The protozoan parasite Trypanosoma cruzi is the causative agent of human American trypanosomiasis, also known as Chagas disease, which in its chronic form can lead to severe debilitation and ultimately death. [1] According to World Health Organization (WHO) estimates, 16 to 18 million people were infected in Latin America in 2005. Only two approved drugs (nifurtimox and benznidazole) are currently used for the treatment of the infection, but both display low efficacy and are associated with severe undesired side effects. [2] The whole genome sequence of Trypanosoma cruzi, completed in 2005, contains genes predicted to encode almost 23 000 proteins. [3] The identification of essential proteins specific to the parasite will hopefully provide attractive new drug targets. Among such targets, the Trypanosoma cruzi trans-sialidase (TcTS) has been extensively studied. TcTS is a glycosylphosphatidylinositol-anchored surface protein that is differentially expressed during the infective developmental stage of the parasite. [4] It belongs to the glycoside hydrolase family 33 (http://afmb.cnrs-mrs.fr/ CAZY) and catalyzes the a-(2,3) transfer of sialic acid residues from host glycoconjugates to the terminal galactosyl units of mucin-like glycoproteins on the surface of the parasite. [5] Extensive sialylation of the T. cruzi surface is pivotal for the establishment of a chronic infection. The crystal structure of TcTS was recently solved, and a two-step, double-displacement mechanism involving a covalent sialylenzyme intermediate was demonstrated (Scheme 1). [6] Tyr342 was identified as the catalytic nucleophile by the use of 3fluorosialyl fluoride 1 as a substrate analogue, which forms a trapped intermediate species (Scheme 1). [7] The fluorine atom at C3 inductively destabilizes the positively charged oxocarbenium ion like transition states, thereby slowing both the formation and the hydrolysis of the covalent intermediate. However, the presence of a good leaving group at C2 allows glycosylation to proceed rapidly and results in the trapping of the sialyl-enzyme intermediate. Compounds of this class therefore have the potential to act as anti-trypanosomal agents if they can be made selective for TcTS over human sialidases and if they form long-lived intermediates.
3-Fluorosialyl fluoride 1 was shown previously to inactivate wild-type TcTS in a time-dependent manner according to the kinetic model illustrated in Scheme 2. [8] However, after removal of excess inactivator, the enzyme rapidly recovered full activity by transglycosylation when incubated with the natural acceptor lactose. Such rapid transglycosylation would render these compounds useless as anti-trypanosomal agents.
Inspection of the three-dimensional structures of TcTS and the only human sialidase as to yet be characterized crystallographically, Neu2, [9] revealed that the TcTS site is more spacious and hydrophobic in the region around C9 of Scheme 1. Mechanism of action of T. cruzi trans-sialidase.