Inhibition of Plasmodium falciparum cysteine proteases by the sugarcane cystatin CaneCPI-4

Malaria is a disease caused by Plasmodium parasites that affects hundreds of millions of people. Plasmodium proteases are involved in invasion, erythrocyte egress and degradation of host proteins. Falcipains are well studied cysteine peptidases located in P. falciparum food vacuoles that participate in hemoglobin degradation. Cystatins are natural cysteine protease inhibitors that are implicated in a wide range of regulatory processes. Here, we report that a cystatin from sugarcane, CaneCPI-4, is selectively internalized into P. falciparum infected erythrocytes and is not processed by the parasite proteolytic machinery. Furthermore, we demonstrated the inhibition of P. falciparum cysteine proteases by CaneCPI-4, suggesting that it can exert inhibitory functions inside the parasites. The inhibition of the proteolytic activity of parasite cells is specific to this cystatin, as the addition of an anti-CaneCPI-4 antibody completely abolished the inhibition. We extended the studies to recombinant falcipain-2 and falcipain-3 and demonstrated that CaneCPI-4 strongly inhibits these enzymes, with IC50 values of 12 nM and 42 nM, respectively. We also demonstrated that CaneCPI-4 decreased the hemozoin formation in the parasites, affecting the parasitemia. Taken together, this study identified a natural molecule as a potential antimalarial that specifically targets falcipains and also contributes to a better understanding of macromolecule acquisition by Plasmodium falciparum infected RBCs.Fundacao de Amparo a Pesquisa do Estado de Sao PauloConselho Nacional de Desenvolvimento Cientifico e TecnologicoUniv Fed Sao Paulo, Dept Biofis, Rua Pedro de Toledo 669, BR-04039032 Sao Paulo, BrazilUniv Fed Sao Carlos, Dept Genet & Evolucao, Sao Carlos, SP, BrazilUniv Fed Sao Paulo, Dept Biociencias, Santos, BrazilUniv Fed Sao Paulo, Dept Biofis, Rua Pedro de Toledo 669, BR-04039032 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biociencias, Santos, BrazilFAPESP: 13/12913-0FAPESP: 09/54598-9FAPESP: 09/53840-0FAPESP: 11/14403-4FAPESP: 12/50475-2FAPESP: 15/06861-3FAPESP: 15/19316-3FAPESP: 16/15298-3CNPq: 473.226/2010-3CNPq: 311745/2013-0Web of Scienc