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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 29, 2001 as doi:10.1096/fj.00-0805fje. |
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* Baker Medical Research Institute, Melbourne, Victoria, Australia 8008;
Department of Biochemistry and Molecular Biology, and
Department of Chemistry, Monash University, Clayton, Victoria, Australia 3800; and
Department of Pharmacology, University of Melbourne, Victoria, Australia 3010
2Correspondence: Baker Medical Research Institute, PO Box 6492, St. Kilda Road Central, Melbourne, Victoria, Australia 8008. E-mail: ian.smith{at}baker.edu.au
SPECIFIC AIMS
We hypothesized that substitution of 
-amino acid residues at the
scissile bond of a peptide substrate with ß-amino acids (containing
an extra carbon in the peptide backbone) would confer resistance to
peptidase cleavage without necessarily abolishing enzyme binding;
indeed, such a stabilized analog may act as a specific inhibitor of the
peptidase. To examine this possibility, we synthesized a series of
ß-amino acid-substituted bradykinin (BK) analogs and monitored both
their degradation by the soluble metalloendopeptidases EC 3.4.24.15
(EP24.15) and their ability to inhibit this enzyme.
PRINCIPAL FINDINGS
1. ß-Glycine substitution of residues near the cleavage site of
bradykinin prevents degradation by EP24.15
Incubation of bradykinin with recombinant rat EP24.15 resulted in
>80% degradation within 1 h, with the generation of
BK1–5 and BK6–9, as
determined by HPLC with on-line mass spectral analysis. Replacement of
residues 5, 6, 7, or 8 with a ß-glycine completely prevented
hydrolysis even after extended (24 h) incubation.
2. Bradykinin analogs containing a ß-glycine at positions 5, 6,
or 7 inhibit cleavage of a specific quenched fluorescent substrate by
EP24.15
As would be expected for an enzyme substrate, bradykinin
efficiently inhibited the cleavage of a specific quenched fluorescent
substrate (QFS:
7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-D-Lys(2,4-dinitrophenyl))by
EP24.15. Concentration-inhibition curves for
ß-Gly5-BK, ß-Gly6-BK,
and ß-Gly7-BK were generated and compared to
bradykinin. As shown in Fig. 1
, both ß-Gly5-BK and
ß-Gly6-BK inhibited EP24.15 with
IC50 values of 
28 µM compared with 7 µM
for BK itself. ß-Gly7-BK was less potent, with
an IC50 > 40 µM. Thus, substitution of
residues either side of the scissile bond with a ß-glycine reduced
affinity for the enzyme by only fourfold, yet completely prevented
cleavage.
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3. Bradykinin analogs containing a ß-congener of the cleavage
site residues (ß-Phe5 or ß-Ser6) are also
resistant to degradation and inhibit EP24.15; the inhibition constant
of the most potent analog (ß-C3-D-Phe5-BK) is only
1.5-fold greater than bradykinin itself
As with the ß-glycine analogs, substitution of the cleavage site
residues with their ß-congeners prevented degradation by EP24.15. In
assays of inhibition of QFS cleavage by EP24.15,
ß-C2-L,D-Phe5-BK and
ß-C3-L-Ser6-BK displayed only moderate affinity for the
enzyme (IC50
35 µM). However, as shown in
Fig. 2
, ß-C3-L-Phe5-BK and
ß-C3-D-Phe5-BK were good inhibitors of EP24.15,
with IC50 values of 20 µM and 12 µM,
respectively. This represents a loss in affinity of less than threefold
relative to bradykinin itself. As would be predicted for substrate
analogs, ß-C3-L-Phe5-BK and
ß-C3-D-Phe5-BK are competitive inhibitors of
EP24.15, as demonstrated by Lineweaver-Burk analysis (Fig. 2
, inset).
The calculated inhibitor constants (Ki) for BK,
ß-C3-L-Phe5-BK, and
ß-C3-D-Phe5-BK are 6.5 µM, 16.2 µM, and 9.7
µM, respectively.
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4. ß-C3-D-Phe5-BK and ß-C3-L-Phe5-BK
act as B2 receptor agonists in pig coronary artery,
although potencies are two to three orders of magnitude less than
bradykinin
Both analogs acted as agonists at the B2
receptor, as assessed by relaxation of pig coronary artery rings, but
with potencies 650- to 3500-fold less than for bradykinin.
Relaxation to each peptide was blocked by HOE140, confirming the
relaxation was mediated by B2 receptors. The
vasodilator response to 1 nM bradykinin was not altered by the presence
of either analog at 10 µM, indicating the lack of significant
antagonist activity.
CONCLUSIONS
The level of any given bioactive peptide within the circulation or within a specified tissue depends not only on the rates of biosynthesis and secretion, but also on the rate of proteolytic degradation. The physiological role of individual metabolizing peptidases can be elucidated through the use of specific synthetic inhibitors, which may also hold potential as therapeutic agents. Specific inhibitors exist for a number of well-characterized peptidases; for example, small, orally active nonpeptidic inhibitors of angiotensin-converting enzyme (ACE) have been developed that have enormous therapeutic value in the treatment of hypertension, but attempts to develop similar inhibitors for other peptidases have been marred by their relative lack of specificity. For example, neutral endopeptidase (NEP) has been implicated in the destruction of enkephalins in the nervous system and atrial natriuretic peptide in the circulation. However, many of the small molecule NEP inhibitors that have been devised also inhibit ACE or another closely related enzyme, endothelin-converting enzyme (ECE). Conversely, the development of specific inhibitors of ECE has been impeded by the similarity of this enzyme to NEP and the frequent cross-inhibition of most small molecule inhibitors.
The challenge of designing a substrate-based peptidase inhibitor lies
in closely mimicking the complex structure of the parent peptide such
that enzyme binding is retained, yet modified sufficiently to prevent
enzymatic cleavage. Although several successful approaches to this
challenge have been developed, such strategies are not universally
applicable and some peptidase targets remain intractable. Recently,
another peptidomimetic approach has received increasing attention:
substitution of -amino acids with ß-amino acids. Rather than a
single carbon atom between the amino and carboxyl termini, ß-amino
acids contain two carbon atoms; the specific side chain may branch off
either the C2 or C3 carbon.
In recent years, ß-amino acids have been successfully used in the
design of agonist and antagonist analogs of several peptides, including
gastrin, somatostatin, integrin, and class I MHC binding peptides. In
some cases, these ß-peptides have been shown to be resistant to
proteolysis, a property heretofore presumed to result from a lack of
enzyme binding. However, the possibility that ß-peptides might retain
affinity for peptidases and thus could potentially function as enzyme
inhibitors has not been adequately addressed prior to our work.
In the present study, we have used the cleavage of bradykinin by the
soluble metallopeptidase EP24.15 as a model system to demonstrate that
ß-amino acid substitution at the cleavage site can prevent peptide
hydrolysis without precluding binding to the enzyme. The most potent
analogs tested, in which the -amino acid amino-terminal to the
scissile bond (Phe5) was replaced by its C3
ß-amino acid congener (L- and D-ß-C3-Phe5),
were completely resistant to hydrolysis yet exhibited affinities for
EP24.15 only twofold lower than bradykinin itself (Fig. 2)
.
Thus, in some cases the introduction of a ß-amino acid can confer
resistance to cleavage with only a modest loss in affinity for the
enzyme.
In the absence of definitive structural studies, it is impossible
to comment on exactly how the introduction of a ß-amino acid will
affect binding of a peptide to a peptidase such as EP24.15. However,
one can speculate that the extra carbon in the peptide backbone
generates a ‘kink’ in the peptide (Fig. 3
) that prevents coordination of the peptide bond carbonyl group with the
catalytic zinc atom in EP24.15. Depending on the exact nature of the
peptide side chains and their interaction with specific subsites in the
peptidase, adequate binding affinity may still be retained. Indeed,
EP24.15 is believed to have an extended active site, wherein residues
distal to the scissile bond are important for substrate binding. Thus,
ß-amino acid-containing substrate analogs such as
ß-C3-D-Phe5-BK may provide the basis for the
development of stable inhibitors of EP24.15, with improvements in
affinity achieved by further modifications, including the introduction
of ß- and other non-natural amino acids.
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The two stereoisomers of ß-C3-Phe5-BK were
similar in their binding affinities for EP24.15 (Fig. 2)
; however,
other results from our laboratory suggest that substrate/inhibitor
stereochemistry may be of more consequence for other peptidases. For
example, the closely related endopeptidase EC 3.4.24.16, which cleaves
bradykinin at the same site and with similar kinetics as EP24.15, was
inhibited by ß-C3-L-Phe5-BK but not by
ß-C3-D-Phe5-BK. Thus, ß-amino acid analogs of
peptide substrates may allow discrimination between peptidases with
very similar substrate specificities for which sufficiently selective
inhibitors are currently lacking.
In conclusion, the present study demonstrates that peptide substrates containing a ß-amino acid at the scissile bond are in some instances capable of inhibiting the peptidase without being cleaved. This observation negates previous assumptions that the resistance of ß-amino acid-containing peptides to hydrolysis results solely from a lack of binding. Incorporation of ß-amino acids thus represents a novel peptidomimetic element with potential utility in the development of specific peptidase inhibitors.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0805fje ; to cite this
article, use FASEB J. (May 29, 2001) 10.1096/fj.00-0805fje 
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