Multivalent Thiosialosides and Their Synergistic Interaction with Pathogenic Sialidases

Abstract

Sialidases (SAs) hydrolyze sialyl residues from glycoconjugates of the eukaryotic cell surface and are virulence factors expressed by pathogenic bacteria, viruses, and parasites. The catalytic domains of SAs are often flanked with carbohydrate‐binding module(s) previously shown to bind sialosides and to enhance enzymatic catalytic efficiency. Herein, non‐hydrolyzable multivalent thiosialosides were designed as probes and inhibitors of V. cholerae, T. cruzi, and S. pneumoniae (NanA) sialidases. NanA was truncated from the catalytic and lectinic domains (NanA‐L and NanA‐C) to probe their respective roles upon interacting with sialylated surfaces and the synthetically designed di‐ and polymeric thiosialosides. The NanA‐L domain was shown to fully drive NanA binding, improving affinity for the thiosialylated surface and compounds by more than two orders of magnitude. Importantly, each thiosialoside grafted onto the polymer was also shown to reduce NanA and NanA‐C catalytic activity with efficiency that was 3000‐fold higher than that of the monovalent thiosialoside reference. These results extend the concept of multivalency for designing potent bacterial and parasitic sialidase inhibitors.

Polyvalent Transition‐State Analogues of Sialyl Substrates Strongly Inhibit Bacterial Sialidases**

“…The inhibitory activity of compounds 1-10 was studied with the substrate 2'-( Table 1 or previously reported). [20] These results are in accordance with previously observed micromolar IC 50 of DANA towards NanA, [45] and confirm the superiority of this transitionstate inhibitor compared with sialoside substrate analogues. Compared with 2, divalent DANA 4 and 5 showed only a moderately improved inhibitory capacity for NanA and NanA-CAT but a strong multivalent effect was observed on BtSA.…”

“…We previously estimated from polymer theory that the selected spacer arms for 4 and 5, bearing 10 and 30 PEG units, attain average distances of 22 and 33 Å, respectively. [20] As previously observed with lectins, this value should match the distance between two protein binding sites for an optimal fit. [28] The α-cyclodextrin (CD) scaffold of compound 7 was selected for a planar orientation of six DANA ligands.…”

“…Previously described thiosialosides 1 and 3 [20] and new compounds 6 and 8 were first designed to compare the potential of the multivalent DANA with these substrate analogues. Three types of DANA clusters were selected to provide potential multivalent inhibitory effects on bacterial SAs ( Figure 1B).…”

“…These sialoclusters displayed submicromolar binding inhibition capacity and inhibitory activity for the pathogenic SA from Vibrio cholerae (VcSA) and Streptococus pneumoniae (NanA). [20] Each clustered thiosialoside showed enhanced inhibitory capacity up to three orders of magnitude compared with relevant monovalent references. Surprisingly, these synergistic inhibitory effects were also observed for NanA catalytic domains alone (CAT), truncated from CBM.…”

Monitoring glycosidase activity for clustered sugar substrates, a study on β-glucuronidase

“…This is in accordance with previous calculations from polymer theory performed with a different set of compounds bearing PEG linkers of similar lengths. 17,34 Compound 5 should therefore cover the distance of around 50Å between two adjacent GUS binding sites, as determined from the crystal structure of E. coli GUS bound to an urea inhibitor (PDB code ¼ 3LPG). 35 Compound 6 was designed from a cubic octameric silsequioxane (COSS) scaffold.…”