Inhibitors of Mammalian and Bacterial Hyaluronidases

• Design, synthesis and structure-activity relationships of inhibitors
• Recombinant expression, purification and characterization of human hyaluronidases

Hyaluronidases are enzymes degrading hyaluronic acid (HA, hyaluronan), an important component of the extracellular matrix. Hyaluronan consists of 1->4-linked b-D-glucuronic acid-(1->3)-b-N-acetyl-D-glucosamine disaccharide units. According to their catalytic mechanism, the hyaluronidases are divided into three major families: (1) endo-b-N-acetylglucosaminidases, including vertebrate hyaluronidases, catalyse the hydrolysis of the b-1,4 glycosidic bond (EC 3.2.1.35); (2) b-eliminases (lyases) include bacterial hyaluronidases and catabolize HA in a b-elimination of the b-1,4 glycosidic bond (EC 4.2.2.1); (3) endo-b-glucuronidases found in parasites like leeches (EC 3.2.1.36). Based on the gene sequences six human hyaluronidase subtypes were identified: Hyal-1, Hyal-2, Hyal-3, Hyal-4, PH-20 and a pseudogene, Phyal1 (Csoka et al., Matrix Biol. 20, 499-508 (2001)). As the physiological and pathophysiological role of hyaluronidases is far from being understood, potent and selective inhibitors are required as pharmacological tools.

The project is aiming at the (structure-based) design, synthesis and characterisation of hyaluronidases inhibitors. De novo design of inhibitors of bacterial hyaluronan lyases, supported by x-ray structures in complex with inhibitors (collaboration with Dr. Mark Jedrzejas, Oakland, CA), resulted in lead compounds with IC50 values in the low micromolar range. In order to apply this successful approach to mammalian hyaluronidases, very recently, the human enzymes Hyal-1, Hyal-2 and PH20 were recombinantly expressed, purified and characterized with respect to biochemical and catalytic properties.

Related tumour pharmacological project:

• Hyaluronidase as an Additive in Cancer Chemotherapy

Selected publications:

• E.S.A. Hofinger, J. Hoechstetter, M. Oettl, G. Bernhardt, A. Buschauer, Isoenzyme-specific differences in the degradation of hyaluronic acid by mammalian-type hyaluronidases. Glycoconj. J. 25 (2), 101-109 (2008)
• E.S.A. Hofinger, G. Bernhardt, A. Buschauer, Kinetics of Hyal1 and PH-20 hyaluronidases: comparison of minimal substrates and analysis of the transglycosylation reaction. Glycobiology 17 (9), 963-971 (2007)
• E.S.A. Hofinger, M. Spickenreither, J. Oschmann, G. Bernhardt, R. Rudolph, A. Buschauer, Recombinant human hyaluronidase Hyal-1: insect cells versus E. coli as expression system and identification of low molecular weight inhibitors. Glycobiology 17 (4), 444-453 (2007).
• M. Spickenreither, S. Braun, G. Bernhardt, S. Dove, A. Buschauer, Novel 6-O-acylated vitamin C derivatives as hyaluronidase inhibitors with selectivity for bacterial lyases. Bioorg. Med. Chem. Lett. 16 (20), 5313-5316 (2006).
• D. J. Rigden, A. Botzki, M. Nukui, R. B. Mewbourne, E. Lamani, S. Braun, E. von Angerer, G. Bernhardt, S. Dove, A. Buschauer, M. J. Jedrzejas, Design of new benzoxazole-2-thione derived inhibitors of Streptococcus pneumoniae hyaluronan lyase: structure of a complex with a 2-phenylindole. Glycobiology 16 (8), 757-765 (2006).
• S. Salmen, J. Hoechstetter, C. Käsbauer, D. H. Paper, G. Bernhardt, A. Buschauer, Sulphated Oligosaccharides as Inhibitors of Hyaluronidases from Bovine Testis, Bee Venom and Streptococcus agalactiae. Planta Med. 71, 727-732 (2005).
• A. Botzki, S. Salmen, G. Bernhardt, A. Buschauer, S. Dove, Structure-Based Design Of Bacterial Hyaluronan Lyase Inhibitors, QSAR Comb. Sci. 24, 458-469 (2005).
• A. Botzki, D. J. Rigden, S. Braun, M. Nukui, S. Salmen, J. Hoechstetter, G. Bernhardt, S. Dove, A. Buschauer, M.J. Jedrzejas, L-ascorbic acid-6-hexadecanoate, a potent hyaluronidase inhibitor: X-ray structure and molecular modeling of enzyme-inhibitor complexes. J. Biol. Chem. 279, 45990-45997 (2004).
• M. Oettl, J. Hoechstetter, I. Asen, G. Bernhardt, A. Buschauer, Comparative characterization of bovine testicular hyaluronidase and a hyaluronate lyase from Streptococcus agalactiae in pharmaceutical preparations, Eur. J. Pharm. Sci. 18, 267-277 (2003).