Carbohydrates and Glycoproteins: Cellular Recognition and Drug Design - New Developments in Medicinal Chemistry - page 144

residues are present as β-galactopyranose linked as linear and branched side chains to the 4 and 6-positions of the
GlcNAc, but in some strains a β-galactofuranose residue can also be linked to the 4-position of the GlcNAc.
Besides being involved in the attachment and subsequent penetration of the parasite into host cells, the sialylated
mucin molecules also serves to mask the presence of the parasite from the host immune response, conferring
resistance to complement and protect against antibody mediated lyses. Therefore, the fundamental role of TcTS
in the cellular invasion process parallel to the fact that it is not present in host cells validate this enzyme as an
important target for design of antichagasic drugs [38, 39].

TcTS: Mechanism of Action and Design of Inhibitors

Recent determined crystal structure of TcTS [33] showed that it consists of two domains: the N -terminal catalytic
domains (residues 1-371) connected through a long α helix (residues 372-394) to a C -terminal lectin-like domain
(residues 395-632), which contains the “shed acute antigen phase” (SAPA), with 12 repeated units of amino acids in
tandem, not necessary for the enzymatic activity [34] (Fig. 11A ). The active site of TcTS has several common
features with microbial sialidases, for example, an arginine triad (Arg35, Arg245 and Arg314), which interacts with
the carboxylate group of sialic acid, besides other important residues essential for stabilization of the transition state
(Tyr342, Glu230) and catalysis (Asp59) [35]. A second site, the sialic acid acceptor site, accommodates the β-
galactose moiety and contains the amino acids Tyr119 and Trp312 that plays a key role in the trans -sialidase activity
of TcTS (Fig. 11B ) [40].

(B)

Arg245

Catalytic domain

Lectin domain

Glu230

Tyr342

Asp96

DANA

DANA

Tyr119

Arg314

(A)

Arg35

Asp59

Figure 11: (A) Crystallographic complex of TcTS with DANA (PDB code 1MS8). (B) Main interactions of DANA with the
active site of TcTS.

TcTS catalyzes the transglycosylation reaction via a bisubstrate double displacement (ping-pong) mechanism
(Scheme 4 ), as the binding of lactose to the enzyme active site occurs only after entrance of the sialic acid. After
binding of sialic acid donor substrate in the active site, the hydroxyl group of Tyr342, assisted by Glu230 acting as
general base, reacts as a nucleophile, with displacement of the protonated group and formation of a covalently bound
TcTS-sialoside intermediate. In the next step, the 3-hydroxyl group of the acceptor galactoside, in the presence of
Asp59 as a base catalyst, performs a nucleophilic substitution, resulting in the retention of configuration of the sialic
acid moiety [41].

Compounds tested as potential TcTS inhibitors can be grouped in two categories, depending on the active site
regions they target, namely sialic acid mimetics and sialic acid donor/acceptor substrate mimetics [41]. In the first
group, the strategy to mimic sialic acid, as utilized in the development of the influenza neuraminidase inhibitors, has
been explored in search of potential TcTS inhibitors. Nevertheless, DANA ( 8 ) (Fig. 12 ), which is a potent inhibitor
of influenza neuraminidase, showed a weak inhibition of TcTS ( K i 1.54 mM) despite its favorable interactions in the
active site. It shows that key amino acid differences, responsible for the different activity of TcTS, are also
responsible for the lower inhibition observed for DANA [35, 42].