Structure-based design of hyaluronidase inhibitors (2004)

Hyaluronan and hyaluronidases have been used in several medical fields for many years and some hyaluronidases play a role in, e.g., meningitis, septicaemia, arthroses and cancer. To further investigate the function of hyaluronan and hyaluronidases in (patho)physiological processes, selective and potent hyaluronidase inhibitors are required. Originated by the lack of such compounds, the main goal of this thesis was the prediction of lead-like structures by de novo ligand design. For de novo ligand design with the programme LUDI, a homology model of S. agalactiae strain 4755 hyaluronidase (hylB4755) was constructed starting from two bacterial hyaluronidases. Screening of the LeadQuest®, Accelrys and the adapted ChemACX (ChemACXF) databases resulted in 1275 hits. 13 out of 19 selected compounds were active on hylB4755 in the milli- and submillimolar range. 1,3-Diacetylbenzimidazole-2-thione was identified to be one of the most potent inhibitors of bacterial hyaluronidases (IC50 values of 5 µM and 160 µM at physiological pH and optimum pH, respectively). To validate the usage of the aforementioned databases in virtual screening, the distribution of six (physico)chemical properties (molecular weight, log P, numbers of H-bond donors and acceptors, numbers of rotatable bonds and of rings) within these databases were analysed with respect to drug-likeness. The analysis revealed that the LeadQuest® databases Vol. 1&amp;2 and Vol. 1-3 are suitable compound selections for virtual screening with drug-like molecules. Furthermore, the raw pre-filtering of the ChemACX database by elimination of reactive compounds and of entities outside a certain molecular weight range resulted in a database with significantly different property distributions, though covering the essential pharmacological space. For de novo design of inhibitors of bovine testicular hyaluronidase (BTH) a homology model of the enzyme based on crystal structures of bee venom hyaluronidase was constructed using MODELLER. Filtering of the Lead-Quest® and the ChemACXF databases resulted in ca. 5500 hits. 5 compounds were selected for testing hyaluronidase inhibition; none of the compounds inhibited BTH. Additionally, a ligand-based approach was performed. The superposition of the active sites of the BTH model, the crystal structures of bee venom hyaluronidase and the bacterial chitinases A and B in complex with inhibitors revealed a very good overlap of the amino acids involved in catalysis and of the co-crystallised ligands. By considering essential substructures mimicking the proposed intermediate of hyaluronic acid hydrolysis and by introducing suitable substituents suggested to interact with amino acids of the active site of the bovine enzyme, two compounds were proposed as potential inhibitors of BTH. Due to the weak inhibition of S. pneumoniae hyaluronidase by vitamin C, the more hydrophobic derivative L-ascorbic acid-6-hexadecanoate was investigated and proved to be a potent inhibitor of hylB4755, S. pneumoniae hyaluronidase and BTH (IC50 values of 4 µM, 100 µM and 56 µM, respectively). The binding mode of L-ascorbic acid-6-hexadecanoate at S. pneumoniae hyaluronidase was determined by X-ray analysis, supporting the hypothesis that additional hydrophobic interactions in the active site contribute to the higher affinity. The potential binding mode of L-ascorbic acid-6-hexadecanoate at BTH was predicted by flexible docking with FlexX suggesting two alternative binding modes. That binding mode seems to be clearly favoured where the long alkyl chain of L-ascorbic acid-6-hexadecanoate favourably interacts with an extended, strongly hydrophobic channel. The analysis of two hyaluronate lyase-inhibitor complexes revealed that the the indole moiety of the inhibitor sulfamic acid 1-decyl-2-(4-sulfamoyloxy-phenyl)-1H-indol-6-yl ester and the vitamin C portion of L-ascorbic acid-6-hexadecanoate bind in exactly the same region of the catalytic site. Additionally, the long aliphatic substituents of both compounds dunk in the same surface crevice. Using the programmes LUDI and GRID, regions where H-bond donor, H-bond acceptor and hydrophobic moieties of inhibitors may interact most favourably were identified and transferred into a 3D pharmacophore model. The analysis of known SAR of 2-phenylindole derivatives with respect to the observed binding mode of sulfamic acid 1-decyl-2-(4-sulfamoyloxy-phenyl)-1H-indol-6-yl ester and to the 3D pharmacophore model led to suggestions about the binding mode of benzoxazole-2-thione derivatives. Based on the superposition of the crystal structure of this bacterial lyase in complex with the co-crystallised indole derivative and a substrate-based hexasaccharide, novel benzoxazole-2-thiones with 3-substituted N-propanoyl groups were predicted as putative hyaluronate lyase inhibitors. This design strategy was confirmed by the activity of the 3-phenylpropanoyl derivative which potently inhibits hylB4755 with an IC50 value of 15 µM.