSA (0.2 mg/mL), 8 µL HA (2 mg/ml), 13 µl H2O. For the investigation of the test compounds 3 µl of the inhibitor solution in dimethylsulfoxide (DMSO) were added to the incubation mixture. The final concentration of DMSO in the incubation mixture was 4 % which is well tolerated by BTH and SagHyal4755 as demonstrated previously^{13, 14, 24}. The decrease in enzymatic activity by DMSO was also found to be acceptable when the recombinant human enzymes were investigated. If not indicated otherwise McIlvaine’s buffer was used as incubation buffer (see above). The enzymatic reaction was started by addition of 10 µL enzyme solution (see Table 3.1 for prepared enzyme solutions). After incubation at 37 °C (incubation times see Table 3.1) the enzymatic reaction was stopped by addition of 200 µl of alkaline cetyltrimethylammonium bromide (CTAB) solution (2.5 % (w/v) CTAB in 0.5 M NaOH), and the plates were incubated for 20 min at room temperature. The turbidity was quantified by measurement of the optical density (OD) at 580 nm in a microplate reader (Tecan Deutschland GmbH, Crailsheim, Germany). The plate was shaken in the reader for 10 s, then the OD was measured after 2 s of settling time by 5 flashes

Chapter 3 Determination of hyaluronidase activity

For the investigation of potential inhibitors, samples without inhibitor and without both inhibitor and enzyme were taken as references. The activities were plotted against the logarithm of the inhibitor concentration, and IC50 ± SEM values were calculated by curve fitting of the experimental data with Sigma Plot 8.0 (SPSS Inc., Chicago, USA).

IC50 values for BTH (Neopermease^®) and SagHyal4755 generally were determined using the turbidimetric assay performed in acryl cuvettes as described previously¹⁹. As poor solubility of the compounds could lead to false positive results in the turbidimetric assay, the terminal solubility was determined prior to the investigation of hyaluronidase inhibition. To determine the solubility of the test compounds, a sample containing 600 µl of citrate-phosphate buffer, 396 µl of water, 300 µl of BSA solution (0.2 mg/ml in water) and 54 µl of a solution of the respective test compound (dissolved in DMSO) at various concentrations was measured at 600 nm. A cuvette filled with water served as reference. Compounds were tested for inhibitory activity at concentrations, where no turbidity was measured.

In Table 3.1 the enzymatic activities, pH values and incubation times employed in the turbidimetric assay are summarized. The incubation periods were adjusted to obtain comparable substrate degradation.

Table 3.1. Enzymatic activities, pH and incubation times employed in the turbidimetric assay.

Enzyme

aAccording to the definition of the International Union of Biochemistry 1 unit (U) of hyaluronidase catalyzes the liberation of 1 µmol N-acetyl-D-glucosamine (NAG) at the reducing ends of sugars per minute under specific conditions. Enzymatic activity was calculated from the formation of the red-colored product per unit time, using standards with known NAG concentration¹¹; ^btested using the turbidimetric assay performed in microtiter plates; ^ctested using the turbidimetric assay using cuvettes as described previously¹⁹.

BTH and SagHyal4755 were used at equiactive concentrations as described by Braun and Salmen^{13, 14}. The enzymatic activity of Hyal-1 used in the turbidimetric assay was in the same range as that of BTH. As small modifications in the enzyme concentration only result in negligible changes of the IC50 values, the obtained IC50

values for Hyal-1, BTH and SagHyal4755 are comparable and conclusions about selectivity can be made. Due to economic reasons, the recombinantly expressed human PH-20 had to be used at significantly lower concentrations possibly leading to lower IC50 values. Thus, for selectivity considerations, representative compounds were re-investigated at equiactive concentrations (chapter 10.3.5).

3.6 References

(1) Duran-Reynals, F., Exaltation de l'activité du virus vaccinal par les extraits de certains organes. CR Séances Soc. Biol. Fil. 1928, 99, 6-7.

(2) Hynes, W. L.; Ferretti, J. J., Assays for hyaluronidase activity. Methods Enzymol. 1994, 235, 606-616.

(3) Stern, M.; Stern, R., An ELISA-like assay for hyaluronidase and hyaluronidase inhibitors.

Matrix 1992, 12 (5), 397-403.

(4) Girish, K. S.; Kemparaju, K., The magic glue hyaluronan and its eraser hyaluronidase: a biological overview. Life Sci. 2007, 80 (21), 1921-1943.

(5) Afify, A. M.; Stern, M.; Guntenhoner, M.; Stern, R., Purification and characterization of human serum hyaluronidase. Arch. Biochem. Biophys. 1993, 305 (2), 434-441.

(6) Jedrzejas, M. J.; Stern, R., Structures of vertebrate hyaluronidases and their unique enzymatic mechanism of hydrolysis. Proteins 2005, 61 (2), 227-238.

(7) Csoka, A. B.; Frost, G. I.; Wong, T.; Stern, R., Purification and microsequencing of hyaluronidase isozymes from human urine. FEBS Lett. 1997, 417 (3), 307-310.

(8) Stern, R.; Jedrzejas, M. J., Hyaluronidases: their genomics, structures, and mechanisms of action. Chem. Rev. 2006, 106 (3), 818-839.

(9) Gacesa, P.; Savitsky, M. J.; Dodgson, K. S.; Olavesen, A. H., A recommended procedure for the estimation of bovine testicular hyaluronidase in the presence of human serum. Anal.

Biochem. 1981, 118 (1), 76-84.

(10) Reissig, J.; Strominger, J.; Leloir, L., A modified colorimetric method for the estimation of N-Acetylamino sugars. J. Biol. Chem. 1955, 217, 959-966.

(11) Muckenschnabel, I.; Bernhardt, G.; Spruss, T.; Dietl, B.; Buschauer, A., Quantitation of hyaluronidases by the Morgan-Elson reaction: comparison of the enzyme activities in the plasma of tumor patients and healthy volunteers. Cancer Lett. 1998, 131 (1), 13-20.

(12) Di Ferrante, N., Turbidimetric measurement of acid mucopolysaccharides and hyaluronidase activity. J. Biol. Chem. 1956, 220 (1), 303-306.

(13) Braun, S. New inhibitors of bacterial hyaluronidase - Synthesis and structure-activity relationships. Doctoral thesis, Regensburg, 2005. http://www.opus-bayern.de/uni-regensburg/volltexte/2006/585/

Chapter 3 Determination of hyaluronidase activity

(14) 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/

(15) Salmen, S.; Hoechstetter, J.; Kasbauer, C.; Paper, D. H.; Bernhardt, G.; Buschauer, A., Sulphated oligosaccharides as inhibitors of hyaluronidases from bovine testis, bee venom and Streptococcus agalactiae. Planta Med. 2005, 71 (8), 727-732.

(16) Rigden, D. J.; Botzki, A.; Nukui, M.; Mewbourne, R. B.; Lamani, E.; Braun, S.; Von Angerer, E.; Bernhardt, G.; Dove, S.; Buschauer, A.; Jedrzejas, M. J., Design of new benzoxazole-2-thione-derived inhibitors of Streptococcus pneumoniae hyaluronan lyase: structure of a complex with a 2-phenylindole. Glycobiology 2006, 16 (8), 757-765.

(17) Botzki, A. Structure-based design of hyaluronidase inhibitors. Doctoral Thesis, Regensburg, 2004. http://www.opus-bayern.de/uni-regensburg/volltexte/2004/378/

(18) Botzki, A.; Rigden, D. J.; Braun, S.; Nukui, M.; Salmen, S.; Hoechstetter, J.; Bernhardt, G.;

Dove, S.; Jedrzejas, M. J.; Buschauer, A., L-Ascorbic acid 6-hexadecanoate, a potent hyaluronidase inhibitor. X-ray structure and molecular modeling of enzyme-inhibitor complexes. J. Biol. Chem. 2004, 279 (44), 45990-45997.

(19) Spickenreither, M.; Braun, S.; Bernhardt, G.; Dove, S.; Buschauer, A., Novel 6-O-acylated vitamin C derivatives as hyaluronidase inhibitors with selectivity for bacterial lyases. Bioorg.

Med. Chem. Lett. 2006, 16 (20), 5313-5316.

(20) Hoechstetter, J. Characterisation of bovine testicular hyaluronidase and a hyaluronate lyase from Streptococcus agalactiae. 2005. http://www.opus-bayern.de/uni-regensburg/volltexte/2005/519/

(21) Hofinger, E. Recombinant expression, purification and characterization of human hyaluronidases. doctoral thesis, Regensburg, 2007. http://www.opus-bayern.de/uni-regensburg/volltexte/2007/788/

(22) Hofinger, E. S.; Spickenreither, M.; Oschmann, J.; Bernhardt, G.; Rudolph, R.; Buschauer, A., Recombinant human hyaluronidase Hyal-1: insect cells versus Escherichia coli as expression system and identification of low molecular weight inhibitors. Glycobiology 2007, 17 (4), 444-453.

(23) Hofinger, E. S. A.; Bernhardt, G.; Buschauer, A., Kinetics of Hyal-1 and PH-20 hyaluronidases: comparison of minimal substrates and analysis of the transglycosylation reaction. Glycobiology 2007, 17 (9), 963-971.

(24) Tung, J. S.; Mark, G. E.; Hollis, G. F., A microplate assay for hyaluronidase and hyaluronidase inhibitors. Anal. Biochem. 1994, 223 (1), 149-152.