| Summary: | 博士 === 國立臺灣大學 === 化學研究所 === 104 === In this thesis, two validated drug targets are focused for structural studies, i.e., human β-galactoside-binding lectins (galectins) and bacterial β-glucuronidases (GUSs), which have been known as key players in tumor progression and drug metabolism, respectively. Because the primary sequence and tertiary structure were remarkably conserved among the galectin and GUS families, developing potent and selective inhibitors for specific members has become a longstanding challenging. We particularly applied multi-disciplinary approaches for structure-based drug development, such as synthetic chemistry, X-ray crystallography, isothermal titration calorimetry, and NMR spectroscopy.
To date, TD139 (3,3ʹ-deoxy-3,3ʹ-bis-(4-[m-fluorophenyl]-1H-1,2,3-triazol- 1-yl)-thio-digalactoside) representing the most potent inhibitor for galectin-3, one of the most prominent galectin family members involved in several pathological processes, has been approved for the clinical trial of idiopathic pulmonary fibrosis. However, the full structural information concerning subsites A–E of galectin and the interactions with TD139 are not currently available. In the first part of this thesis, we studied the binding contributions of these subsites in galectins-1, -3, and -7 with several sialylated/sulfated disaccharides and three thio-digalactoside (TDG) derivatives, including TDG, TD139, and TAZTDG (3-deoxy-3-(4-[m-fluorophenyl]-1H-1,2,3-triazol-1-yl)-thio- digalactoside). Surprisingly, we found that the fluorophenyl group of TAZTDG preferentially bound to subsite B in galectin-3, whereas the same group favored binding at subsite E in galectins-1 and -7. The characterized dual binding modes demonstrate how binding potency, reflected in decreased Kd values of the TDG-derived inhibitors from μM to nM levels, is improved. The resulting information offers insights into the development of selective inhibitors for individual galectins.
In the second part, we focused on microbial GUSs, which interfere with xenobiotic detoxification and thus impact human health. Currently several selective inhibitors (such as ASN 03273363) for bacterial GUSs are known to rely on the unique loop that is located near the enzyme active site and is found only in microbial enzymes. However, there are two crucial problems associated with ASN 03273363. One is that the bacterial loop exhibits significant sequence variation, whilst the other is the presence of more than half of bacterial GUSs lacking this loop (NL-GUSs). To solve these problems, we developed a series of uronic isofagomine (IFG) derivatives as transition-state analogues, which directly interact with the conserved catalytic residues of GUSs. The results indicated that substituents introduced at the C1-position play an important role in determining the selectivity between bacterial and mammalian GUSs. Moreover, a combinatorial method was applied to rapidly generate and screen a variety of uronic IFG derivatives. Among them, an irreversible inhibitor was identified to target the non-catalytic cysteine that is conserved in several NL-GUSs, but is absent in both the human and loop-containing bacterial GUS. The selectivity is up to 2–3 orders of magnitude greater for bacterial NL-GUSs (IC50 in the range of nM) than for HsGUS (IC50 of μM).
|
|---|
