Luiz Juliano
Luiz Juliano - Biophysics Department – Escola Paulista de Medicina (ljuliano@terra.com.br)
Interface studies of cancer with the hydrolytic activities and inhibition of Kallikrein Related Peptidases (KLKs).
Human kallikrein-related peptidases (KLKs) form a cluster of 15 serine peptidase genes of the chymotrypsin-like S1A family clan PA(S) with a high degree of identity and are localized at the human chromosome locus 19q13.4. KLK4, KLK5, KLK6, and KLK7 often have elevated expression levels in ovarian cancer, particularly in the epithelial form. Similarly, the prostatic KLK2, KLK3 and KLK4 are suggested to play important roles in prostate cancer. In particular, prostate specific antigen (or KLK3) is the current clinically used biomarker for this disease, although its functional role in the underlying cancer biology is not yet fully understood.
We have identified synthetic substrates and inhibitors for the proteases particularly related to KLKs (Del Nery E, et al., 1995, Melo RL et al., 2001; Andrade et al., 2010; 2011). Detailed analysis of KLK6 substrate specificity using panels of internally quenched fluorescent peptides showed the preference of the enzyme for amino acids at both sites of arginine for which KLK6 is highly specific and natural substrates were identified (Angelo et al., 2006). Similar work is in progress with KLK7 and KLK5, and particularly relevant was the finding of the peptide sequence N-L-Y-↓-R-V-E (↓cleavage site) as the major susceptible peptide to hydrolysis by KLK7 which is present in semaphorin -6C and that was also found as one of the most significantly affected proteins by KLK4-7 over expression in the human ovarian cancer cell line OV-MZ-6 (Shahinian et al., 2014). We observed (work in progress) for recombinant KLK5 restricted activity as an arginyl-hydrolase with preferences for serine at P1’ and P2’ positions of the substrates. Interestingly, this was also one of the three most prevalent amino acids found in the KLK4-7 degradome (Shahinian et al., 2014). Several non-natural basic amino acids were synthesized and incorporated in short peptides and resulted in efficient competitive inhibitors for KLK1 (Melo et al., 2001). The same compounds and others that are being prepared will be assayed as inhibitors of KLK4, 5 6 and 7 and evaluated for their activities on ovarian and prostate cancer cells.
References
- Andrade D, et al, 2010. Arch Biochem Biophys. 498, 74-82.
- Andrade D, et al, 2011, Biochimie 93, 1701-1709.
- Angelo PF, et al., J Biol Chem. 281, 3116-3126.
- Del Nery , E. et al., 1995. Biochem J. 312, 233-238.
- Melo. RL, 2001, Biochemistry, 40, 5226-5232.
- Shahinian, H, et al., 2014. Molecular Oncology 8, 68-82.
