Novel inhibition scaffolds targeting human cysteine cathepsins

J. Benýšek1, Z. Kovářová1, M. Mishra1, J. Brynda1,2, M. Buša1, J. Srp1, P. Řezáčová1,2, M. Horn1 and M. Mareš1*

1 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 16610 Prague, Czech Republic

2 Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, 16610 Prague, Czech Republic


This work is focused on novel structural motives for inhibition of two model human cysteine proteases, namely cathepsin K and cathepsin V. Cathepsin V is overexpressed in breast and colorectal adenomas and adenocarcinomas and is a potential oncology target while cathepsin K is expressed in osteoclasts and has been evaluated as a target for osteoporosis. We demonstrated that cathepsin V is effectively inhibited by PCPI3 (potato cysteine protease inhibitor), a 20 kDa protein isolated from potato tubers which belongs to the plant Kunitz-type inhibitor family. Also, we identified synthetic peptidomimetic compounds containing a nitrile reactive warhead as potent inhibitors of cathepsin K. Both cathepsins and their active-site mutants were produced in the form of recombinant proteins in the P. pastoris expression system and purified. Inhibitor-cathepsin complexes were prepared and subjected to high-throughput crystallization screening. A preliminary crystallographic model of the complex of PCPI3-cathepsin V is presented.