This report provides evidence for the in vitro schistosomicidal activity of cardamonin and demonstrated, for the first time, that this chalcone is highly effective in inhibiting S. mansoni ATP diphosphohydrolase, opening the route to further studies of chalcones as prototypes for new S. mansoni ATP diphosphohydrolase inhibitors.

BackgroundChagas disease kills 2.5 thousand people per year of 15 million persons infected in Latin America. The disease is caused by the protozoan, Trypanosome cruzi, and vectored by triatomine insects, including Panstrongylus megistus, an important vector in Brazil. Medicines treating Chagas disease have unpleasant side effects and may be ineffective, therefore, alternative control techniques are required. Knowledge of the T. cruzi interactions with the triatomine host needs extending and new targets/strategies for control identified. Serine and cysteine peptidases play vital roles in protozoan life cycles including invasion and entry of T. cruzi into host cells. Peptidase inhibitors are, therefore, promising targets for disease control.MethodsSDS PAGE and chromatograpy detected and isolated a P. megistus serpin which was peptide sequenced by mass spectrometry. A full amino acid sequence was obtained from the cDNA and compared with other insect serpins. Reverse transcription PCR analysis measured serpin transcripts of P. megistus tissues with and without T. cruzi infection. Serpin homology modeling used the Swiss Model and Swiss-PDB viewer programmes.ResultsThe P. megistus serpin (PMSRP1) has a ca. 40 kDa molecular mass with 404 amino acid residues. A reactive site loop contains a highly conserved hinge region but, based on sequence alignment, the normal cleavage site for serine proteases at P1-P1′ was translocated to the putative position P4′-P5′. A small peptide obtained corresponded to the C-terminal 40 amino acid region. The secondary structure of PMSRP1 indicated nine α-helices and three β-sheets, similar to other serpins. PMSRP1 transcripts occurred in all tested tissues but were highest in the fat body and hemocytes. Levels of mRNA encoding PMSRP1 were significantly modulated in the hemocytes and stomach by T. cruzi infection indicating a role for PMSRP1 in the parasite interactions with P. megistus.ConclusionsFor the first time, a constitutively expressed serpin has been characterized from the hemolymph of a triatomine. This opens up new research avenues into the roles of serine peptidases in the T. cruzi/P. megistus association. Initial experiments indicate a role for PMSRP1 in T. cruzi interactions with P. megistus and will lead to further functional studies of this molecule.

Proteases have received enormous interest from the research and medical communities because of their significant roles in several human diseases. Some examples include the involvement of thrombin in thrombosis, HIV-1 protease in Acquired Immune Deficiency Syndrome, cruzain in Trypanosoma cruzi infection, and membrane-type 1 matrix metalloproteinase in tumor invasion and metastasis. Many efforts has been undertaken to design effective inhibitors featuring potent inhibitory activity, specificity, and metabolic stability to those proteases involved in such pathologies. Protease inhibitors usually target the active site, but some of them act by other inhibitory mechanisms. The understanding of the structure-function relationships of proteases and inhibitors has an impact on new inhibitor drugs designing. In this paper, the structures of four proteases (thrombin, HIV-protease, cruzain, and a matrix metalloproteinase) are briefly reviewed, and used as examples of the importance of proteases for the development of new treatment strategies, leading to a longer and healthier life.

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