Abstract
There is increasing interest in developing peptides for pharmacological intervention with pathophysiological functions of serine proteases. From phage-displayed peptide libraries, we previously isolated peptidylic inhibitors of urokinase-type plasminogen activator, a potential target for intervention with cancer invasion. The two peptides, upain-1 (CSWRGLENHRMC) and mupain-1 (CPAYSRYLDC), are competitive inhibitors of human and murine urokinase-type plasminogen activator, respectively. Both have an Arg as the P1 residue, inserting into the S1 pocket in the active site of the enzymes, but their specificity depends to a large extent on interactions outside the enzymes' active sites, so-called exosite interactions. Here we describe upain-2 (CSWRGLENHAAC) and the synthesis of a number of upain-2 and mupain-1 variants in which the P1 Arg was substituted with novel non-natural Arg analogs and achieved considerable improvement in the affinity of the peptides to their targets. Using chimeras of human and murine urokinase-type plasminogen activator as well as X-ray crystallography, we delineated the relative contribution of the P1 residue and exosite interactions to the affinity and specificity of the inhibitors for their target enzyme. The effect of inserting a particular non-natural amino acid into the P1 position is determined by the fact that changes in interactions of the P1 residue in the S1 pocket lead to changed exosite interactions and vice versa. These findings are of general interest when the affinities and specificities of serine protease inhibitors to be used for pharmacological intervention are considered and could pave the way for potential drug candidates for the treatment of cancer.
Footnotes
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This work was supported by the National Natural Science Foundation of China [Grants 30811130467, 30973567, 30770429]; the Danish National Research Foundation [Grant 26-331-6]; the Lundbeck Foundation [Grant R19-A2173]; the Danish Cancer Society [Grant DP 07043, DP 08001]; the Danish Research Agency [Grant 272-06-0518]; and the Novo Nordisk Foundation [Grant R114-A11382].
All authors are affiliated with the Danish-Chinese Centre for Proteases and Cancer (http://www.proteasesandcancer.org).
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.111.072280.
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ABBREVIATIONS:
- uPA
- urokinase-type plasminogen activator
- Fmoc
- 9H-fluoren-9-ylmethoxycarbonyl
- Dap
- l-2,3-diaminopropionic acid
- Boc
- di-tert-butyl dicarbonate
- HPLC
- high-performance liquid chromatography
- HBTU
- N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide
- TFA
- trifluoroacetic acid
- HOBt
- N-hydroxybenzotriazole
- HOAt
- 1-hydroxy-7-azabenzotriazole
- DIPEA
- N,N-diisopropylethylamine
- NMP
- N-methyl-2-pyrrolidone
- Alloc
- allyloxycarbonyl
- HRMS
- high-resolution mass spectrometry
- ES
- electrospray
- Cbz
- carboxybenzyl
- Fmoc-OSu
- 9-fluorenylmethoxycarbonyl-N-hydroxysuccinimide
- MS
- mass spectrometry
- wt
- wild-type
- S-2444
- pyro-Glu-Gly-Arg-p-nitroanilide
- PDB
- Protein Data Bank.
- Received March 19, 2011.
- Accepted June 30, 2011.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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