Prohormone-substrate peptide sequence recognition by peptidylglycine α-amidating monooxygenase and its reflection in increased glycolate inhibitor potency

Article abstract of DOI:10.1016/j.bmcl.2012.10.004

The interactions of nineteen peptide substrates and fifteen analogous peptidomimetic glycolate inhibitors with human peptidylglycine α-amidating monooxygenase (PAM) have been investigated. The substrates and inhibitors are the prohormones of calcitonin an

Full text of DOI:10.1016/j.bmcl.2012.10.004

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7015–7018
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Prohormone-substrate peptide sequence recognition by peptidylglycine
a-amidating monooxygenase and its reflection in increased glycolate
inhibitor potency
⇑
Kelly M. Morris, Feihua Cao, Hideki Onagi, Timothy M. Altamore, Allan B. Gamble, Christopher J. Easton
ARC Centre of Excellence for Free Radical Chemistry and Biotechnology, Research School of Chemistry, Australian National University, Canberra ACT 0200, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 July 2012
Revised 27 September 2012
Accepted 1 October 2012
Available online 11 October 2012
The interactions of nineteen peptide substrates and fifteen analogous peptidomimetic glycolate inhibi-
tors with human peptidylglycine -amidating monooxygenase (PAM) have been investigated. The sub-
a
strates and inhibitors are the prohormones of calcitonin and oxytocin and their analogues. PAM both
secreted into the medium by and extracted from DMS53 small lung carcinoma cells has been studied.
The results show that recognition of the prooxytocin and procalcitonin peptide sequences by the enzyme
extends more than four and five amino acid residues, respectively, from their C-termini. This substrate
sequence recognition is mirrored by increased inhibitor potency with increased peptide length in the gly-
colate peptidomimetics. Substitution of the C-terminal penultimate glycine and proline residues of pro-
oxytocin and procalcitonin and their analogues with phenylalanine increases the enzyme binding affinity.
However, this changes the binding mode from one that depends on peptide sequence recognition to
another primarily determined by the phenylalanine moiety, for both the substrates and analogous glyco-
late inhibitors.
Keywords:
Peptidylglycine a-amidating
monooxygenase
Prohormones
Peptide substrates
Glycolate inhibitors
Human cancer
Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
More than half of all the bioactive peptide hormones require
C-terminal amidation for full biological activity, the catalysis of
Potency and selectivity are important features of inhibitors. Po-
tent inhibitors are required to achieve low drug doses, whilst selec-
tivity is important for avoiding generic side effects. Micromolar
glycolate inhibitors of bovine PAM were reported by May and co-
workers.13,14 We reinvestigated compounds of this class for their
inability to form radical intermediates through interaction with
PHM,15 and recently reported their selective inhibition, at nanomo-
lar concentrations, of PAM extracted from small cell lung carcinoma
cells.16 On this basis, glycolate PAM inhibitors were chosen as a fo-
cus of this study. PAM has been shown to recognise the C-terminal
penultimate amino acid of peptidyl substrates.14,17–19 Further,
Tamburini et al.,20 found that prohormones and their C-terminal
pentapeptide mimics show equal binding affinity with PAM, indi-
cating that recognition by the enzyme is based entirely on this re-
gion of the substrates. We decided to explore this further by
measuring the apparent Km values of the N-acetylated amino acid,
and di-, tri- and tetra-peptide analogues 1 and 3a–c (Fig. 1) of the
C-terminus of the oxytocin prohormone (3d), as well as that of pro-
oxytocin (3d) itself, in competitive assays with (R)-Tyr-(S)-Val-Gly,
as substrates of PAM. Since it has also been shown that PAM binds
preferentially to peptides that contain a phenylalanine residue at
the C-terminal penultimate position,19 the study has been extended
to include the analogous phenylalanine derivatives 4a–d. Similarly,
the di-, tri-, tetra- and penta-peptide analogues 7a–d of the C-ter-
minus of the calcitonin prohormone (7e), as well as procalcitonin
(7e) itself, and the analogous phenylalanine derivatives 8a–e have
which is accomplished by peptidylglycine
genase (PAM).1 Being a bifunctional enzyme, PAM consists of two
subunits: peptidylglycine -hydroxylating monooxygenase (PHM)
that initiates formation of a radical at a substrate’s C-terminal gly-
cine -carbon to then give an -hydroxylated glycine derivative;
a-amidating monooxy-
a
a
a
and peptidylamidoglycolate lyase (PAL) which catalyzes cleavage
of this hydroxyglycine intermediate to give the amidated product
with consequent release of glyoxylate (Scheme 1). Apart from their
ubiquitous use in nature, these amidated peptides have been impli-
cated in a number of pathological conditions such as inflammation,2
asthma,3 arthritis4 and cancer.5–12 Calcitonin, for example, is a
C-terminal amidated neuropeptide that has been found to inhibit
apoptosis of prostate cancer cell lines, thus promoting growth of
the cancer.5,6 Oxytocin, another amidated peptide, which normally
induces contractions during labour as well as the milk let-down re-
sponse during breast-feeding in mammalian species, is produced by
a number of cancer cell lines7 and is known to induce metastasis of
cancer masses.8–11 This role of C-terminal amidated peptides in
pathological conditions has led to interest in drug development
based on the production of inhibitors that regulate PAM activ-
ity.13–30
⇑
Corresponding author.
E-mail address: easton@rsc.anu.edu.au (C.J. Easton).
0960-894X/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.004

7016
K. M. Morris et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7015–7018
PAM), and that secreted into the medium by those cells (medium
PAM).16 The DMS53 cell line is available from the American Type
Culture Collection and known to produce both calcitonin and oxy-
tocin.7 The glycine derivatives 1, 3a–c, 4a–c, 7a–d and 8a–d were
either obtained commercially or prepared by acetylation of the
corresponding peptides. The peptidomimetic glycolates 5a–c, 6a–
c, 9a–c and 10a–c were synthesized by treatment of the corre-
sponding, appropriately protected, amino acids and peptides with
either benzyl bromoacetate or tert-butyl bromoacetate, followed
by hydrogenation or hydrolysis to remove the protecting groups.
The glycolate 2 was purchased. Prooxytocin (3d) and procalcitonin
(7e) and the analogous phenylalanine derivatives 4d and 8e, as
well as the corresponding glycolates 5d and 6d, were prepared
using solid-phase peptide synthesis. Enzyme Km,app and IC50 values
were determined in competitive assays using the tripeptide
(R)-Tyr-(S)-Val-Gly as the substrate.16 The results are shown in Ta-
ble 1, with standard deviations from experiments performed at
least in triplicate, and analysed at least in duplicate. Further details
are provided as Supplementary data.
Scheme 1. Biosynthesis of C-terminal amidated peptide hormones catalyzed by
PAM.
been investigated. In addition, the corresponding glycolate pepti-
domimetics 2, 5a–d, 6a–d, 9a–c and 10a–c have been studied.
We15 and others13 have determined that compounds of this class
are competitive inhibitors of PAM. We now find that the potency
of inhibition of PAM by the glycolates 2, 5a–d, 6a–d, 9a–c and
10a–c reflects the prohormone-substrate sequence recognition
apparent from the results with the glycine derivatives 1, 3a–d,
4a–d, 7a–e and 8aÀe.
Comparison of the apparent Km values of the N-acetylated amino
acid, and di-, tri- and tetra-peptides 1 and 3a–c, and of prooxytocin
(3d), with the cellular PAM, shows increased binding affinity with
increased peptide length (Km,app 1 > 3a > 3b > 3c > 3d). The value
for prooxytocin (3d) is more than an order of magnitude less than
that seen even with the acylated tetrapeptide 3c, indicating that
The PAM used in these experiments was cellular enzyme ex-
tracted from DMS53 small cell lung carcinoma cells (cellular
Figure 1. Substrates and inhibitors of peptidylglycine a-amidating monooxygenase used in this study. All amino acid residues have the a-(S)-configuration.

K. M. Morris et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7015–7018
7017
Table 1
Km,app and IC50 valuesa for interactions of prooxytocin (3d) and procalcitonin (7e) and related substrates and inhibitors with PAM
Substrates
Km,app
(
lM)
Inhibitors
IC50
(
lM)
Substrates
Km,app
(l
M)
Inhibitors
IC50 (lM)
Cellular
PAM
Medium
PAM
Cellular
PAM
Medium
PAM
Cellular
PAM
Medium
PAM
Cellular
PAM
Medium
PAM
Oxytocin analogues
1
7500 500
1600
860 80
660 80
9900 2000
1200 90
860 150
550 100
2
1100 170
210 40
1100 200
1800 350
3a
3b
3c
3d
3
5a
5b
5c
5d
4a
4b
4c
4d
6
8
6
5
1
1
0.3
0.8
16
11 0.7
7
27
3
6a
6b
6c
6d
5
3
6
2
0.3
0.6
0.3
0.3
11
9
38 0.4
23
2
0.1
120
76
7
5
1
200
180
71
8
4
7
0.2
2
36
3
190
8
12
4
Calcitonin analogues
7a
7b
7c
7d
7e
980 150
5000 450
3000 600
1000 100
200 25
880 90
4000 770
3000 500
960 180
200 40
9a
9b
9c
600 100
370 50
980 150
500 30
290 40
1100 170
8a
8b
8c
8d
8e
6000 900
6500 780
200 40
10a
10b
10c
1100 200
350 60
560 50
1600 290
370 50
180
40
8
5
5
0.2
0.2
35
18
14
7
3
4
160
4
8
a
Due to the limitations of comparing Km,app and IC50 values, and in interpreting Km,app values as a measure of binding affinity, conclusions drawn in this manuscript are
based on patterns observed independently in the Km,app values of the substrates 1, 3a–d, 4a–d, 7a–e and 8a–e and the IC50 values of the inhibitors 2, 5a–d, 6a–d, 9a–c and
10a–c.
substrate recognition extends more than four amino acid residues
from the C-terminus. These findings are consistent with earlier
observations31 that ‘relatively short model substrates cannot mimic
that full range of enzyme-substrate contacts that exist in the PHM/
PAM active site for longer peptide substrates’. An identical trend of
peptide sequence recognition is observed with compounds 1 and
3a–c and the medium PAM. The peptidomimetic glycolate pro-
oxytocin analogues 2 and 5a–d display this same pattern with the
cellular PAM (IC 2 > 5a > 5b > 5c > 5d), showing that their potency
as PAM inhibitors follows the relationship of amino acid sequence
recognition apparent from the data obtained for the corresponding
substrates 1 and 3a–d. Analogous behaviour is observed with the
glycolates 2 and 5a–d and the medium PAM. This correlation holds
even though, in common with the results of earlier studies and their
interpretation,16 the glycolates 5a–d are more potent inhibitors of
the cellular PAM than of the enzyme secreted into the medium,
and they also show greater binding affinity than the corresponding
substrates 3a–d for the cellular protein. Further, the glycolates
5a–d selectively inhibit the cellular PAM, compared to the enzyme
in the medium, whereas the substrates 1 and 3a–d do not exhibit
this behaviour pattern.
Differences between the trends apparent for the prooxytocin-re-
lated substrates 1, 3a–d and 4a–d, and inhibitors 2, 5a–d and 6a–d,
and the results obtained for the corresponding procalcitonin-re-
lated compounds 7a–e, 8a–e, 9a–c and 10a–c are most likely due
to the impact of the interactions of the N-terminal free amino group
of the latter compounds with PAM. Whereas the N-acetylated ana-
logues 1, 2, 3a–c, 4a–c, 5a–c and 6a–c of prooxytocin (3d) retain an
amide bond at the N-terminus in place of a peptide bond of pro-
oxytocin (3d), in place of a peptide bond of procalcitonin (7e) the
procalcitonin analogues 7a–d, 8a–d, 9a–c and 10a–c instead have
an amino group, that will be protonated under the assay conditions.
This is likely to have the greatest impact on the binding affinities of
the shorter compounds 7a,b, 8a,b, 9a,b and 10a,b. Even so, the
Km,app values of the tetra- and penta-peptides 7c and 7d, and of pro-
calcitonin (7e), with both the cellular and the medium PAM, show
increased binding affinity with increased peptide length (Km,app
7c > 7d > 7e), with the value for procalcitonin (7e) being about
one fifth of that seen with the pentapeptide 7d with PAM from
either source. This shows that recognition of the substrate calcito-
nin (7e) extends more than five amino acid residues from the C-ter-
minus, even further than previously thought.19
The prooxytocin-related substrates 4a–d having phenylalanine
instead of glycine as the C-terminal penultimate amino acid resi-
due do not show the sequence recognition seen with the glycine
derivatives 1 and 3a–d. They all bind to the cellular PAM with
If considered in isolation, the pattern of increased binding affin-
ity with increased peptide length observed with the procalcitonin-
related phenylalanine derivatives 8a–e could be interpreted to be
the consequence of recognition of the lengthening peptide se-
quence. However, in the context of the widely different Km,app val-
ues of the substrates 4a and 8a, and the IC50 values of the inhibitors
6a and 10a, it is clear that this is not the case. The binding affinities
of the free amines 8a and 10a are much less than those of the cor-
responding N-acetylated but otherwise identical compounds 4a
and 6a, showing that the protonated amino groups of the amines
8a and 10a cause their relatively weak binding. Comparison of
the Km,app values shows that there is no recognition of the extended
peptide sequence with the phenylalanine derivatives 4a and 8c–e.
Instead, the close similarity between the data for the procalcitonin-
related species 8c–e and the prooxytocin analogues 4a–d appears
to be a consequence of all these compounds binding to PAM in a
similar way, determined in each case mainly by recognition of
the phenylalanine component.
Km,app values between 5–8 lM, and show similar but slightly re-
duced binding affinities for the medium PAM. The high binding
affinities of the phenylalanine derivatives 4a–d are consistent with
the earlier report of the preferential binding of such species to
PAM.19 However, the contrast between the relative lack of impact
of the N-terminal portion of these compounds 4a–d on the Km,app
values and the sequence recognition displayed by the glycine
derivatives 1 and 3a–d indicates that the two groups of compounds
adopt different binding modes, dominated by the phenylalanine
portion and peptide sequence, respectively.
The phenylalanylglycolates 6a–d are all low micromolar inhib-
itors of PAM with binding affinities very similar to those of the
analogous substrates 4a–d. Combined with the lack of any effect
of structural differences between the glycolates 6a–d on the IC50
values, this indicates that, in common with the substrates 4a–d,
the strength of their association with the enzyme is primarily
determined by the interactions with the phenylalanine portion.
The inhibitors 6a–d do not exhibit peptide sequence recognition
but, nevertheless, their affinity for the enzyme closely parallels
that of the corresponding substrates 4a–d.
Given the complexity associated with the N-terminal amino
groups of the peptidylglycolates 9a–c and 10a–c, it is not feasible
to draw any general conclusions from the potency of these com-
pounds as PAM inhibitors. Nevertheless, overall the results of this
study show that peptide sequence recognition is an important as-
pect of the interactions of the prohormone substrates prooxytocin

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We gratefully acknowledge the support received for this work
from the Australian Research Council, and the assistance of
Dr. M. J. Gresser and Mr. J. Kerr with glycolate syntheses and
Dr. H.-K. Kim with culturing DMS53 cells.
Supplementary data
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