A selective and potent inhibitor of neuraminidases, a hydrolase that is responsible for processing sialylated glycoconjugates, is a promising drug candidate for various infective diseases. The current study demonstrates that the use of an aglycone-focused library of 2-difluoromethylphenyl -sialosides is an effective technique to find potent and selective mechanism-based labeling reagents for neuraminidases. The focused library was constructed from a 4-azide-2-difluoromethylphenyl sialoside (2) and an alkyne-terminated compound library by a click reaction. The focused library showed different inhibition patterns for two neuraminidases, Vibrio cholerae neuraminidase (VCNA) and human neuraminidase 2 (hNeu2), and the most potent inhibitors for each neuraminidase were selected. A kinetic analysis of the selected inhibitors demonstrated that the modification of the aglycone moiety improved the K I value with little change in the t 1/2 value of the enzyme activity relative to the basic skeleton (2).

A potent inhibitor for Vibrio cholerae neuraminidase (VCNA) was developed by using a novel two-step strategy, a target amino acid validation using mechanism-based labeling information, and a potent inhibitor search using a focused library. The labeling information suggested the hidden dynamics of a loop structure of VCNA, which can be a potential target of the novel inhibitor. A focused library composed of 187 compounds was prepared from a 9-azide derivative of 2,3-dehydro-N-acetylneuraminic acid (DANA) to interrupt the function of the loop of the labeled residues. Inhibitor 3 c showed potent inhibition properties and was the strongest inhibitor with FANA, a N-trifluoroacetyl derivative of DANA. Validation studies of the inhibitor with a detergent and a Lineweaver-Burk plot suggested that the 9-substitution group would interact hydrophobically with the target loop moiety, adding a noncompetitive inhibition property to the DANA skeleton. This information enabled us to design compound 4 having the combined structure of 3 c and FANA. Compound 4 showed the most potent inhibition (K(i) =73 nM, mixed inhibition) of VCNA with high selectivity among the tested viral, bacterial, and mammal neuraminidases.

A macrocyclic mechanism-based inhibitor for neuraminidases (NAs) bearing a 2-difluoromethylphenyl aglycone and a linker between the aglycone and C-9 positions of sialic acid was synthesized and evaluated. The macrocyclic structure was designed to keep the aglycone moiety in the active site of the neuraminidase after cleavage of the glycoside bond. When Vibrio chorelae neuraminidase (VCNA) was treated with a similar acyclic derivative in the presence of detergent, the irreversible inhibition property was disabled. In contrast, this macrocyclic compound acted as an irreversible inhibitor for VCNA in the presence of detergent. Inhibition assay for various NAs using this macrocyclic compound revealed that the irreversible inhibition property depends on the k(cat) of the neuraminidase treated. NAs having small k(cat) values, such as Influenza viruses, Clostridium, Trypanosoma cruzi, and Human, were also inhibited irreversibly. However, Salmonella typhimurium NA, which has an extremely high k(cat) , was not affected irreversibly by the inhibitor. Interestingly, in contrast to common k(cat) inhibitors, the irreversibility of inhibition by this macrocyclic compound is inversely proportional to the k(cat) of the target neuraminidase.