Importance of the aromatic residue at position 6 of [Nle10]neurokinin A(4-10) for binding to the NK-2 receptor and receptor activation

Article abstract of DOI:10.1021/jm9807151

Steric and electrostatic requirements at position 6 of [Nle¹⁰]NKA(4- 10), a full agonist of NK-2 receptors, for molecular recognition by the receptor were studied. Two series of peptide analogues, (a) p-substituted analogues, [p-X-Phe⁶

Full text of DOI:10.1021/jm9807151

3004
J . Med. Chem. 1999, 42, 3004-3007
Im p or ta n ce of th e Ar om a tic Resid u e a t P osition 6 of [Nle10]Neu r ok in in A(4-10)
for Bin d in g to th e NK-2 Recep tor a n d Recep tor Activa tion
Dmitry S. Gembitsky,†,§ Mark Murnin,†,§ Ferenc L. O¨ tvo¨s,† J ames Allen,‡ Richard F. Murphy,† and
Sa´ndor Lovas*,†
Department of Biomedical Sciences, Creighton University School of Medicine, 2500 California Plaza,
Omaha, Nebraska 68178-0405, and Biomedical Sciences Research Centre, Coleraine BT52 1SA, Ireland
Received March 17, 1999
Steric and electrostatic requirements at position 6 of [Nle10]NKA(4-10), a full agonist of NK-2
receptors, for molecular recognition by the receptor were studied. Two series of peptide
analogues, (a) p-substituted analogues, [p-X-Phe6,Nle10]NKA(4-10), where X ) F, Cl, Br, I,
NH2, NO2, and (b) [D-Phe6,Nle10]NKA(4-10), [Trp6,Nle10]NKA(4-10), and [Chex-Ala6,Nle10]-
NKA(4-10), were synthesized, and their biological activity was examined. Competition binding
experiments with [3H]NKA were performed using cloned human NK-2 receptors expressed in
CHO cells. Antagonistic and agonistic properties of the analogues were studied using an in
vitro functional assay with hamster tracheal rings. The rank order of potency of agonists was
[Nle10]NKA(4-10) ≈ [p-F-Phe6,Nle10]NKA(4-10) > [p-NH2-Phe6,Nle10]NKA(4-10) > [p-Cl-Phe6,-
Nle10]NKA(4-10) > [p-NO2-Phe6,Nle10]NKA(4-10) > [Trp6,Nle10]NKA(4-10). Size and planarity
of the aromatic side chain were crucially important for the biological activity, whereas electron-
donating and electron-withdrawing properties of the para-substituent were less important.
The results favor the hypothesis that weakly polar π-π interactions exist between the aromatic
group and the receptor.
In tr od u ction
Molecular dynamics simulations of the structures of
both NKA and 1 in water, using a simulated annealing
method, demonstrate that the aromatic ring of Phe6 is
located on the surface of the folded peptide backbone,
forming a hydrophobic patch.9 Weakly polar interac-
tions, aromatic-aromatic (π-π) and amino-aromatic
(N-π), make a significant contribution to the stability
of protein-ligand complexes.10,11 The peptide-binding
site of the human NK-2 receptor includes hydrophobic
residues on the extracellular third of helices 3, 5, 6, and
7, close to the lipid bilayer interface.12,13 Consequently,
the Phe6 aromatic side chain of 1 could be involved in
weakly polar interactions with a putative hydrophobic
pocket in the NK-2 receptor.
NKA belongs to the tachykinin family of oligopeptides,
including SP and NKB, which have 10-12 amino acid
residues and share the common C-terminal sequence
Phe-Xaa-Gly-Leu-MetNH2.1 They are widely distributed
in the central nervous system as well as in peripheral
organs and tissues. The term “tachykinins” reflects their
contractile effect on smooth muscles.2,3 SP, NKA, and
NKB interact with at least three receptor subtypes. The
rank order of potency for tachykinin receptors is SP >
NKA > NKB for the NK-1 receptor, NKA > NKB > SP
for the NK-2 receptor, and NKB > NKA > SP for the
NK-3 receptor.4 The receptors have been cloned and are
members of the superfamily of G-protein-coupled recep-
tors with seven putative membrane-spanning seg-
ments.5
The C-terminal heptapeptide, NKA(4-10), contains
the minimal sequence required for NK-2 receptor acti-
vation.6 Met10fNle replacement enhances selectivity of
NKA(4-10) for the NK-2 receptor but with some reduc-
tion of potency.7,8 The replacement also prevents oxida-
tion of Met10, and consequently, [Nle10]NKA(4-10) (1)
is more stable than NKA(4-10) and is the parent
compound for this study.
In the present study, the importance of the aromatic
residue is further investigated by examining steric and
electrostatic requirements at position 6 of 1 both for
binding to the NK-2 receptor and for receptor activation.
For this purpose, the following peptides were synthe-
sized: (a) para-substituted analogues, [p-X-Phe6,Nle10]-
NKA(4-10), where X ) F, Cl, Br, I, NH2, NO2, and (b)
analogues in which the Phe6 was replaced with a
nonaromatic structure (Chex-Ala) and with different
aromatic amino acids, D-Phe and Trp (Table 1). The
substituents can be classified as either electron donating
(NH2) or electron withdrawing (NO2 and halogens). The
substituents should not only increase the van der Waals
volumes of the Phe6 derivatives but also perturb the
electrostatic field around the aromatic ring. Replace-
ments by D-amino acids introduce a conformational
change in the peptide backbone and are therefore
suitable for examination of the contribution of individual
amino acids to the secondary structure of peptides
required for activity.14-16
A Phe residue at position 6 is common to all decapep-
tide tachykinins and is found at corresponding positions
7 in peptides containing 11 amino acid residues and 8
in kassinin. The Phe residue is crucially important since
its replacement with either L-Ala or 1-amino-1-cyclo-
hexyl carboxylic acid eliminates binding of NKA to all
three receptor subtypes.6,8
† Creighton University School of Medicine.
‡ Biomedical Sciences Research Centre.
§ Made an equal contribution.
10.1021/jm9807151 CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/25/1999

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 3005
Ta ble 1. Analysis of [Nle10]NKA(4-10) Analoguesa
amino acid composition
peptide
Ser
Asp
Xb
Val
Gly
Leu
Nle
MS results
2, [p-F-Phe6,Nle10]NKA(4-10)
3, [p-Cl-Phe6,Nle10]NKA(4-10)
4, [p-Br-Phe6,Nle10]NKA(4-10)
5, [p-I-Phe6,Nle10]NKA(4-10)
6, [p-NH2-Phe6,Nle10]NKA(4-10)
7, [p-NO2-Phe6,Nle10]NKA(4-10)
8, [Chex-Ala6,Nle10]NKA(4-10)
9, [Trp6,Nle10]NKA(4-10)
0.82 (1)
0.88 (1)
0.94 (1)
0.83 (1)
0.74 (1)
0.71 (1)
0.75 (1)
0.85 (1)
0.73 (1)
1.02 (1)
1.01 (1)
0.99 (1)
0.99 (1)
1.03 (1)
0.98 (1)
1.01 (1)
0.96 (1)
1.05 (1)
0.95 (1)
0.92 (1)
ndc (1)
nd (1)
0.79 (1)
nd (1)
1.01 (1)
- (1)
1.01 (1)
0.99 (1)
0.95 (1)
1.04 (1)
1.05 (1)
1.12 (1)
1.08 (1)
1.09 (1)
0.99 (1)
1.02 (1)
1.10 (1)
0.97 (1)
0.95 (1)
0.99 (1)
1.09 (1)
1.05 (1)
1.05 (1)
1.00 (1)
1.04 (1)
1.10 (1)
1.06 (1)
0.99 (1)
0.98 (1)
1.12 (1)
1.11 (1)
1.05 (1)
1.06 (1)
1.12 (1)
1.02 (1)
0.97 (1)
1.09 (1)
0.97 (1)
1.10 (1)
1.08 (1)
1.05 (1)
1.08 (1)
1.02 (1)
767.0 (766.0)
783.0 (783.5)
827.3 (827.0)
875.0 (875.0)
764.0 (764.0)
794.0 (793.0)
754.5 (754.5)
788.0 (788.0)
749.0 (749.0)
10, [D-Phe6,Nle10]NKA(4-10)
a
b
Numbers in parentheses are the expected number of residues or the calculated masses. Amino acid replacement at position 6. c nd,
not determined.
Ta ble 2. Competition Binding Potencies of Natural
Ta ble 3. Absolute Electronegativity (ø) of the Para-Substituent
in the Aromatic Ring of Phe6 in [Nle10]NKA(4-10) Analogues
and Volume Difference between the Parent Residue and Its
Replacements
Neurokinins, [Nle10]NKA(4-10), and [Nle10]NKA(4-10)
Analogues
peptide
log IC50 ( SEM (n ) 3)
Ki (nM)
residue
øa
∆V (Å3)
NKA
1
NKB
2
SP
6
3
7
9
-8.77 ( 0.17
-7.51 ( 0.19
-6.99 ( 0.24
-6.97 ( 0.07
-6.61 ( 0.20
-6.43 ( 0.14
-5.46 ( 0.05
-
0.71
12.7
42.2
44.2
102
p-F-Phe
p-Cl-Phe
p-Br-Phe
p-I-Phe
p-NH2-Phe
p-NO2-Phe
Chex-Ala
Trp
10.41
8.31
7.6
6.76
6.07
6.2
1.6
11.7
16.8
28
11.6
16.9
38.5
35.8
156
1422
>2000
>2000
>2000
-
-
-
10
-
a
Absolute electronegativity values were determined by Pear-
son.22
Human recombinant NK-2 receptors are used to
screen the receptor binding activity of the analogues.
The effects of para-substituents and replacements of
Phe6 on receptor activation are assessed by measuring
contraction of hamster tracheal rings in vitro. The
biological data could then be correlated with the topo-
graphic features of residues at position 6 of [Nle10]NKA-
(4-10) analogues using molecular modeling.
that (116.99 ( 3.01) of 1. The KA values were 70.3 (
15.7 and 126 ( 42.78 nM for 2 and 1, respectively. The
efficacy of 6, 95.77 ( 2.73, was significantly lower than
that of 1, and the KA was 663.1 ( 27.54 nM. The
efficacy, 97.78 ( 2.73, of 3 was also significantly lower
than that of 1. The KA was 2250 ( 71 nM, corresponding
with a reduction in potency. Similarly, 7 caused smaller
maximal contraction (efficacy 84.6 ( 4.64) than did 1.
The analogue still displayed agonism; the KA was 2490
( 77 nM. 4 and 5 displayed neither agonism nor
antagonism.
Resu lts
[3H]NKA bound to membranes prepared from CHO
cells expressing the NK-2 receptor in a saturable and
displaceable manner with Kd of 0.71 nM (Table 2). The
Hill coefficient was close to 1.0 in all experiments. The
cloned receptors showed the correct rank order of
potency for natural neurokinins (Table 2). 1 inhibited
the binding of [3H]NKA to the NK-2 receptor with an
inhibitory constant (Ki) of 12.7 nM.
The rank order of potency of the synthetic analogues,
parent peptide, and natural neurokinins, in binding to
the NK-2 receptor, is shown in Table 2. In general,
substitutions of hydrogen in the para-position of the
aromatic ring of Phe6 led to a reduction of binding, but
p-F substitution resulted in only a moderate decrease,
with Ki still in the nanomolar range. The next most
potent analogue was 6 with Ki of 0.15 µM. Substitution
with Cl gave rise to an analogue with Ki of 1.42 µM.
Introduction of a nitro group was even more detrimental
to binding. Analogues with the p-Br or p-I substitution,
at concentrations up to 0.1 mM, did not compete with
[3H]NKA for binding to the NK-2 receptors.
Replacement of Phe6 with either D-Phe or Trp resulted
in a pronounced decrease in the binding affinity (Table
2). 8 did not inhibit [3H]NKA binding to the NK-2
receptors at concentrations up to 0.1 mM. 9 had some
agonism, but the efficacy (56.64 ( 6.24) was reduced to
nearly one-half that of the parent peptide. The KA of
this partial agonist was 941 ( 30 nM. 10 had no
agonism but had low antagonist activity (data not
shown). Replacement with Chex-Ala led to an analogue
devoid of both agonism and antagonism.
Calculated volume difference (∆V) between the Phe6
residue and its replacements is shown in Table 3.
Discu ssion
The substituent at the para-position of the aromatic
ring of Phe6 in 1 appears to be crucially important for
interactions with the NK-2 receptor. It was assumed
that para-substitution would not change the overall
backbone conformation. This hypothesis was proven
when ∆V and ø of substituents were correlated with
biological activity of analogues. The binding affinity and
agonism of analogues with p-halogen substitution seem
to correlate inversely with the van der Waals volume
of the halogens. The similarity of receptor activity of 2
with that of 1 (Table 2) is probably a reflection of the
Introduction of F in the para-position of the aromatic
ring of Phe6 had no significant influence on the ability
of the peptide to stimulate contractile activity of tracheal
rings. Moreover, curves, reflecting agonistic activity of
1 and of 2, were virtually superimposable. The efficacy
(114.38 ( 4.81) of 2 was not significantly different from

3006 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Brief Articles
small size of F (Table 3). The absence of receptor activity
following substitution with Br, but not with smaller
halogens, indicates that the substituent must be smaller
than Br to allow the “hydrophobic patch” of the peptide9
to fit a putative hydrophobic pocket in the receptor.
Con clu sion
The results show that the size of the para-substituent
in the aromatic ring at position 6 of [Nle10]NKA(4-10)
analogues is more important than electron-donating or
-withdrawing properties. The planarity of the aromatic
ring in Phe derivatives and Trp appears to be conducive
to peptide activity, indicating a complementary planar
pocket in the NK-2 receptor. The results also favor the
hypothesis that the Phe6 residue binds to the receptor
through weakly polar π-π interactions.
Significant differences in the binding affinity and
agonism between 2 and 3 indicate that a 10-fold
increase approximately in the size of the substituted
group (Table 3) can have a detrimental effect on
biological activity. It might have been expected that the
higher electronegativity of F compared with Cl (Table
3) would have an influence on the peptide binding and
agonism. The observation, however, that 7 is less potent
than 6 indicates that the electronegativity of the sub-
stituent is unlikely to be crucially important for binding
and receptor activation (Tables 2 and 3). Escher and co-
workers17 similarly demonstrated that [p-NO2-Phe7,-
Nle11]SP was less potent than [p-NH2-Phe7,Nle11]SP,
confirming the primary importance of the size of the
para-substituent for activation of NK-1 receptors in
guinea pig ileum.
p-NH2-Phe6 has a ∆V close to that of p-Cl-Phe6, and
the ∆V of p-NO2-Phe6 is close to that of p-Br-Phe6 (Table
3). 6, however, is more potent than 3, and 7 is active
despite the ∆V of p-NO2-Phe being as large as that of
p-Br-Phe. Evidently, NH2 and NO2 groups have more
planar structures than Cl and Br, respectively, and
assuming planarity of the binding pocket in the receptor
(see below) enhanced affinity of peptides with NH2 and
NO2 substitution might be expected.
Exp er im en ta l Section
Ma ter ia ls. Boc-Asp(OBzl)-OH, Boc-Leu-OH, Boc-Met-OH,
Boc-Nle-OH, Boc-Phe-OH, Boc-D-Phe-OH, Boc-Ser(Bzl)-OH,
Boc-Trp-OH, Boc-Val-OH, Boc-p-Bzl-NH2-Phe-OH, Boc-p-F-
Phe-OH, Boc-p-Cl-Phe-OH, Boc-p-I-Phe-OH, Boc-p-NO2-Phe-
OH, and p-methylbenzhydrylamine resin were purchased from
Bachem California Inc. (Torrance, CA). Boc-p-Br-Phe-OH and
Boc-Ala-OH were from Aldrich Chemical Co. (St. Louis, MO).
TFMSA was from Applied Biosystems/Perkin-Elmer (Foster
City, CA). NKA, NKB, and SP were purchased from Peninsula
Laboratories Inc. (Belmont, CA). [4,5-3H-Leu9]Neurokinin A
([3H]NKA; specific activity 170 Ci/mmol) was purchased from
Cambridge Research Biochemicals (London, U.K.). Human
recombinant NK-2 receptors expressed in CHO cell mem-
branes were obtained from Biosignal Inc. (Montreal, Canada).
Tris-HCl, Hepes (enzyme grade), DIC, TFA, DMF (peptide
synthesis grade), DCM, acetonitrile, and triethylamine were
purchased from Fisher Scientific (St. Louis, MO), and phe-
noxybenzamine (PBZ) was purchased from SmithKline Bee-
cham Pharmaceuticals (Brentford, U.K.). Other chemicals
were from Sigma Chemical Co. (St. Louis, MO).
P ep tid e Syn th esis. Peptides were synthesized on a 430A
Applied Biosystems peptide synthesizer (Perkin-Elmer, Foster
City, CA). Syntheses were performed on a 0.5-mmol scale on
p-methylbenzhydrylamine resin. The reactive side chains in
Asp and Ser were protected with a â-benzyl group. The amino
group of p-NH2-Phe was protected with a benzyloxycarbonyl
group. N-R-Boc-protected amino acid derivatives were coupled
as their 1-hydroxybenzotriazole active esters. Removal of N-R-
Boc groups was performed in 50% TFA/DCM for 2 min and
then 30% TFA in DCM for 17 min. DIEA was used to
neutralize the resin. The resin was washed with DCM and
NMP. Side chain deprotection and cleavage of peptides from
the resin were achieved in a single-step reaction by stirring
the peptide-resin in a solution of trifluoromethanesulfonic acid/
thioanisole/ethanedithiol/trifluoroacetic acid (2:2:1:20, v/v/v/
v) for 30 min at -10 °C followed by 90 min at room
temperature.
The total loss of peptide activity in the functional
assay, when the aromatic side chain at position 6 was
replaced with a cyclohexyl group, favors the hypothesis
that weakly polar π-π interactions exist between the
Phe6 residue and the receptor. Huang and co-workers13
identified 15 NK-2 receptor residues which are involved
in the binding of peptide ligands; 10 are aromatic. The
attractive orientation in the π-π interaction is either
edge-to-face or offset geometry, whereas face-to-face
geometry leads to a repulsive orientation.11 Thus, the
π-π interactions should be facilitated by planarity as
well as by rigidity of the binding pocket of the receptor.
This concept is supported by our results.
HP LC Exp er im en ts. For analytical HPLC, Vydac 218TP54
columns (C18, 5-mm particle size, 250 mm × 4.6 mm; The
Separations Group, Hesperia, CA) were irrigated for 35 min
at 1 mL/min with a 3-60% linear gradient of 0.09% TFA in
CH3CN added to 0.1% TFA in H2O. A220 of effluent fractions
was recorded. For preparative HPLC, the above solvents were
used at a flow rate of 4.5 mL/min, on a Vydac 218TPB1015
coulmn (C18, 15-mm particle size, 500 mm × 10 mm) packed
in-house.
Ma ss Sp ectr om etr y. FAB-MS analyses were carried out
by the Midwest Center for Mass Spectrometry (Department
of Chemistry, University of Nebraska-Lincoln).
Am in o Acid An a lysis. The hydrolysates of purified pep-
tides (6 N HCl, 110 °C, 24 h) were analyzed using a Waters
AccQTag amino acid analysis system.
Liga n d Bin d in g Assa y. Binding assays were performed
with a cytoplasmic membrane fraction of CHO cells which had
been transfected with human NK-2 receptor cDNA and
selected for stable expression of NK-2 receptors.18 The incuba-
tion media consisted of 20 mM Hepes buffer, pH 7.4, supple-
mented with 2 mM MnCl2, 0.01% bovine serum albumin
(protease-free), 4 µg/mL leupeptin, 4 µg/mL chymostatin, 0.1
mM bestatin, and 10 µM phosphoramidon. For competition
The retention of some, although low, peptide agonism
when Phe6 is replaced by Trp may be due to the planar
nature of the Trp side chain. By contrast, the cyclohexyl
ring in 8 adopts a chair conformation in energy mini-
mization. This structure may not be fully complemen-
tary with the hydrophobic pocket and might only
partially occupy the receptor-essential volume. Simi-
larly, replacement of the Phe6 with p-Br-Phe is not
conducive to a planar side group conformation, since it
causes total loss of peptide binding even though the ∆V
of 4 is much lower than the ∆V of 9.
The reduction in binding affinity and loss of all
agonism but acquisition of some antagonism, when
L-Phe6 is replaced by D-Phe, may be a reflection of the
totally different conformation of 10 from that of 1. The
D-Phe might interact with groups in the receptor other
than those which could interact with an L-amino acid
at position 6. The D-Phe appears to confer antagonism
on the peptide with concomitant loss of agonism.

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 3007
(7) Regoli, D.; Nguyen, Q. T.; J akie, D. Neurokinin receptor subtypes
characterized by biological assays. Life Sci. 1994, 54, 2035-2047.
(8) Rovero, S.; Pestellini, V.; Rhaleb, N. E.; Dion, S.; Rouissi, N.;
Tousignant, C.; Telemaque, S.; Drapeau, G.; Regoli, D. Structure-
activity studies of neurokinin A. Neuropeptides 1989, 13, 263-
270.
(9) Lovas, S.; Smith, D. D.; Murphy, R. F. Simulated annealing
studies of some neurokinin A analogues. In Peptides 1992;
Schneider, C. H., Eberle, A. N., Eds.; ESCOM: Leiden, 1993;
pp 515-516.
(10) Burley, S. K.; Petsko, G. A. Weakly polar interactions in proteins.
Adv. Protein Chem. 1988, 39, 125-189.
(11) Hunter, C. A.; Sanders, J . K. M. The nature of π-π interactions.
J . Am. Chem. Soc. 1990, 112, 5525-5534.
(12) Bhogal, N.; Donnelly, D.; Findlay, J . B. The ligand binding site
of the neurokinin 2 receptor. J . Biol. Chem. 1994, 269, 27269-
27274.
(13) Huang, R. C.; Vicario, P. P.; Strader, C. D.; Fong, T. M.
Identification of residues involved in ligand binding to the
neurokinin-2 receptor. Biochemistry 1995, 34, 10048-10055.
(14) Moore, G. J .; Smith, J . R.; Baylis, B. W.; Matsoukas, J . M. Design
and pharmacology of peptide mimetics. Adv. Pharmacol. 1995,
33, 91-140.
(15) Paterson, S. J .; Kosterlitz, H. W.; Varvek, R. J .; Stewart, J . M.
Effect of D-amino acid substitution in dynorphin A(1-9) on
binding at the mu-, delta- and kappa-sites. Neuropeptides 1984,
5, 177-180.
(16) Rinscheid, R. K.; Ardati, A.; Monsma, F. J .; Civelli, O. Structure-
activity relationship studies on the novel neuropeptide orphanin
FQ. J . Biol. Chem. 1996, 271, 14163-14168.
studies, the membrane suspension (protein concentration 15.6
µg/mL) was incubated for 60 min at 27 °C with 1 nM [3H]NKA
and various concentrations of the peptides to be tested.
Nonspecific binding was determined in the presence of a 1000-
fold molar excess of unlabeled NKA. The reaction was termi-
nated by a rapid filtration through Whatman GF/B glass filters
presoaked in 0.3% poly(ethylenimine), using a Brandel cell
harvester (Biomedical R&D Laboratories Inc., Gaithersburg,
MD). Then the filters were washed three times with ice-cold
50 mM Tris-HCl buffer, pH 7.4. The bound radioactivity was
measured with a 1209 Rackbeta liquid scintillation counter
(Wallac Inc., Gaithersburg, MD). Data were analyzed using
GraphPad Prizm software (GraphPad Software Inc., San
Diego, CA).
Assa y w ith Isola ted Ha m ster Tr a ch ea l Rin g. Tracheae
were obtained from male Syrian hamsters (90-120 g) which
had been sacrificed by CO2 intoxication. The tracheal rings
(diameter 3-4 mm) were rapidly prepared and suspended at
37 °C in an organ bath containing 4 mL of Krebs solution19
supplemented with 10 µM phosphoramidon and 1 µM in-
domethacin. The contractile activity was recorded by isometric
transducers after a 60-min equilibration period which followed
two or more reproducible responses to 1 mM carbachol.
Cumulative responses to increasing concentrations of peptides
were registered. Agonist dissociation constants (KA) and rela-
tive efficacies were determined by the method of partial
irreversible blockade with the alkylating agent PBZ as de-
scribed elsewhere.20,21
(17) Escher, E.; Couture, R.; Champagne, G.; Mizhari, J .; Regoli, D.
Synthesis and biological activities of photoaffinity labeling
analogues of substance P. J . Med. Chem., 1982, 25, 470-475.
(18) Gembitsky, D. S.; Lovas, S.; Murphy, R. F. Development of a
high throughput functional assay for structure-activity studies
of neurokinin A analogs. J . Biomol. Screening 1998, 3, 183-
188.
Volu m e Ca lcu la tion . The differences in the van der Waals
volumes (∆V) of the parent residue and its replacements were
determined using the SYBYL molecular modeling package
(Tripos Inc., St. Louis, MO).
Ack n ow led gm en t. This work was supported by the
Cancer and Smoking Related Diseases Research Pro-
gram (LB595) of the State of Nebraska and the Car-
penter Endowed Chair in Biochemistry, Creighton
University. We are grateful to Dr. Donald R. Babin for
performing amino acid analysis. Mass spectral deter-
minations were made at the Midwest Center for Mass
Spectrometry, University of Nebraska-Lincoln.
(19) Mizrahi, J .; Dion, S.; D′Orleans-Fuste, P.; Escher, E.; Drapeau,
G.; Regoli, D. Tachykinin receptors in smooth muscles: a study
with agonists (substance P, neurokinin A) and antagonists. Eur.
J . Pharmacol., 1985, 118, 25-36.
(20) Forchgott, R. F.; Bursztyn, P. Comparison of dissociation
constants and of relative efficacies of selected agonists acting
on parasympathetic receptors. Ann. N. Y. Acad. Sci. 1967, 114,
882-899.
(21) Patacchini, R.; Astolfi, M.; Quartara, L.; Rovero, P.; Giachetti,
A.; Maggi, C. A. Further evidence for the existence of NK2
tachykinin receptor subtypes. Br. J . Pharmacol. 1991, 104, 91-
96.
(22) Pearson, R. G. Absolute electronegativity and hardness: ap-
plication to organic chemistry. J . Org. Chem. 1989, 54, 1423-
1430.
(23) Abbreviations used for amino acids and peptides follow the
recommendations of the IUPAC-IUB Commission of Biochemi-
cal Nomenclature (Eur. J . Biochem. 1984, 138, 9-37). Other
abbreviations: Boc, tert-butyloxycarbonyl; Chex, cyclohexyl;
CHO, Chinese hamster ovary; DCM, dichloromethane; DIC,
diisopropylcarbodiimide; DMF, N,N-dimethylformamide; [3H]-
NKA, [4,5-3H-Leu9]neurokinin A; NKA, neurokinin A; NKB,
neurokinin B; NMP, N-methylpyrolidone; PBZ, phenoxyben-
zamine; SP, substance P; TFA, trifluoroacetic acid; TFMSA,
trifluoromethanesulfonic acid.
Refer en ces
(1) Erspamer, V. The tachykinin peptide family. Trends Neurosci.
1981, 4, 269-297.
(2) Erspamer, V.; Melchiorri, P. Active polypeptides of the amphib-
ian skin and their synthetic analogues. Pure Appl. Chem. 1973,
35, 463-494.
(3) Osakada, F.; Kubo, K.; Goto, K.; Kanazava, I.; Mukekata, E. The
contractile activities of neurokinin A, B and related peptides on
smooth muscles. Eur. J . Pharmacol. 1986, 120, 201-208.
(4) Maggi, C. A. The mammalian tachykinin receptors. Gen. Phar-
macol. 1995, 26, 911-944.
(5) Nakanishi, S. Mammalian tachykinin receptors. Annu. Rev.
Neurosci. 1991, 14, 123-136.
(6) Regoli, D.; Rhaleb, N. E.; Dion, S.; Tousignant, C.; Rouissi, N.;
J ukis, D.; Drapeau, G. Neurokinin A: a pharmacological study.
Pharmacol. Res. 1990, 22, 1-14.
J M9807151