PACE4 is a prostate and ovarian cancer validated target, which belongs to the proprotein convertase’s (PCs) family of serine proteases. We have developed a potent and selective inhibitor, Multi-Leu, with the following structure: Ac-LLLLRVKR-NH2. The PCs active site is conserved within the family, which make the designing of the selective inhibitors difficult. In order to benefit from a difference in S3 pocket of PACE4 and furin (the other member of PC family), we modified the P3 Val of our Multi-leu (ML) inhibitor to target PACE4 Asp160 vs. furin Glu256. The other interaction in S3 is β-sheet like hydrogen bonding of P3 Val with Gly158 of PACE4. This is a general attribute of proteases substrates to form a β-sheet with the active site residues. The β-branched amino acids like Val and Ile are known to enhance this interaction in the β-sheets. We designed β-branched basic amino acids to consider both mentioned interactions in S3. We examined different basic amino acids in P3 to optimize the side chain length. We found Apg as the most potent in the series so we synthesized our β-branched analogues based on it. With iso-Abg in P3, we obtained more selective compound than ML (30fold vs. 20fold) while increasing the potency 7 times. We analyzed H bonding between Ala or Val backbone and Gly158 PACE4 in S3, utilizing MD simulations. We found that in the case of Val, the backbone H-bond is shorter and the resulting conformation of the inhibitor is closer to the conformation of antiparallel beta-sheets which results in stronger binding. In summary, we described the introduction of a basic β-branched residue instead of Val in P3 of a PACE4 peptide inhibitor, ML, which results in better selectivity and increased potency. In addition, we showed that β-branching of P3 is important for formation of β-sheet conformation of peptide inhibitor in the active site.

β-Sheet propensity of competitive peptide inhibitor’s residue is crucial in binding to proteases: PACE4 inhibitors as a case study

253rd National Meeting (2017)

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