Metabolic stability of peptidomimetics: N-methyl and aza heptapeptide analogs of a PKB/Akt inhibitor

Article abstract of DOI:10.1111/j.1747-0285.2011.01207.x

Linear peptides suffer from poor pharmacokinetic and pharmacodynamic properties. Peptidomimetics are designed to overcome these pharmacological drawbacks while maintaining the biological effects of the parent peptides. Aza-peptides, in which an alpha carbon is replaced with nitrogen, are promising peptidomimetic analogs; however, little is known about the stability of these analogs toward enzymatic degradation. We performed systematic aza and N-methyl scans of a PKB/Akt inhibitor, PTR6154. We evaluated the stability of the aza-scan and N-methyl scan libraries toward enzymatic degradation by trypsin/chymotrypsin. Our results indicate that the modification site is important for metabolic stability and that aza-peptides have a more global effect than N-methylation, affecting cleavage sites distant from the modification site.

Full text of DOI:10.1111/j.1747-0285.2011.01207.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01207.x
Chem Biol Drug Des 2011; 78: 887–892
Research Letter
Metabolic Stability of Peptidomimetics: N-Methyl
and Aza Heptapeptide Analogs of a PKB⁄Akt
Inhibitor
Yftah Tal-Gan1, , Noam S. Freeman1,
,
enhance the biological effects of natural peptides while, at the
Shoshana Klein2, Alexander Levitzki2 and
Chaim Gilon1,*
same time, overcoming these undesirable properties. Many types of
local and global modifications have been developed in attempts to
obtain peptidomimetics with improved pharmacological properties
(1–4). The local modifications include Na-methylated derivatives and
aza-peptides.
1Institute of Chemistry, The Hebrew University of Jerusalem, 91904
Jerusalem, Israel
2Unit of Cellular Signaling, Department of Biological Chemistry, The
Alexander Silberman Institute of Life Sciences, The Hebrew
University of Jerusalem, 91904 Jerusalem, Israel
*Corresponding author: Chaim Gilon, gilon@vms.huji.ac.il
Both authors contributed equally to this work.
Na-methylation (Figure 1) is a powerful and common tool for struc-
ture–activity relationship studies. It is often used to examine the
effects of local backbone modifications and hydrogen bonding on
the potency of a known peptide sequence. N-methylation has been
shown to improve important pharmacological parameters such as
lipophilicity (5), bioavailability (5–10), proteolytic stability (4,6–
9,11,12), conformational rigidity (7,8,13), and duration of action
(6,7). N-methylation may also result in enhanced potency (7–9,14–
16), new receptor subtype selectivity (9,12,17–21), and conversion
of an agonist into an antagonist (6). N-methylation induces local
backbone constraints caused by steric hindrance (6–8,22). However,
the reduced number of potential hydrogen bonds formed by the
backbone and the lower tendency to exhibit trans conformations
may result in subsequent secondary structural changes (23). Hence,
a single N-methylation may influence the ability of the peptide to
recognize its binding site, as well as its selectivity and its pharma-
cological properties (7–9,11,12,16,18–21,24).
àThe abbreviations for amino acids are according to the IUPAC-IUB
Commission of Biochemical Nomenclature, http://
www.chem.qmul.ac.uk/iupac/AminoAcid/.
Linear peptides suffer from poor pharmacokinetic
and pharmacodynamic properties. Peptidomimet-
ics are designed to overcome these pharmacologi-
cal drawbacks while maintaining the biological
effects of the parent peptides. Aza-peptides, in
which an alpha carbon is replaced with nitrogen,
are promising peptidomimetic analogs; however,
little is known about the stability of these analogs
toward enzymatic degradation. We performed sys-
tematic aza and N-methyl scans of a PKB ⁄ Akt
inhibitor, PTR6154. We evaluated the stability of
the aza-scan and N-methyl scan libraries toward
enzymatic degradation by trypsin ⁄ chymotrypsin.
Our results indicate that the modification site is
important for metabolic stability and that aza-pep-
tides have a more global effect than N-methyla-
tion, affecting cleavage sites distant from the
modification site.
The synthesis of N-methyl peptides is well established (25–31), and
these peptidomimetics often display favorable pharmacokinetic
properties. N-methylation is usually one of the first modifications
applied when attempting to enhance the proteolytic resistance of a
bioactive peptide (4,32–34). There are several examples of system-
atic N-methylation scans of active peptides to obtain superior ana-
logs that exhibit longer duration of action (5,11,12,35,36).
Key words: aza-peptide, chymotrypsin, N-methylation, peptidomi-
metics, PKB ⁄ Akt, trypsin
Aza-peptides are peptides in which one (or more) of the a-carbons
bearing the side chain residues has been replaced by a nitrogen
atom (Figure 1). Aza-amino acid residues impart special conforma-
tional properties to the peptide structure, owing to the loss of stere-
ogenicity and the reduction in flexibility of the parent linear peptide
(37). Insertion of an aza-amino acid residue imparts both local and
global effects. Locally, the substitution of a Ca by N results in the
potential for additional hydrogen bonding and affects the acidity of
the neighboring amide N-H bonds (38). A global reduction in the flex-
ibility of aza-peptides results from the generation of hydrazide and
urea structural elements and from lone pair repulsion of the adjacent
amines. Notably, this reduced flexibility has been shown to induce
Received 6 February 2011, revised 14 July 2011 and accepted for publi-
cation 2 August 2011
Abbreviationsà: ACN, acetonitrile; MALDI, matrix-assisted laser
desorption ionization; MS, mass spectrometry; PKB ⁄ Akt, Protein kinase
B; RP-HPLC, reverse-phase high-pressure liquid chromatography; TDW,
triple-distilled water; TFA, trifluoroacetic acid; TOF, time of flight.
Linear peptides have critical drawbacks as drug candidates; typi-
cally, rapid metabolism by proteolysis, nonselective receptor binding
and poor bioavailability. Peptidomimetics are designed to retain or
887

Tal-Gan et al.
N-terminus
C-terminus
O
O
O
R
N
H
R
H
R
H
H
H
N
N
N
N
N
N
H
H
O
O
O
Peptide
Aza-Peptide
N-Methylated peptide
R: side chain
Figure 1: Peptide, N-Methylated peptide and aza-peptide.
b-turn conformations (39,40). The replacement of a particular Ca by
N in a biologically active peptide will probably affect its overall con-
formation and hence its absorption, transport, distribution, enzyme or
receptor binding, and metabolic stability. Aza-peptides have been
shown to be promising modifications for the generation of peptidom-
imetic drug leads (38,41,42) and powerful tools for structure–activity
relationship studies. The systematic replacement of amino acid resi-
dues in a given peptide with their aza-counterparts (aza-scan) has
been recently introduced as an attractive method for identifying bio-
logically active conformations (40).
Our results verified that aza-peptides are resistant to Trypsin ⁄ Chy-
motrypsin degradation; thus, aza-peptides are useful peptidomimet-
ics for drug design, despite their synthetic challenges.
Results and Discussion
The synthesis of the N-methyl PTR6154 scan and the aza PTR6154
scan libraries has been previously reported (50,54). All the pepti-
domimetics, as well as the parent peptide, PTR6154, were incu-
bated at 37 ꢀC in a mixture of trypsin and chymotrypsin and their
degradation was monitored by HPLC and mass spectrometry (MS)
(Tables 1 and 2 and Figures 2 and 3). The N-methyl and aza deriva-
tives were named according to the modified residue (e.g., N-
MeArg1 corresponds to N-methylation on Arg 1 and AzaArg1 corre-
sponds to replacement of Arg 1 with its aza-counterpart).
Aza-peptides are highly promising serine protease inhibitors (42).
Owing to the reduced electrophilicity of the aza residue carbonyl
group, upon interaction with aza-peptides, serine proteases form sta-
ble carbamoyl enzyme adducts that undergo very slow deacylation.
Aza-peptides have been presumed to be resistant to enzymatic deg-
radation, but this assumption is based on a few specific examples
(43–46). To the best of our knowledge, no systematic assessment of
the metabolic stability of an aza-scan library has ever been reported.
Moreover, the stability of aza-peptide analogs has not been com-
pared to the stability of more common peptidomimetic structures.
The N-methyl library
Methylation of the terminal residues of the peptide, distant from the
trypsin and chymotrypsin cleavage sites, resulted in a notable
decrease in resistance to trypsin ⁄ chymotrypsin. N-MeArg1 and N-
MeHol7 were completely degraded within 30 min, compared to the
parent PTR6154 that was completely degraded only after 120 min
(Figure 2), suggesting that the methylation of these residues results
in peptide conformations with higher affinities for the protease bind-
ing site. N-MeAla2, in which proline was replaced by N-methyl ala-
nine (50), was less resistant to degradation than PTR6154 (Figure 2).
This result can be accounted for by the conformational freedom
gained by the replacement of the rigid proline residue with the less
rigid N-methyl alanine residue, resulting in an extended peptide
backbone conformation that is more accessible to peptidase binding.
On the other hand, methylation of the inner amino acids resulted in
enhanced resistance toward enzymatic degradation, either because
peptidase binding was inhibited or because of inherent stabilization
of the peptide bonds at the cleavage sites. Full degradation of these
peptides was not observed even after 4 h (Figure 2).
Trypsin and chymotrypsin are serine proteases formed in the pancreas
and have essential roles in protein and peptide digestion (47,48). The
peptide binding sites of both enzymes consist of His and Ser residues;
however, the specificity of each enzyme is attributed to the nature of
its active site pocket. Trypsin has a negatively charged pocket, which
stabilizes positive charge and thus cleaves peptide bonds after posi-
tively charged Arg and Lys residues. Chymotrypsin has a hydrophobic
pocket that fits aromatic residues and thus cleaves peptide bonds
after the aromatic amino acids residues Trp, Tyr, or Phe (47,48).
Because of their peptide digestion activity, trypsin and chymotrypsin
are commonly used for in vitro evaluation and comparison of the met-
abolic stability of peptides and peptidomimetic analogs (12,49–51).
Recently, we reported an extensive structure–activity relationship
study of the protein kinase B (PKB ⁄ Akt) peptide inhibitor, PTR6154
(Arg-Pro-Arg-Nva-Tyr-Dap-Hol) introduced by Litman et al. (52). Our
peptidomimetic structure-activity relationship studies included N-
methyl peptides, peptoids, backbone cyclic peptides, and aza-pep-
tides (50,53,54). Because the parent peptide, PTR6154, contains
both Arg and Tyr residues, it is a good model for a Trypsin ⁄ Chymo-
trypsin stability assay. Here, we present a systematic study of the
metabolic stability of the aza-peptide and Na-methylated peptide
scan libraries of PTR6154. Unlike previous studies of peptidomimetic
stability, we evaluated the stability of all of the aza and N-methyl
PTR6154 derivatives, without reference to their biological activity.
All of the N-methyl peptides were evaluated as PKB ⁄ Akt inhibitors
(50). N-MeArg1 and N-MeAla2 maintained potency, as compared
with the parent peptide PTR6154. All the other modified peptides
had 10-fold reduced potencies compared to the parent PTR6154.
The unstable analog, N-MeHol7, was no less bioactive than the
most stable analog, N-MeTyr5. Thus, there was no correlation
between bioactivity and stability.
To obtain further insight regarding the resistance of the different
analogs to trypsin ⁄ chymotrypsin, the cleavage products were ana-
888
Chem Biol Drug Des 2011; 78: 887–892

Metabolic Stability of Peptidomimetics
Table 1: Fragmentation of the N-methyl scan library after degra-
dation by trypsin ⁄ chymotrypsina
Table 2: Fragmentation of the aza-scan library after degradation
by trypsin ⁄ chymotrypsina
Name
Deduced sequence of fragment
Observed MH+
Name
Deduced sequence of fragment
Observed MH+
PTR6154
H-Arg-Pro-Arg-Nva-Tyr-Dap-Hol-NH2
902.67
PTR6154
H-Arg-Pro-Arg-Nva-Tyr-Dap-Hol-NH2
H-Arg-Pro-Arg-Nva-Tyr-OH
H-Dap-Hol-NH2
H-Arg-Pro-Arg-OH
H-Nva-Tyr-Dap-Hol-NH2
H-AzaArg-Pro-Arg-Nva-Tyr-Dap-Hol-NH2
H-Pro-Arg-Nva-Tyr-Dap-Hol-NH2
H-AzaArg-OH
H-Pro-Arg-Nva-Tyr-OH
H-Dap-Hol-NH2
H-Pro-Arg-OH
H-Nva-Tyr-Dap-Hol-NH2
H-Arg-AzaPro-Arg-Nva-Tyr-Dap-Hol-NH2
H-Arg-AzaPro-Arg-Nva-Tyr-OH
H-Dap-Hol-NH2
902.67
Fragment 1 H-Arg-Pro-Arg-Nva-Tyr-OH
Fragment 2 H-Dap-Hol-NH2
Fragment 3 H-Arg-Pro-Arg-OH
Fragment 4 H-Nva-Tyr-Dap-Hol-NH2
690.43
Fragment 1
Fragment 2
Fragment 3
Fragment 4
AzaArg1
Fragment 1
Fragment 2
Fragment 3
Fragment 4
Fragment 5
Fragment 6
AzaPro2
Fragment 1
Fragment 2
Fragment 3
Fragment 4
AzaArg3
Fragment 1
Fragment 2
AzaNva4
690.43
b
b
428.66
493.52
916.45
428.66
493.52
903.48
N-MeArg1
H-N(Me)Arg-Pro-Arg-Nva-Tyr-Dap-Hol-NH2
Fragment 1 H-N(Me)Arg-Pro-Arg-Nva-Tyr-OH
Fragment 2 H-Dap-Hol-NH2
Fragment 3 H-N(Me)Arg-Pro-Arg-OH
Fragment 4 H-Nva-Tyr-Dap-Hol-NH2
704.55
746.36
b
b
442.36
493.63
890.48
534.45
b
b
N-MeAla2
H-Arg-N(Me)Ala-Arg-Nva-Tyr-Dap-Hol-NH2
Fragment 1 H-Arg-N(Me)Ala-Arg-Nva-Tyr-OH
Fragment 2 H-Dap-Hol-NH2
Fragment 3 H-Arg-N(Me)Ala-Arg-OH
Fragment 4 H-Nva-Tyr-Dap-Hol-NH2
678.52
493.44
903.35
b
416.32
493.47
916.13
691.38
b
N-MeArg3
H-Arg-Pro-N(Me)Arg-Nva-Tyr-Dap-Hol-NH2
H-Arg-AzaPro-Arg-OH
H-Nva-Tyr-Dap-Hol-NH2
429.66
493.58
903.59
Fragment 1 H-Arg-Pro-N(Me)Arg-Nva-Tyr-OH
Fragment 2 H-Dap-Hol-NH2
N-MeNva4
Fragment 1 H-Arg-Pro-Arg-N(Me)Nva-Tyr-OH
Fragment 2 H-Dap-Hol-NH2
Fragment 3 H-Arg-Pro-Arg-OH
Fragment 4 H-N(Me)Nva-Tyr-Dap-Hol-NH2
N-MeTyr5
Fragment 1 H-Arg-Pro-Arg-OH
Fragment 2 H-Nva-N(Me)Tyr-Dap-Hol-NH2
N-MeDap6
Fragment 1 H-Arg-Pro-Arg-Nva-Tyr-OH
Fragment 2 H-N(Me)Dap-Hol-NH2
Fragment 3 H-Arg-Pro-Arg-OH
Fragment 4 H-Nva-Tyr-N(Me)Dap-Hol-NH2
704.59
b
H-Arg-Pro-AzaArg-Nva-Tyr-Dap-Hol-NH2
H-Arg-Pro-AzaArg-Nva-Tyr-OH
H-Dap-Hol-NH2
H-Arg-Pro-Arg-AzaNva-Tyr-Dap-Hol-NH2
H-Arg-Pro-Arg-AzaNva-Tyr-OH
H-Dap-Hol-NH2
H-Arg-Pro-Arg-Nva-AzaTyr-Dap-Hol-NH2
H-Arg-Pro-Arg-Nva-Tyr-Dap-AzaHol-NH2
H-Arg-Pro-Arg-Nva-Tyr-OH
H-Dap-AzaHol-NH2
H-Arg-Pro-Arg-N(Me)Nva-Tyr-Dap-Hol-NH2
916.20
691.25
b
704.34
b
903.82
428.35
507.30
Fragment 1
Fragment 2
AzaTyr5
691.54
b
H-Arg-Pro-Arg-Nva-N(Me)Tyr-Dap-Hol-NH2 916.35
903.60
903.60
428.29
507.41
916.47
AzaHol7
Fragment 1
Fragment 2
Fragment 3
Fragment 4
690.77
b
H-Arg-Pro-Arg-Nva-Tyr-N(Me)Dap-Hol-NH2
690.47
H-Arg-Pro-Arg-OH
H-Nva-Tyr-Dap-AzaHol-NH2
428.32
494.63
b
428.61
507.31
916.67
aFragments were identified by mass spectrometry.
bComplementary fragment.
Bold letters emphasize the modified residue.
N-MeHol7
H-Arg-Pro-Arg-Nva-Tyr-Dap-N(Me)Hol-NH2
Fragment 1 H-Arg-Pro-Arg-Nva-Tyr-OH
Fragment 2 H-Dap-N(Me)Hol-NH2
Fragment 3 H-Arg-Pro-Arg-OH
Fragment 4 H-Nva-Tyr-Dap-N(Me)Hol-NH2
690.36
b
428.60
507.46
aza-counterparts, were less resistant to trypsin ⁄ chymotrypsin than
PTR6154, suggesting that these analogs had better affinities for the
proteases. The other aza PTR6154 analogs exhibited enhanced
resistance toward trypsin ⁄ chymotrypsin degradation (Figure 3),
which could be owing to reduced affinity for the protease or
enhanced stability of the bond to be cleaved.
aFragments were identified by mass spectrometry.
bComplementary fragment.
Bold letters emphasize the modified residue.
lyzed using MS. As expected, PTR6154 had two major cleavage
sites for trypsin ⁄ chymotrypsin: Arg3-Nva4 and Tyr5-Dap6 (Table 1).
Interestingly, the analogs N-MeArg3 and N-MeTyr5, in which the N-
methylation precedes these cleavage sites, were completely resis-
tant to cleavage – N-MeArg3 was not cleaved between Arg3-Nva4
and N-MeTyr5 was not cleaved between Tyr5-Dap6. On the other
hand, the analog N-MeNva4 was not resistant to cleavage between
Arg3-Nva4, and similarly, N-MeDap6 was not resistant to cleavage
between Tyr5-Dap6. Thus, in the case of PTR6154, N-methylation of
the amide bond adjacent to the cleavage site conferred more resis-
tance toward enzymatic degradation by trypsin ⁄ chymotrypsin than
N-methylation of the cleaved amide bond itself.
The entire aza PTR6154 scan library showed reduced potency as
PKB ⁄ Akt inhibitors, compared to the parent peptide, PTR6154 (54).
With the exception of AzaArg3, which did not inhibit PKB ⁄ Akt at all
at the concentrations tested, all of the modified peptides had simi-
lar potencies, 15-fold less potent than PTR6154. Again, there was
no correlation between bioactivity and stability.
As we anticipated, MS analysis of the aza PTR6154 analogs indi-
cated the same two cleavage sites as in the N-methyl library,
namely between Arg3-Nva4 and between Tyr5-Dap6 (Table 2). The
analogs AzaArg3 and AzaNva4 appeared to be completely resistant
toward cleavage at the Arg3-Nva4 site. Notably, AzaTyr5 showed
complete resistance at both cleavage sites. These results suggest
that aza-amino acid residues are likely to induce resistance toward
enzymatic degradation of neighboring peptide bonds and may also
affect the entire secondary structure of the peptide.
The aza library
The aza PTR6154 scan library gave a similar stability pattern to the
N-methyl PTR6154 scan library. Peptides AzaArg1 and AzaHol7, in
which the terminal residues of PTR6154 were replaced by their
Chem Biol Drug Des 2011; 78: 887–892
889

Tal-Gan et al.
N-methyl versus aza modifications
toward cleavage (Figures 2 and 3). We expect that modifications
that resulted in reduced binding affinity to the protease would tend
to slow down (but not completely inhibit) degradation. Complete
resistance toward cleavage at specific cleavage sites is more likely
to be attributed to inherent stabilization of the relevant bonds. The
inherent stabilization effect of N-methylation was localized, affect-
ing only adjacent trypsin ⁄ chymotrypsin cleavage sites. Aza-amino
acid insertion, on the other hand, caused a more global effect, sta-
bilizing cleavage sites distant from the modification sites (Tables 1
and 2). This reflects the different natures of the modifications.
Methylation affects the ability to form hydrogen bonds at the meth-
ylation site itself and causes localized steric effects. Aza modifica-
tion affects the acidity and hence the ability to form hydrogen
bonds on both sides of the modification (38). In addition, resonance
effects lead to significant changes in conformation (39,40).
Both the N-methyl and aza-scan libraries showed similar patterns;
modifying the N or C terminal residues resulted in reduced resis-
tance, whereas modifying the inner residues enhanced resistance
Degradation of the N-methyl scan library
100%
80%
PTR6154
N-MeArg1
N-MeAla2
N-MeArg3
N-MeNva4
N-MeTyr5
N-MeDap6
N-MeHol7
60%
40%
20%
0%
0
30
60
90
120
180
240
Conclusions
Time (min)
Figure 2: Comparison of the degradation rates of the N-methyl
scan library towards Trypsin and Chymotrypsin enzymatic cleavage.
The effects of N-methylation and aza-amino acid insertion on the
resistance of peptides toward enzymatic degradation by trypsin and
chymotrypsin were assessed (Figure 4). Two libraries, an N-methyl
scan library and an aza-peptide scan library, were evaluated and
compared to the parental linear peptide, PTR6154. Both libraries
showed similar patterns; modifying the N or C terminal residues
resulted in reduced resistance toward degradation, whereas modify-
ing the inner residues resulted in enhanced resistance toward deg-
radation. These results imply that backbone modifications can lead
to conformational changes that impair stability and that the modifi-
cation site is important for obtaining enhanced stability. N-methyla-
tion has a more local effect than aza-amino acid insertion, which
results in a more global effect. Thus, N-methylation only affected
cleavage sites adjacent to the modification, whereas aza-amino acid
insertion also affected cleavage sites distant from the modification
site.
Degradation of the aza scan library
100%
80%
PTR6154
AzaArg1
60%
40%
20%
0%
AzaPro2
AzaArg3
AzaNva4
AzaTyr5
AzaHol7
0
30
60
90
120
180
240
Time (min)
Figure 3: Comparison of the degradation rates of the aza scan
We saw no correlation between peptide stability and in vitro activ-
library towards Trypsin and Chymotrypsin enzymatic cleavage.
ity. Nonetheless, we expect that enhanced stability to peptidases
Replacement Replacement
Replacement
by nitrogen
increased
metabolic
stability
(no cleavage
of Arg3
or Tyr5 sites)
by nitrogen
increased
metabolic
stability
by nitrogen
increased
metabolic
stability
Replacement
by nitrogen
increased
metabolic
stability
Replacement
by nitrogen
reduced
metabolic
stability
Replacement
by nitrogen
reduced
metabolic
stability
(no cleavage (no cleavage
of Arg3
site)
of Arg3
site)
O
O
C
O
O
O
O
O
N
CH C
NH2 CH
(CH2)3
NH CH C NH CH C NH CH C NH CH C NH CH C NH2
(CH2)3
NH
CH2
CH2
CH3
CH2
CH2
NH2
CH2
CH2
NH
Methylation
CH CH3
CH3
C
NH
C
NH
increased
metabolic
stability
OH
Methylation
N(Me)Ala
reduced
Methylation
reduced
metabolic
stability
Methylation
increased
metabolic
stability
(no cleavage
of Arg3
Methylation Methylation
NH2
NH2
Methylation
reduced
increased
metabolic
stability
increased
metabolic
stability
(no cleavage
of Tyr5
metabolic
stability
metabolic
stability
site)
site)
Figure 4: Schematic representation of the effects of N-methylation and aza amino acid insertion on metabolic stability. Modification of
the inner residues resulted in increased resistance to trypsin ⁄ chymotrypsin cleavage whereas modification of the C or N terminal residues
resulted in reduced resistance to cleavage.
890
Chem Biol Drug Des 2011; 78: 887–892

Metabolic Stability of Peptidomimetics
will be a critical parameter in vivo. Therefore, it is important to
optimize the stability of peptide drug leads, even if the improved
stability comes at the expense of slightly reduced activity. The
results presented here should be considered in the use of N-methyl
or aza modification with the aim of turning a linear parental pep-
tide into a drug lead.
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on a RP-18 column (5 l 250 · 4.6 mm, 110 ꢀ), Eluents A (0.05%
TFA in TDW), and B (ACN) were used in a linear gradient (95%
A fi 5% A in 35 min).
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Trypsin stability assay
The trypsin stability assay was conducted as described by Pakkala
et al. (49). 400 lL of each peptide (1 mM) dissolved in 200 mM
NH4HCO3 buffer solution (pH 8) were mixed with 1 lL of trypsin
and chymotrypsin (porcine pancreas, Biological Industries Israel, Beit
Haemek LTD) solution (2.5 mg ⁄ mL). The peptides were incubated at
37 ꢀC, and 30 lL samples were taken every 30 min and mixed with
30 lL of 2% TFA and 30% ACN in water. Samples were analyzed
by HPLC and by MALDI-TOF MS.
Acknowledgments
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mers;15:1615–1622.
A.L. was supported by grants from The European Commission (Pro-
kinase Consortium), the Prostate Cancer Foundation (USA), and the
Goldhirsh Foundation (USA).
15. Ron D., Gilon C., Hanani M., Vromen A., Selinger Z., Chorev M.
(1992) N-methylated analogs of Ac[Nle28,31]CCK(26-33): synthe-
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substance P receptor and its action is antagonized by enkepha-
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