Polyarginines are potent furin inhibitors

The ubiquitous serine endoprotease furin has been implicated in the activat ion of bacterial toxins and viral glycoproteins as well as in the metastati c progression of certain tumors, Although high molecular mass bioengineered serpin inhibitors have been well characterized, no small nontoxic nanomola r inhibitors have been reported to date. Here we describe the identificatio n of such inhibitors using positional scanning amidated and acetylated synt hetic L- and D hexapeptide combinatorial libraries, The results indicated t hat L-Arg or L-Lys in all positions generated the most potent inhibitors. H owever, further investigation revealed that the peptide terminating groups hindered inhibition. Consequently, a series of non-amidated and acetylated polyarginines was synthesized. The most potent inhibitor identified, nona-L -arginine, had a K-i for furin of 40 nM. The K-i, values for the related co nvertases PACE4 and prohormone con vertase-1 (PC1) were 110 nM and 2.5 muM respectively. Although nona-L-arginine was cleaved by furin, the major prod ucts after a 6-h incubation at 37 degreesC were hexa-and hepta-L-arginines, both of which retained the great majority of their potency and specificity against furin, Hexa-D-arginine was as potent and specific a furin inhibito r as hexa-L-arginine (K-i values of hexa-D-arginine: 106 nM, 580 nM, and 13 .2 muM for furin, PACE4, and PC1, respectively). PC2 was not inhibited by a ny polyarginine tested; indeed, PC2 showed an increase in activity of up to 140% of the control in the presence of L-polyarginines, Data are also pres ented that show extended subsite recognition by furin and PC2, Whereas N-te rminal acetylation was found to reduce the inhibitory potency of the L-hexa peptide LLRVKR against furin 8-fold, C-terminal amidation reduced the poten cy <2-fold. Conversely, N-terminal acetylation increased the potency agains t PC2 nearly 8-fold, whereas C-terminal amidation of the same peptide incre ased the potency by a factor of 1,6, Our data indicate that non-acetylated, poly-D-arginine-derived molecules may represent excellent lead compounds f or the development of therapeutically useful furin inhibitors.