(Received for publication, April 3, 1995; and in revised form, June 7, 1995)
From the
Several hundred phosphinic peptides having the general formula
Z-Phe
(PO
CH
)
Xaa`-Yaa`-Zaa`,
where Xaa` = Gly or Ala and Yaa` and Zaa` represent 20 different
amino acids, have been synthesized by the combinatorial chemistry
approach. Peptide mixtures or individual peptides were evaluated for
their ability to inhibit the rat brain zinc endopeptidases 24-15 and
24-16. Numerous phosphinic peptides of this series act as potent (K
in the nanomolar range) mixed
inhibitors of these two peptidases. However, our systematic and
comparative strategy led us to delineate the residues located in
P
and P
positions of the inhibitors that are
preferred by these two peptidases. Thus, endopeptidase 24-15 exhibits a
marked preference for inhibitors containing a basic residue (Arg or
Lys) in the P
position, while 24-16 prefers a proline in
this position. The P
position has less influence on the
inhibitory potency and selectivity, both peptidases preferring a
hydrophobic residue at this position. On the basis of these
observations, we have prepared highly potent and selective inhibitors
of endopeptidase 24-15. The
Z-
Phe
(PO
CH
)
Ala-Arg-Met
compound (mixture of the four diastereoisomers) displays a K
value of 70 pM for
endopeptidase 24-15. The most selective inhibitor of endopeptidase
24-15 in this series,
Z-
Phe
(PO
CH
)
Ala-Arg-Phe,
exhibits a K
value of 0.160 nM and is more than 3 orders of magnitude less potent toward
endopeptidase 24-16 (K
= 530
nM). Furthermore, at 1 µM this selective
inhibitor is unable to affect the activity of several other zinc
peptidases, namely endopeptidase 24-11, angiotensin-converting enzyme,
aminopeptidase M, leucine aminopeptidase, and carboxypeptidases A and
B. Therefore,
Z-
Phe
(PO
CH
)
Ala-Arg-Phe
can be considered as the most potent and specific inhibitor of
endopeptidase 24-15 developed to date. This new inhibitor should be
useful in assessing the contribution of this proteolytic activity in
the physiological inactivation of neuropeptides known to be hydrolyzed,
at least in vitro, by endopeptidase 24-15. Our study also
demonstrates that the combinatorial chemistry approach leading to the
development of phosphinic peptide libraries is a powerful strategy for
discovering highly potent and selective inhibitors of zinc
metalloproteases and should find a broader application in studies of
this important class of enzymes.
The endopeptidase 24-15 (EC 3.4.24.15) ()belongs to
the zinc metalloprotease family (1) and resembles a peptidase
previously purified from rabbit brain by Camargo et
al.(2) . Later, endopeptidase 24-15 was named thimet
oligopeptidase with respect to the thiol and metal dependence of its
catalytic activity(3, 4, 5) . Molecular
cloning of the cDNA of endopeptidase 24-15 revealed a HEXXH
motif, which characterizes peptidases belonging to this family, and a
cysteine residue, lying on the C-terminal side of the second histidine
of the zinc binding motif(6) . This cysteine residue has been
proposed to be responsible for activation of the enzyme by
2-mercaptoethanol or dithiothreitol, as well as for inhibition of the
enzyme activity by thiol reactive reagents(6) .
24-15 displays several biochemical and physicochemical properties (for a review, see (7) ) in common with another zinc-containing metallopeptidase, endopeptidase 24-16 (EC 3.4.24.16)(8) . Interestingly, these two peptidases have the ability to hydrolyze numerous bioactive or synthetic peptides at the same cleavage site, suggesting that they have a closely related active site(7, 8, 9) .
We previously reported
that phosphodiepryl03, a phosphonamide peptide, acts as a potent mixed
inhibitor of 24-15 and 24-16, with K values in the nanomolar range(10) . This inhibitor
was shown to be unable to block the activity of several other zinc
peptidases such as endopeptidase 24-11, angiotensin-converting enzyme,
aminopeptidase M, leucine aminopeptidase, and carboxypeptidases A and
B. We particularly studied the effect of this inhibitor in vivo on the neurotensin catabolism, since we previously established
that 24-15 and 24-16 participated in vitro in the inactivation
of this neuropeptide(11, 12) . We established that
phosphodiepryl03 prevented neurotensin degradation in vivo in
vascularly perfused dog ileum(13) . More recently, several
phosphodiepryl03 analogues were also proved to be potent but still
acting as mixed inhibitors of 24-15 and 24-16(14) . The most
potent analogue of this series drastically potentiated
neurotensin-induced antinociceptive effects in hot plate-tested mice
after i.c.v. administration and enhanced the neurotensin-induced
contraction of isolated longitudinal smooth muscle from guinea pig
ileum(14) . However, the delineation of the respective
contribution of these enzymes in the neurotensin degradation will
depend on the development of highly selective inhibitors able to
discriminate between 24-15 and 24-16. To this end, a systematic
approach was thus devised to find such potent and selective inhibitors.
Peptides containing a phosphinic bond (PO
CH
)
instead of a phosphonamide bond (PO
NH) were selected
because the former are more chemically stable than the latter.
Furthermore, with bacterial collagenase, a zinc metalloprotease, we
recently demonstrated that the phosphinic peptide inhibitors have
nearly the same potency as the corresponding parent phosphonamide
peptide inhibitors(15) .
In this paper, we demonstrate that
the synthesis, by combinatorial chemistry, of several hundred different
phosphinic peptides having the general formula
Z-Phe
(PO
CH
)
Xaa`-Yaa`-Zaa`
has led to the discovery of both highly potent and selective inhibitors
of 24-15.
Diisopropyl fluorophosphate-treated carboxypeptidases A and
B, leucine aminopeptidase, and angiotensin-converting enzyme were from
Sigma. Endopeptidase 24.11 was purified and kindly provided by Drs. P.
Crine and G. Boileau (Département de Biochimie,
Université de Montréal,
Canada). All the Fmoc-amino acid derivatives, the
Mcc-Pro-Leu-Gly-ProLys(Dnp) synthetic substrate and the
2-chlorotrityl resin were from Novabiochem.
which takes into account mutual depletion of enzyme and
inhibitor(19, 20) . All the values reported for K were reproducible within ±5%.
The
first Fmoc amino acids were attached to the 2-chlorotrityl resin
according to Barlos et al.(21) . The degree of
substitution of each resin sample was determined according to
Meienhofer et al.(22) , but using = 7040 M
cm
at 300 nm (23) . The Fmoc groups were removed with 50% piperidine in
dimethylformamide. Coupling of the next residue was achieved using the
2-(1H-benzotriazol-1-yl)1,1,3,3-tetramethyluronium
hexafluorophosphate/diisopropylethylamine in situ strategy.
Typically, 3 equivalents of Fmoc amino acid and 4 equivalents of
diisopropylethylamine in dimethylformamide or N-methylpyrrolidone were added to the resin, and the reaction
was allowed to proceed for 30 min. The coupling of
Z-
Phe
(PO
-CH
)
XaaOH
was achieved using 1.5 equivalents of this block. Fully protected
peptides were cleaved from 2-chlorotrityl resin with a mixture of
glacial acetic acid, trifluoroethanol, and dichloromethane (2/2/6)
during 2 h. Solutions of protected peptides were dried in
vacuo. Protective groups were removed by the action of
trifluoroacetic acid containing 5% H
O, 5% thioanisol, 5%
phenol, 2.5% ethanedithiol, and 22.5% of dichloromethane. Solutions of
deprotected peptides were concentrated in vacuo, and peptides
were treated with 2.5 equivalents of NaHCO
in water.
Aqueous solutions were repeatedly extracted with cold diethylether and
then lyophilized. Peptide mixture homogeneity was checked by amino acid
analysis, allowing us also to determine concentrations of these
solutions. Single peptides were purified by preparative HPLC (Vydac
218TP1022 column) performed on a Gilson system equipped with a variable
wavelength detector. Peptide purities were checked by TLC, analytical
HPLC (Vydac 218TP104 column), and mass spectrometry.
Figure 1:
Preparation of phosphinic peptide
mixtures of general formula
Z-Phe
(PO
CH
)Gly-Yaa`-Zaa`
by combinatorial chemistry.
The protocol used
to synthesize the mixtures of phosphinic peptides of formula
Z-Phe
(PO
CH
)-
Ala-Yaa`-Zaa`
was similar to that one described for the phosphinic peptide mixtures
containing the
Z-
Phe
(PO
CH
)Gly moiety.
However, in this case due to the presence of both
Phe
and
Ala residues in these peptides, the final 20
different mixtures each contain 80 different phosphinic peptides.
Attribution of the L (R configuration) or D (S configuration) stereochemistry of the pseudophenylalanine residue was determined according to the procedure previously described(24) .
We previously reported the ability of a phosphonamide
peptide, phenylethyl-(PO
-NH)Gly-Pro-Nle
(phosphodiepryl03), to act as a potent mixed inhibitor of both 24-16
and 24-15(10) . The structure of this inhibitor, which is
thought to encompass the S
to S
subsites of
the catalytic site of these enzymes, was therefore used as the starting
point of a strategy aimed at developing fully specific blockers of
24-15. Preliminary experiments examined the putative influence of 1)
substitution of the phosphonamide surrogate by a phosphinic group; 2)
stereochemical modifications introduced at the P
,
P
, and P
positions; and 3) esterification of
the C terminus of the inhibitor. Table 1indicates that the
phosphinic peptide appears slightly less potent than the corresponding
phosphonamide peptide (compounds 2 and 1). Furthermore, introduction of
D-amino acids at the P
, P
, and P
positions drastically reduces the potency of the inhibitors
toward both peptidases (see compounds 3, 4, and 5). Finally,
esterification of the C-terminal carboxyl group decreases the inhibitor
affinity, indicating that the binding of these phosphorus peptides
requires a free C-terminal carboxylate (compounds 2 and 6). Based on
these preliminary observations, the search for new compounds was
undertaken by checking systematically the role played by the P
and P
positions in the potency and selectivity of
the inhibitors.
Figure 2:
Influence of the P2` position (Yaa`) for
the inhibition of 24-15 (upper part) and 24-16 (lower
part) by inhibitor mixtures of general formula
Z-Phe
(PO
CH
)Gly-Yaa`-Zaa`
(Zaa` contains a mixture of 20 different amino acids). The
concentration of phosphinic peptides in each mixture was 500
nM.
Figure 3:
Selectivity factor
K
The development of phosphinic peptide chemistry by solid
phase synthesis, in conjunction with the combinatorial chemistry
approach (26, 27) , makes it possible to prepare
rapidly a huge number of different phosphinic peptides. All these
phosphinic peptides, as good analogues of the substrates of zinc
metalloproteases in the transition state, are expected to be potent
inhibitors of this enzyme family. In fact, as demonstrated in this
study, but also in previous work(15) , phosphinic peptides
provided that they contain the right amino acid sequence, which ensures
an optimal recognition of these inhibitors by the target zinc
metalloprotease, are highly potent inhibitors of this class of
proteases. In this work, we have identified a phosphinic sample
(Z-Phe
(PO
CH
)
Ala-Arg-Met,
a mixture of four distereoisomers) displaying a K
value of 70 pM. Thus, the phosphinic peptides of this
sample are by far much more potent inhibitors of 24-15 than the
previously reported carboxyalkyl (25, 28) or
hydroxamate (29) peptide inhibitors, all exhibiting K
values in the range of 10-100 nM.
The other important aspect of the approach is the possibility of
optimizing the inhibitor selectivity by screening the peptide mixtures
with different proteases. The power of the approach is well illustrated
in this work by the recognition of the clear preference of 24-15 for an
arginine or lysine residue in the P position of the
inhibitor, two residues that are much less tolerated at the same
position by 24-16. 24-16 prefers a proline residue in this position
than a basic one. Interestingly, the proline residue is also well
accommodated by 24-15. This result may explain why, for a long time, it
has been stated that 24-15 has a preference for proline in the P
position(28) . This lack of selectivity, displayed by the
two peptidases toward the recognition of peptide inhibitor with proline
in the P
position, accounts for the fact that all the
phosphinic peptides having the general formula
Z-Phe-
(PO
CH
)Gly-Pro-Zaa` behave as mixed
inhibitors of these two peptidases (data not shown). Such results are
also consistent with the view that these two endopeptidases have
closely related active sites, and that even with a systematic approach,
the development of highly selective inhibitors of these enzymes still
remains a challenge. Thus, identification of selective inhibitors of
24-16 will require investigating the influence of the P
and
P
positions in these inhibitors on the selectivity.
Nevertheless, Z-Phe-
(PO
CH
)Ala-Arg-Phe,
which is 3 orders of magnitude more potent for 24-15 than for 24-16 and
unable to block several other zinc endopeptidases, is to date the most
specific inhibitor of 24-15.
The marked preference of 24-15 for
inhibitors having an Arg residue in the P position
probably explains the efficiency of this enzyme in cleaving bioactive
peptides displaying such a structural feature. In this respect, it can
be mentioned that the best specificity constant (k
/K
ratio) for rat 24-15
has been observed for the hydrolysis of dynorphin
A
(3, 9) . In this peptide
(Tyr
-Gly-Gly-Phe-Leu-Arg-Arg-Ile
), the cleavage
occurs between the Leu and Arg residues. Thus, in the bound state, the
Arg
of this peptide probably interacts with the S
subsite of 24-15. Hydrolysis of several bioactive peptides
possessing a C-terminal sequence like Xaa-Arg-XaaOH are now under
investigation to confirm the effect of the P
arginine
residue on the 24-15 specificity.
Among other factors that may
influence the specificity of 24-15, the presence of a free C-terminal
carboxylate group has been previously pointed
out(28, 30) . Our data confirm the importance of a
free C-terminal carboxylate group in the P position of the
inhibitor (Table 1). It should be noticed that the carboxyalkyl
peptide inhibitors developed by Orlowski et al.(25) do not contain a free carboxylate group at the
P
position since this group is protected by a p-aminobenzoate moiety. Therefore, the free carboxylate group
of these inhibitors is borne by the para-amino-benzoate group, which
likely interacts with the S
or S
subsites of
24-15(30) . Altogether, these data suggested that there are
several cationic loci in the active site of 24-15 and explain the
ability of this endopeptidase to cleave peptidyl bonds located three,
four, or five residues from the C-terminal free carboxylate group of a
peptide.
Inhibition of 24-15 by the CPP-AAF-pAB inhibitor triggers
an increased recovery of luteinizing hormone-releasing hormone in
vivo(31) and potentiated the luteinizing
hormone-releasing hormone-induced release of plasmatic luteinizing
hormone and follicle-stimulating hormone(32) . Subsequent
studies reported the increased antinociceptive properties of dynorphin
A and leucine enkephalin-Arg-Gly-Leu after
administration of this inhibitor(33) . Finally, intravenous
infusion of CPP-AAF-pAB rapidly slowed down the arterial pressure of
normotensive rats(34) , indicating that 24-15 could play a role
in the control of the pressor response in mammals. However, these data
are still controversial since it was recently reported that CPP-AAF-pAB
could undergo proteolytic
cleavage(35, 36, 37) . This produces a
catabolite that potently inhibits angiotensin-converting enzyme, whose
role in blood pressure response has been well documented. Therefore,
the potent and selective 24-15 inhibitors reported here represent novel
tools for reexamining the reel contribution of this enzyme in the
control of the above physiological processes. These molecules should
also help us to establish the relative contribution of 24-15 and 24-16 in vivo in the neurotensin
degradation(13, 14) .
This new series of inhibitors might also constitute valuable probes for evaluating the degree of similarity between 24-15 and the family of proteins that exhibit a high percentage of sequence identity with the 24-15(38, 39) . In this connection, it will be of interest to determine the ability of the present inhibitors to block the peptidase activity of porcine-soluble angiotensin-binding protein, a protein which was recently shown to share a 65% sequence identity with porcine 24-15 (40) .
To our knowledge, our study is the
first example of the development of phosphinic peptide libraries and
their successful use for discovering potent and selective inhibitors of
zinc metalloproteases. Recently, a synthetic procedure for developing
peptide libraries containing a phosphonate group (POO) was
published(41) , but no results were given for the potency of
these phosphonate peptides as zinc metalloprotease inhibitors. However,
for bacterial collagenase and mammalian 24-15 and 24-16, it should be
mentioned that phosphonate peptides have been found to be extremely
poor inhibitors of these proteases(14, 42) . A similar
result was reported for thermolysin(43) . More recently,
phosphinic peptides were also proved to be potent inhibitors of the
astacin protease, another zinc metalloprotease(44) . Thus, in
our opinion, development of phosphinic peptide libraries should be an
important future approach for discovering potent and selective
inhibitors of other zinc metalloproteases(45) , a family of
proteases that has rapidly grown in the last few years and is of great
interest.