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Fig. 3. Inhibition constants of multi-Leu analogues against furin and PACE4.The proteolytic stability of peptides in absence or presence of prostate carcinoma cells(6000 DU145 cells) was determined by RP-HPLC analysis (Figure 4).Fig. 4. Proteolytic stability of selected multi-Leu analogues.ConclusionOur results showed that position P6 (Leu 3 ) of inhibitors based on Ac-LLLLRVKR-NH 2sequence is crucial for their activity. Modification of this position by D-Leu, Nle resulted insuppression of inhibitory potency. On the other hand, substitution of position P8 orintroduction of triazole ring between P8 and P7 (Leu 1 -Leu 2 ) did not affect peptide activity.On the other hand, replacement of the Leu 1 residue by D-Leu or Nle, Nle 4 substitution orintroduction of triazole ring increase proteolytic stability of resulting peptides (in absenceof prostate carcinoma cells), as compared to control multi-Leu peptide. In regard to cellpenetration properties of two derivatives of multi-Leu, the FACS analysis showed thatreplacement of -Ala by PEG8 fragment greatly decrease penetration properties ofresulting peptide (FITC-PEG8-ML). It is worth it to emphasize, that results obtained fromMTT analysis showed that the antiproliferative effect of PEG8-ML on DU145 issignificantly lower than ML peptide suggesting a role of intracellular PACE4 in tumorproliferation.AcknowledgmentsThis work is supported by research grants to RD from the Canadian Institutes of Health Research(CIHR) and the Ministère du Développement Économique, de l'Innovation et de l'Exportation(MDEIE) du Québec.References1. Fugère, M., Day, R. Trends Pharmacol. Sci. 26, 294-301 (2005).2. Day, R., Fugère, M., Neugebauer, W.A. International patent application no. PCT/CA2009/000935;Jan 14, 2010.3. Fugère, M., Limperis, P.C., Beaulieu-Audy, V., et al. J. Biol. Chem. 277, 7648-7656 (2002).467