*Article* **Novel Cysteine Protease Inhibitor Derived from the** *Haementeria vizottoi* **Leech: Recombinant Expression, Purification, and Characterization**

**Débora do Carmo Linhares 1, Fernanda Faria 2,\*, Roberto Tadashi Kodama 3, Adriane Michele Xavier Prado Amorim 2, Fernanda Calheta Vieira Portaro 3, Dilza Trevisan-Silva 4, Karla Fernanda Ferraz <sup>2</sup> and Ana Marisa Chudzinski-Tavassi 2,4,\***


**Abstract:** Cathepsin L (CatL) is a lysosomal cysteine protease primarily involved in the terminal degradation of intracellular and endocytosed proteins. More specifically, in humans, CatL has been implicated in cancer progression and metastasis, as well as coronary artery diseases and others. Given this, the search for potent CatL inhibitors is of great importance. In the search for new molecules to perform proteolytic activity regulation, salivary secretions from hematophagous animals have been an important source, as they present protease inhibitors that evolved to disable host proteases. Based on the transcriptome of the *Haementeria vizzotoi* leech, the cDNA of Cystatin-Hv was selected for this study. Cystatin-Hv was expressed in *Pichia pastoris* and purified by two chromatographic steps. The kinetic results using human CatL indicated that Cystatin-Hv, in its recombinant form, is a potent inhibitor of this protease, with a Ki value of 7.9 nM. Consequently, the present study describes, for the first time, the attainment and the biochemical characterization of a recombinant cystatin from leeches as a potent CatL inhibitor. While searching out for new molecules of therapeutic interest, this leech cystatin opens up possibilities for the future use of this molecule in studies involving cellular and in vivo models.

**Keywords:** leech; *Haementeria vizottoi*; cysteine proteases inhibitor; recombinant cystatin; cathepsin L

**Key Contribution:** Cystatin-Hv is the first cysteine protease inhibitor described from leeches and obtained in its recombinant form, allowing its biochemical characterization. It was discovered in the transcriptome of the salivary complexes from *Haementeria vizottoi* leeches, obtained in recombinant form and characterized biochemically. It was able to strongly inhibit the human cathepsin L with a Ki of 7.9 nM.
