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J. Biol. Chem., Vol. 281, Issue 11, 7437-7444, March 17, 2006

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Selective Inhibition of Fibroblast Activation Protein Protease Based on Dipeptide Substrate Specificity^*

Conrad Yap Edosada, Clifford Quan, Christian Wiesmann^¶, Thuy Tran, Dan Sutherlin, Mark Reynolds, J. Michael Elliott^||, Helga Raab^||, Wayne Fairbrother^¶, and Beni B. Wolf¹

From the Departments of Molecular Oncology, Medicinal Chemistry, ^¶Protein Engineering, and ^||Protein Chemistry, Genentech, Inc., South San Francisco, California 94080

Fibroblast activation protein (FAP) is a transmembrane serine peptidase that belongs to the prolyl peptidase family. FAP has been implicated in cancer; however, its specific role remains elusive because inhibitors that distinguish FAP from other prolyl peptidases like dipeptidyl peptidase-4 (DPP-4) have not been developed. To identify peptide motifs for FAP-selective inhibitor design, we used P₂-Pro₁ and acetyl (Ac)-P₂-Pro₁ dipeptide substrate libraries, where P₂ was varied and substrate hydrolysis occurs between Pro₁ and a fluorescent leaving group. With the P₂-Pro₁ library, FAP preferred Ile, Pro, or Arg at the P₂ residue; however, DPP-4 showed broad reactivity against this library, precluding selectivity. By contrast, with the Ac-P₂-Pro₁ library, FAP cleaved only Ac-Gly-Pro, whereas DPP-4 showed little reactivity with all substrates. FAP also cleaved formyl-, benzyloxycarbonyl-, biotinyl-, and peptidyl-Gly-Pro substrates, which DPP-4 cleaved poorly, suggesting an N-acyl-Gly-Pro motif for inhibitor design. Therefore, we synthesized and tested the compound Ac-Gly-prolineboronic acid, which inhibited FAP with a K_i of 23 ± 3 nM. This was 9- to 5400-fold lower than the K_i values for other prolyl peptidases, including DPP-4, DPP-7, DPP-8, DPP-9, prolyl oligopeptidase, and acylpeptide hydrolase. These results identify Ac-Gly-BoroPro as a FAP-selective inhibitor and suggest that N-acyl-Gly-Pro-based inhibitors will allow testing of FAP as a therapeutic target.

Received for publication, October 12, 2005 , and in revised form, December 15, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1.

¹ To whom correspondence should be addressed: Genentech, Inc., 1 DNA Way-MS42, South San Francisco, CA 94080. Tel.: 650-467-1954; Fax: 650-225-6327; E-mail: bbwolf{at}gene.com.

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