Structural and biological effects of a b2- or b3-amino acid insertionin a peptide Application to molecular recognition of substance P by the neurokinin-1 receptor Sandrine Sagan, Thierry
Trang 1Structural and biological effects of a b2- or b3-amino acid insertion
in a peptide
Application to molecular recognition of substance P by the neurokinin-1 receptor
Sandrine Sagan, Thierry Milcent, Rachel Ponsinet, Odile Convert, Olivier Tasseau, Ge´rard Chassaing, Solange Lavielle and Olivier Lequin
UMR 7613 CNRS-Paris 6, Universite´ Pierre et Marie Curie, Paris, France
Molecular mechanics calculations on conformers of
Ac-HGly-NHMe, Ac-b2-HAla-NHMe and Ac-b3
-HAla-NHMe indicate that low-energy conformations of the
b-amino acids backbone, corresponding to gauche rotamers
around the Ca–Cb bond, may overlap canonical backbone
conformers observed for a-amino acids Therefore,
Sub-stance P (SP) was used as a model peptide to analyse the
structural and biological consequences of the substitution of
Phe7 and Phe8 by (R)-b2-HPhe and of Gly9 by HGly (R)-b2
-HAla or (S)-b3-HAla [(R)-b2-HAla9]SP has
pharmacolo-gical potency similar to that of SP while [HGly9]SP and
[(S)-b3-HAla9]SP show a 30- to 50-fold decrease in biological
activities The three analogues modified at position 9 are
more resistant to degradation by angiotensin converting
enzyme than SP and [Ala9]SP NMR analysis of these SP
analogues suggest that a b-amino acid insertion in position 9
does not affect the overall backbone conformation Alto-gether these data suggest that [HGly9]SP, [(S)-b3-HAla9]SP and [(R)-b2-HAla9]SP could adopt backbone conforma-tions similar to that of SP, [Ala9]SP and [Pro9]SP In con-trast, incorporation of b2-HPhe in position 7 and 8 of SP led
to peptides that are almost devoid of biological activity Thus, a b-amino acid could replace an a-amino acid within the sequence of a bioactive peptide provided that the addi-tional methylene group does not cause steric hindrance and does not confine orientations of the side chain to regions of space different from those permitted in the a-amino acid Keywords: b2- and b3-amino acid; secondary structure; molecular mechanics calculations; substance P; neurokinin-1 receptor
Bioactive a-peptides often present conformational
equili-briums in solution but probably adopt one structure (or
family of related structures) when bound to their receptor
[1] A plethora of studies has been conducted on chemical
modifications of a-amino acids to stabilize this so-called
bioactive conformation that may be present as a minor
conformer in solution, and/or to design peptidomimetics
[2–8] The corresponding b-amino acids (Fig 1), should be
better building blocks to design peptidomimetics as
b-peptides are more resistant to degradation by mammalian
enzymes [9] However, each b-amino acid insertion in a
peptide sequence introduces additional degrees of confor-mational flexibility with the rotation around the Ca–Cb bond [10] Previous quantum mechanics calculations on protected b-dipeptides (mimics of tetra-b-peptides) have permitted the identification of many low-energy conforma-tions, namely six- and eight-membered ring hydrogen-bonded structures (C6, C8), extended structures (Ex) and helical structures (He) [11] The C6structure was found to be the most stable conformation by ab initio calculation [12] The Ex and He conformations observed for b-sheet, H14 and H12 helices (14- and 12-membered ring hydrogen-bonded structures) were less stable in the gas phase but gain stabilization in polar solvents [13] All these structures have also been found experimentally in b-peptides either in solution and/or in the solid state [14–17] An additional helix corresponding to the formation of successive , 10-, 12-hydrogen-bonded structures (C12/C10/C12) has also been detected [18]
The wide use of b-amino acids is prevented by the small number of enantiomerically pure b2-amino acids commer-cially available and the cost of synthesizing b3-amino acids Therefore, the design of heterooligomers made of both a- and b-amino acids could overcome this limitation, assuming that these chimeric a, b-peptides may keep the molecular recognition properties of a-peptides and the biological stability of b-peptides Only a few data have been reported in the literature on the structural properties of these heterooligomers However, it has been shown that the c-turn or C structure found with a-amino peptides can be
Correspondence to: O Lequin, UMR 7613 Paris 6-CNRS,
Laboratoire Structure et Fonction de Mole´cules Bioactives,
Universite´ Pierre et Marie Curie, Paris, France.
Fax: + 33 1 44 27 71 50, Tel.: + 33 1 44 27 26 78,
E-mail: lequin@ccr.jussieu.fr
Abbreviations: NK-1, neurokinin-1; SP, substance P
(H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH 2 ); NKA, neurokinin A;
HGly, homoglycine; b2-HAla, b2-homoalanine; b3-HAla, b3
-homo-alanine; b 2 -HPhe, b 2 -homophenylalanine; Aib (aMeAla),
a-amino-isobutyric acid; Sar, sarcosine (N-MeGly); ACE, angiotensin
converting enzyme (E.C 3.4.15.1); PtdIns, phosphatidyl inositol;
PLC, phospholipase C; CHO, Chinese hamster ovary; CSD, chemical
shift deviation; Ex, extended structures; He, helical structures.
Enzyme: angiotensin converting enzyme (EC 3.4.15.1).
(Received 3 October 2002, revised 20 December 2002,
accepted 9 January 2003)
Trang 2stabilized as a C8 conformer in cyclic tetrapeptides and
pentapeptides containing one b-amino acid [19], whereas the
introduction of two adjacent b-amino acids induced a
10-membered hydrogen-bonded turn (C10) analogous to the
b-turn made of a-amino acids [20] This propensity of
b-amino acids to mimic canonical structures observed with
a-peptides (oligomers of a-amino acids) has recently been
applied to the syntheses of linear tetrapeptides of
somato-statin [20] and of cyclic tetrapeptide and pentapeptide
analogues of the RGD sequence [19] With the cyclic
compounds it is uncertain whether the C8 conformation
observed with a single b-amino acid is enforced by the cyclic
constraints The C10 conformation, initially identified in
cyclic b-peptides containing adjacent b2-, b3-amino acids,
has also been identified in solution with a linear
b-tetra-peptide analogue of somatostatin [20] These few examples
suggested that there might be spatial overlaps between some
three-dimensional structures of a-amino acids and those of
b-amino acids and posed the following questions Which
structures of b-amino acids overlap the canonical structures
of a-amino acids? Subsequently, is it possible to substitute
one a-amino acid in the sequence of a linear peptide for one
b-amino acid without drastically affecting the recognition
properties of the resulting peptide? To try to answer these
questions we chose SP (RPKPQQFFGLM-NH2) and the
glycine residue in position 9 of SP that has been extensively
analysed in structure–activity relationship studies In terms
of peptide affinity for the NK-1 receptor and biological
activity, this achiral amino acid can be favourably replaced
by sarcosine or proline, whereas a b-II¢ turn constraint
around residues 9 and 10 confers antagonist properties
[22,23] Therefore, this position associated with a plausible
conformational flexibility is considered as a switch point
between agonist and antagonist structures [21–23]
By molecular mechanics calculations the conformers of
Ac-HGly-NHMe, Ac-b2-HAla-NHMe and Ac-b3
-HAla-NHMe have been generated and compared to the
canonical structures of the corresponding a-amino acid
Ac-Gly-NHMe The corresponding SP analogues
substi-tuted in position 9 by these b-amino acids have been
synthesized and their conformational preferences analysed
by NMR spectroscopy These substituted SP analogues
were tested for their resistance to enzymatic degradation,
their affinity for the human NK-1 receptor and their
potency to stimulate adenylate cyclase and
phospho-lipase C (PLC) in CHO cells transfected with the human
NK-1 receptor [24,25] Modelling and superimposition of
the conformers of the different b- and a-amino acids
inserted in position 9 of SP was performed to analyze
structure–activity relationships
Experimental procedures Molecular mechanics calculations N-acetyl N¢-methyl amide derivatives of HGly, b2-HAla and
b3-HAla and of a-amino acids were built using INSIGHTII
(Accelrys Inc.) Molecular mechanics calculations were performed with theDISCOVERprogram and AMBER force field [26] The electrostatic potential energy was calculated with a distance-dependent dielectric screening of 4Ær and no cut-off was used
Minimum-energy conformers of b- and a-amino acids were generated by molecular dynamics at high tempera-ture followed by energy minimization Two thousand structures were generated by molecular dynamics at
1000 K, saving snapshots every 2 ps The time step used was 1 fs and the temperature was controlled by direct velocity scaling Each structure was then submitted to
2 ps of dynamics at 300 K and minimized using steepest descent, conjugate gradient and Newton–Raphson algo-rithms until the gradient was less than 0.001 kcalÆ mol)1ÆA˚)1
Conformational grid searches of b-amino acids were initially carried out at intervals of 30 for each torsion angle /, h, and w and were subsequently refined by varying /, w angles in intervals of 10 and setting h angle to) 60 ± 10 and 60 ± 10 Each /, h, and w-value was fixed by applying
a harmonic potential and the structures were minimized (adiabatic relaxation)
NMR spectroscopy Lyophilized peptides were dissolved in 550 lL of methanol (C2H3OH or C2H3O2H) at 1–2 mMconcentration NMR experiments were recorded at 298 K and 278 K on Bruker Avance spectrometers at a1H frequency of 500 MHz and were processed with the XWIN-NMR software 1D spectra were acquired over 16 K data points using a spectral width
of 5000 Hz Solvent suppression was achieved by presatu-ration during the relaxation delay or with a WATERGATE sequence [27] Proton assignments were obtained from the analysis of TOCSY (20 and 80 ms isotropic mixing times) [28] and NOESY (400 ms mixing time) experiments [29] Typically 512 t1 increments were acquired over a spectral width of 5000 Hz Prior to Fourier transformation in t2and
t1, the time domain data were multiplied by a 60–90 shifted square sinebell function and zero-filled Baseline distortions were corrected with a fifth-order polynomial.1H-13C HSQC experiments were recorded using pulsed field gradients for coherence selection [30]
Fig 1 Schematic representation of a-, b2- and
b 3 -amino acids with the torsion angles /, h and w For the b2and b3-amino acid substi-tuted-SP analogues, the methyl side chain of b-HAla exhibits the same orientation as the one in [Ala9]SP (CIP’s rule a-amino acid:
S configuration, thus (R) for b 2 -HAla and (S) for b 3 -HAla) (CIP, Cahn–Ingold–Prelog.)
Trang 3The chemical shift deviations of Ha and Ca were
calculated using random coil values determined in methanol
[31] and water [32], respectively No sequence correction
(proline effect, for example) was applied for this calculation
as only position 9 in the sequence of these analogues is
modified, the RPKPQQFF and LM-NH2 domains being
constant
Enzymatic degradation
Enzymatic cleavage of SP, [Ala9]SP, [HGly9]SP, [(R)-b2
-HAla9]SP and [(S)-b3-HAla9]SP by ACE was performed
as described previously, with slight modifications [33]
Briefly, peptides (10 nmol) were incubated in 100 lL Tris/
HCl 50 mM pH 8.3, NaCl 300 mM, with 0.01 U rat lung
ACE (Fluka) at 37C for 60 min Degradation was
stopped by addition of 1 lL trifluoroacetic acid and
followed by HPLC using a RP8 Lichrospher100 column in
isocratic mode (72% H2O, 28% acetonitrile, 0.072%
trifluoroacetic acid) at a flow rate of 1.5 mLÆmin)1
Retention times were 7.3 min for SP, 8.6 min for [Ala9]SP,
7.5 min for [HGly9]SP, 7.1 min for [(R)-b2-HAla9]SP and
7.7 min for [(S)-b3-HAla9]SP Enzymatic assays were
performed twice and yielded similar results All assays
were done in parallel experiments with control at t¼ 0 for
each peptide The percentage of degradation was calculated
by comparing the area of the peaks of the intact peptide at
t¼ 0 and t ¼ 60 min
Binding assays
Binding assays were carried out at 22C with either
[3H][Pro9]SP (0.2–0.5 nM, 65 CiÆmmol)1) for 100 min or
[3H]propionyl[Met(O2)11]SP(7–11) (2–5 nM, 95 CiÆ mmol)1)
for 80 min on whole CHO cells expressing the human
NK-1 receptor (6 pmolÆmg protein)1) as described
previously [24], in 200 lL Krebs-Ringer phosphate
solution consisting of 120 mM NaCl, 4.8 mM KCl,
1.2 mM CaCl2, 1.2 mM MgSO4 and 15.6 mM NaH2PO4,
pH 7.2 and containing 0.04% bovine serum albumin
(w/w), 0.6% glucose (w/v), 10 mMphenylmethanesulfonyl
fluoride SP peptide analogues (stored at )20 C in
water at a concentration of 1 mM) were diluted in the
binding buffer to the desired concentration just prior to
the assay
Phospholipase C and adenylate cyclase assays
PtdIns hydrolysis and cAMP accumulation were
deter-mined as described previously [25] Briefly, CHO cells
expressing the human NK-1 receptor (6 pmolÆmg
pro-tein)1) were labelled with [3H]adenine (0.2 lCi per well)
or [3H]inositol (0.5 lCi per well) for 15–24 h PtdIns
hydrolysis assay was performed in 500 lL Krebs-Ringer
phosphate buffer containing 10 mM LiCl and the peptide
to be tested for 10 min The accumulation of cAMP was
performed in 500 lL Krebs-Ringer phosphate buffer
containing 1 mM 3-isobutyl-1-methylxanthine and the
peptide to be tested for 10 min SP peptide analogues
(stored at )20 C in water at a concentration of 1 mM)
were diluted in the assay buffer to the desired
concen-tration just prior to the assay
Results Molecular modelling The conformational properties of b-amino acids and b-pep-tides have already been extensively analysed by ab initio quantum calculations [11,13] and molecular mechanics [12]
In the present study minimum-energy conformations of model b-peptides (Ac-HGly-NHMe, Ac-b2-HAla-NHMe and Ac-b3-HAla-NHMe) were first generated by molecular mechanics using AMBER force field The results were compared with those obtained previously from ab initio molecular orbital (MO) calculations and molecular mechan-ics calculations [11–13] The potential energy surfaces accessible to b-amino acids have plenty of local minima The minimum-energy conformers (data not shown) are similar to those obtained with CHARMm 23.1 force field [12]: six- and eight-hydrogen-bonded conformations with the methyl group in axial or equatorial orientations (conformers C6eq, C6ax, C8eqand C8ax), extended structure (Ex) with no hydrogen bonds and helical structures (H12and
H14) The C6and C8structures were found to be the most stable conformations Most low-energy structures corres-pond to h torsion angles around 180, 60 and)60 Then, in order to compare the conformational spaces of a and b-amino acids, grid searches were performed and the generated structures were superimposed with canonical conformations of a-amino acids The atoms used in the rmsd calculation are those involved in the two peptide bonds and the two N- and C-terminal methyl groups, in order to compare the backbone orientations on both sides
of the b-amino acid The systematic fits indicate that structures with h torsion angle above 90 have large rmsd values In particular, the low-energy extended structures, corresponding to h 180, cannot overlap any conforma-tion of a-amino acids In order to visualize on the potential energy surfaces the conformers of b-amino acids that fit with canonical conformations of a-amino acids, two (/) w) Ramachandran maps corresponding to h torsion angle values of +60, gauche(+), or)60, gauche(–), were calculated (Fig 2) The calculated conformers are only a part of the representative statistical ensemble, due to the h angle restriction HGly has a wide range of accessible conformations, whereas b3-HAla has a more limited conformational space (Fig 2) For both gauche(–) and gauche(+) conformers, regions of the (/ – w) diagram can overlap canonical b-sheet, a-helix and reverse c-turn conformations found in a-amino acids (rmsd lower than 0.05 nm) The gauche(+) conformers which fit to a-helical, C7 and b-sheet conformations have approximate (/, w) torsion angles of ()100, )110) ()130, 20) and ()170, 80), respectively, and correspond to favourable regions For the gauche(–) conformers, the conformations that fit to a-helix have high energies and only two low-energy regions
of the diagram can fit to C7 or b-sheet conformations, with (/, w) torsion angles of ()30, 120) and ()70, 180), respectively
Secondary structure of the SP analogues [(R)-b2-HAla9]SP, [(S)-b3-HAla9]SP as well as [(R)-b2 -HPhe7]SP and [(R)-b2-HPhe8]SP have been synthesized
Trang 4by solid phase methodology and obtained with purities and
yields comparable to SP and [Pro9]SP [34]
Due to its inherent flexibility, SP is largely unstructured in
water but helical conformation of the 4–8 domain is induced
in lower dielectric constant solvents such as methanol
[35,36] The1H-NMR spectra of all the SP analogues in
methanol are relatively well-dispersed and have been
completely assigned using conventional techniques [37]
The chemical shifts of a carbon (Ca) have been assigned
from HSQC spectra (data not shown) The chemical shift
deviations of Ha protons and Ca carbons (CSDHa and
CSDCa), corresponding to differences between observed
chemical shifts and random coil values, are commonly used
to detect secondary structures in peptides and proteins [38]
They are reported in Fig 3
Stronger upfield shifts of Ha (Fig 3A) and downfield
shifts of Caresonances (Fig 3B) in the 4–8 sequence are
observed for [Aib9]SP compared to SP The positive variation observed for CSDCain methanol is weak when compared with CSDHa Because the random coil values of
Ca were determined in water, it is possible that Ca chemical shifts are sensitive to solvent variation, causing an under-estimation of calculated CSDs These CSDHaand CSDCa variations demonstrate the formation of more stable and abundant helical structures for [Aib9]SP than for SP By restrained molecular dynamics, [Aib9]SP has been shown to adopt a stable helix from residues 4–10 while the 4–8 domain of SP adopts a more flexible helix [36] Taking SP as
a reference, CSDHaand CSDCaindicate that the introduc-tion of one methyl (Ala) or two methyl groups (Aib) on Ca carbon of Gly9 increases progressively the 4–8 domain folding into helical structures An opposite effect due to the helix breaker property of Pro is observed for [Pro9]SP, this decrease in helical structure is limited to the adjacent Phe8
Fig 2 (/ – w) maps of b-amino acids with indication of the conformational space common with that of a-amino acids The (/ ) w) maps corresponding to h-values of )60 and 60 are indicated for HGly (A), b 2 -HAla (B) and
b3-HAla (C) The contours are drawn within
21 kJÆmol)1(5 kcalÆmol)1) of the global minimum and are gradually coloured from blue to red (the energy difference between each level is 2.1 kJÆmol)1) The structures that exhibit rmsd values smaller than 0.05 nm with canonical structures of a-amino acids are indicated with squares of different colours: blue, a-helix; green, reverse c-turn; red, anti-parallel b-sheet The corresponding (/, w) values of the canonical structures are ( )57, )47) ()90, 68), and ()139, 135).
Trang 5residue Homologation of Gly9 in [HGly9]SP does not
decrease the helix proportion Methylation of HGly in
position two, in [b2-HAla9]SP, has no effect on the helical
structure when compared to SP, whereas methylation of
HGly in position three, in [b3-HAla9]SP, induces a helical
folding of the 4–8 domain similar in amplitude to that
observed for [Ala9]SP Whatever the chemical modifications
carried out in position 9, the3JNH-Hacoupling constant of
Gln5 remains close to 5 Hz, indicating that Gln5 always
adopts a helical conformation This invariable folding of
Gln5 is related to the helix initiator propensity of Pro4 and
decreases from Gln5 to Gly9 In methanol, the helical
structures in the 4–8 domain increase with [Pro9]SP < SP
[HGly9]SP [b2-HAla9] < [b3-HAla9]SP < [Ala9]SP
< [Aib9]SP The presence of helical structures was
con-firmed by the observation of NN (i, i + 1), aN (i, i + 2), aN
(i, i + 3), aN (i, i + 4) and ab (i, i + 3) NOEs along the 5–8
domain of SP and [Aib9]SP The aN (i, i + 4) and ab (i, i
+ 3) NOEs were not detected in [b2-HAla9]SP, [b3
-HAla9]SP and [HGly9]SP, indicating more flexible helical
structures
The C-terminal part of SP undergoes a complex
con-formational equilibrium between more or less extended
structures On the basis of CSDHa, mono- or di-methylation
of Gly9 (Ala, Aib) appears to induce helical structure for
Leu10 This effect is also observed for [b3-HAla9]SP The analysis of coupling constants and NOEs indicate that the local conformation of residue 9 is not well-defined in the b-amino acid-substituted SP analogues
The lack of NOEs prevented three-dimensional structure calculations by restrained molecular dynamics Neverthe-less, the similar patterns of Ha and Ca CSDs suggest that the different SP analogues may adopt conformations of their peptide backbone close to that of SP, which has been previously described [36] The decreased number of observed NOEs in [HGly9]SP, [b2-HAla9] and [b3-HAla9]SP
is consistent with a higher flexibility around the b-amino acid-substituted position
Potency of the SP analogues The binding potencies of these analogues for the two specific binding sites, NK-1M and NK-1m, associated with the human NK-1 receptor have been measured with transfected CHO cells [24,25] The more abundant binding site NK-1M (85%) is labelled by [3H][Pro9]SP and is coupled to cAMP production, whereas the less abundant binding site NK-1m (15%) is labelled by [3H]propionyl[Met(O2)11]SP(7–11) and associated with the production of inositol phosphates The binding and agonist potencies of the SP analogues are
Fig 3 Chemical shift deviations of Ha (A) and Ca (B) for the SP analogues modified in position 9 The CSD Ca are not shown for [Pro9]SP Residue X
is either Gly (SP), Ala, Pro, Aib, HGly, b 2 -HAla or b 3 -HAla.
Trang 6expressed as Kifor NK-1M (major site) and NK-1m (minor
site), and EC50values for the cAMP pathway and for the
inositol phosphates pathway
SP, [Ala9]SP and [Pro9]SP are almost equipotent at the
major binding site NK-1M (Kibetween 0.64 nMand 1.6 nM
and EC50 values between 4.8 and 8 nM) [HGly9]SP and
[(S)-b3-HAla9]SP are 30 to 45 times less potent than SP (Ki
and EC50values) [(R)-b2-HAla9]SP is 10 times more potent
than the corresponding b3-analogue and is only three to five
times less potent than SP (Table 1)
Regarding the minor binding site NK-1m, SP and
[Pro9]SP show a 10-fold increase in affinity compared to
their affinity for the NK-1M binding site, with Ki in the
subnanomolar range (0.13 nM) and EC50 values in the
nanomolar range (1.0 and 0.7 nM, respectively) Surprisingly,
[Ala9]SP exhibits one of the highest affinity ever found for
the NK-1m specific binding site (Ki¼ 7 pM), as it is almost
20 times more potent than SP and [Pro9]SP and even 10
times more potent than [Gly9(wCH2CH2)(S)Leu10]SP
[HGly9]SP and [(S)-b3-HAla9]SP present similar Kivalues
for the NK-1m specific binding sites (3.2 and 2.3 nM,
respectively), being 20–30 times less potent than SP [(R)-b2
-HAla9]SP is only 4.5 times less potent than SP and
[Pro9]SP, as observed for the NK-1M binding site The
EC50 values for inositol phosphates production of these
three b-amino acids-substituted SP analogues [HGly9]SP,
[(S)-b3-HAla9]SP and [(R)-b2-HAla9]SP are almost
identi-cal (EC50 2 nM) For comparison, the affinities and
potencies of different analogues of SP substituted at
position(s) 9 or/and 10 are also reported in Tables 1, i.e
[Aib9]SP [36], [Pro10]SP, [Gly9w(CH2CH2)Gly10]SP,
[Gly9w(CH2CH2)(S)Leu10]SP and [Gly9w(CH2CH2)
(R)Leu10]SP
In contrast, [(R)-b2-HPhe7]SP and [(R)-b2-HPhe8]SP
are very weak competitors for NK-1M and NK-1m
specific binding sites, being 2000 times less potent than
SP
Table 1 Affinities of b-amino acid-containing peptide analogues of SP for the NK-1M (labelled with [3H][Pro9]SP) and the NK-1m (labelled with [3H]propionyl[Met(O 2 )11]SP(7–11)) binding sites and their related potency to stimulate adenylate cyclase and phospholipase C in CHO cells expressing the human NK-1 receptor All experiments have been performed in triplicate in at least three independent experiments Numbers in parentheses refer
to structures in Fig 5.
Peptide K i , NK-1M (n M ) EC 50 , adenylate cyclase K i , NK-1m (n M ) EC 50 , phospholipase C
SP (1)a 1.6 ± 0.4 8 ± 2 0.13 ± 0.02 1.0 ± 0.2
Propionyl[Met(O 2 )11]SP(7–11)a 1900 ± 450 >5000 10 ± 2 37 ± 4
[Pro9]SP (2) a 1.1 ± 0.1 10 ± 2 0.13 ± 0.02 0.7 ± 0.1
[Aib9]SP (3) b 44 ± 4 125 ± 30 c 3.8 ± 0.4 5.5 ± 1.5
[Pro10]SP 24 ± 2 375 ± 50 3.7 ± 0.5 3.0 ± 1.0
[Gly9(YCH 2 CH 2 )Gly10]SP (4) 190 ± 30 1250 ± 50 3.0 ± 0.7 1.2 ± 0.5
[Gly9(YCH 2 CH 2 )(S)Leu10]SP (5) 2.6 ± 0.5 24 ± 6 0.07 ± 0.01 0.6 ± 0.1
[Gly9(YCH 2 CH 2 )(R)Leu10]SP (6) 73 ± 8.5 1130 ± 370 4.6 ± 0.8 2.5 ± 0.7
[Ala9]SP (7) 0.64 ± 0.070 4.8 ± 1.2 0.0070 ± 0.00065 0.69 ± 0.03
[HGly9]SP (8) 64 ± 3.5 290 ± 74 3.2 ± 0.4 2.0 ± 0.2
[(R)-b2-HAla9]SP (9) 5.2 ± 0.70 40 ± 5 0.54 ± 0.12 2.0 ± 0.4
[(S)-b3-HAla9]SP (10) 53 ± 6 360 ± 55 2.3 ± 0.3 2.9 ± 0.7
[(R)-b 2 -HPhe8]SP 2400 ± 300 >10 000 240 ± 25 215 ± 25
[(R)-b2-HPhe7]SP 2500 ± 400 >10 000 230 ± 30 210 ± 30
a, b
results already published in [24] and [36], respectively.cEfficacy 73% that of [Pro9]SP taken as the peptide of reference.
Fig 4 Relation between (A) affinity for the NK-1M binding site and potency to activate adenylate cyclase and (B) affinity for the NK-1m binding site and potency to activate phospholipase C of b-amino acid-containing peptide analogues Symbols are the experimental results obtained with data in Table 1 Dotted lines represent theoretical values obtained from equations previously determined with 53 (A) and 22 (B)
SP analogues, respectively [39].
Trang 7We have previously established that a good correlation
exists between EC50values and the corresponding Kivalues,
i.e EC50 for cAMP production and Ki for the NK-1M
binding site (log(EC50)¼ 0.8 log(Ki) – 0.6), and EC50for
inositol phosphates production and Kifor the NK-1m site
(log (EC50)¼ 0.9 log (Ki) –1.0), respectively [39] The data
obtained in this study (Fig 4) square with the equations
determined previously with the exception of SP, [Pro9]SP,
[Ala9]SP, [Gly9w(CH2CH2)(S)Leu10]SP and [(R)-b2
-HAla9]SP, which show apparently abnormal behaviour in
their affinity for the NK-1m binding and their potency to
stimulate PLC (Fig 4B) Whatever the affinity measured
for the NK-1m binding site for these agonists (from 7 pMto
0.54 nM), the corresponding potency to stimulate PLC is
always close to 1 nM (from 0.6 nM to 2 nM), the highest
EC50/Kiratio being close to 100 for [Ala9]SP (Table 1) This
apparent discrepancy between the varying affinities and the
nonvarying response of these agonists could be explained by
the number of receptors to be occupied by these agonists to
get activation of the second messenger cascade as reported
[40]
Enzymatic degradation of SP analogues
ACE is a dipeptidyl carboxypeptidase known to hydrolyze
the peptide bonds between residues 8–9 and 9–10 of SP [33]
The peptide cleavage after one hour incubation with ACE
was monitored by HPLC Percentages of degradation were
found to be 54 ± 1% for SP, 55 ± 3% for [Ala9]SP,
33 ± 5% for [HGly9]SP, 26 ± 3% for [b2-HAla9] and
14.5 ± 2.5% for [b3-HAla9], thus showing that these b-amino acid-containing peptides have increased stability towards cleavage by ACE compared to the corresponding a-peptides
Discussion Computational studies confirm that the conformational space of b-amino acids is larger than that of a-amino acids, but low-energy conformations of the b-amino acids backbone, corresponding to gauche rotamers around the Ca–Cb bond, can overlap canonical backbone conformers observed for a-amino acids Therefore the addition of a backbone methylene group could have minor effects on the overall conformation and biological activity of the peptide
Thus, SP was used as a model peptide to analyse the structural and biological consequences of a single b-amino acid incorporation When Phe7 or Phe8 are replaced by
b2-HPhe, the corresponding analogues are weak competi-tors of specific NK-1 binding sites These amino acids are in the helical domain of SP which extends from residues 4–8 [35] Any modification of Phe7 causes a dramatic loss in receptor affinity for the corresponding peptide [21,41] Indeed, the backbone conformation, the aromatic ring and the orientation (v1and v2) of this phenylalanine have to be conserved for full biological potencies of the peptide [41] It
is possible that the side chain of b2-HPhe may not fit in the binding subsite devoted to the aromatic ring of Phe7 Yet, molecular calculations indicate that low-energy structures of
Fig 5 Schematic representation of the amino acid sequence 9–10 of the SP analogues 1–10 (pharmacological data in Table 1) Rectangles under analogues indicate compounds that have affinity for the two (NK-1M/NK-1m) binding sites similar to those of SP Ovals under analogues point out compounds compared to SP that lose at least a factor 20 in affinity for the two (NK-1M/NK-1m) binding sites (1) SP; (2) [Pro9]SP; (3) [Aib9]SP; (4) [Gly9(YCH 2 CH 2 )Gly10]SP; (5) [Gly9(YCH 2 CH 2 )(S)Leu10]SP; (6) [Gly9(YCH 2 CH 2 )(R)Leu10]SP; (7) [Ala9]SP; (8) [HGly9]SP; (9) [(R)-b 2 -HAla9]SP; (10) [(S)-b3-HAla9]SP.
Trang 8b2-HPhe could fit the bioactive conformation of Phe7
around the peptide bond and the aromatic ring Because
Ca-methylation of Phe7 leads to an inactive compound [36],
it is likely that the additional methylene group of b2-HPhe
causes steric hindrance within the receptor as does the
methyl group in Ca-MePhe Although position 8 of SP can
accept larger aromatic substituents than position 7 [41],
Ca-methylation is also prohibited, suggesting that steric
hindrance can again be evoked to explain the lack of affinity
of [(R)-b2-HPhe8]SP for the NK-1 receptor
Gly9 in the sequence of SP constitutes a hinge between
the helical domain and the C-terminal residues which adopt
more or less extended conformations, Phe7, Phe8, Leu10,
and Met11 being key elements of the SP pharmacophore
[21] N-methylation of Gly9, i.e [Sar9]SP, as well as proline
substitution, i.e [Pro9]SP, yield potent and selective NK-1 agonists [21] Therefore Gly9 should be a more favourable candidate for b-amino acid substitution than Phe7 or Phe8 The SP analogues modified in position 9 are schematically represented in Fig 5 Their binding potencies for the two binding sites (NK-1M and NK-1m) associated with the NK-1 receptor in CHO transfected cells [24,25] have been classified in two groups In one group, peptides are as potent
as SP, or even more potent, whatever the binding site considered, i.e [Pro9]SP, [Gly9(wCH2CH2)(S)Leu10]
SP, [Ala9]SP and [b2-HAla]SP In the second group, SP analogues are more than 20 times less potent than SP, whatever the binding site considered, i.e [Aib9]SP, [Gly9(wCH2CH2)Gly10]SP, [Gly9(wCH2CH2)(R)Leu10]SP, [HGly9]SP and [b3-HAla9]SP Previous structure–activity
A
B
C
Fig 6 Superposition of selected conformers of
b 2
-HAla (A), b 3
-HAla (B) and Aib (C) with Pro The (/, w) values of the selected Pro conformer are ( )72, 153) The low-energy conformers of b-HAla generated in the grid calculation have been selected on the basis of the best fit with Pro They correspond to (/, h, w) angles of ( )110, 70, 100) The energy differences relative to the global energy mini-mum are 0.88 and 0.86 kcalÆmol)1for b2-HAla and b 3 -HAla, respectively Nitrogen atoms are coloured in blue, oxygens in red, hydrogens in grey Carbon atoms of Pro are coloured in cyan, those of other amino acids in green The side chain methyl carbon of b-HAla and the pro R methyl carbon of Aib are coloured in magenta.
Trang 9relationship studies [42] have established that the leucine
side chain orientation is crucial for full binding potency,
[Gly9(wCH2CH2)(S)Leu10]SP being even more potent than
SP whereas [Gly9(wCH2CH2)(R)Leu10]SP is a weak
com-petitor at the NK-1 receptor (at that time NK-1M and
NK-1m binding sites were not differentiated) The amide
bond between residues 9 and 10 is not involved per se in any
stabilizing interaction within the NK-1 receptor because
[Gly9(wCH2CH2)(S)Leu10]SP is as potent as SP More
important is the length of the spacer between residues 8 and
10 Indeed, [Gly9(wCH2CH2)Gly10]SP is a weak
compet-itor of specific NK-1 binding sites while homologation
of one carbon, such as [Gly9(wCH2CH2CH2)Gly10]SP
(D Loeuillet & S Lavielle, unpublished data), led to
an analogue completely devoid of binding potency for
NK-1 binding sites Interestingly, the presence of an amide
bond such as in the homologated analogues [HGly9]SP,
[b2-HAla9]SP and [b3-HAla9]SP restores part of the
potency to recognize the NK-1 receptor In view of this
observation it can be proposed that the improved affinity
is indicative of an energetically favoured bioactive
conformation stabilized by the amide function In
[Gly9(wCH2CH2CH2)Gly10]SP, the backbone of the
ami-nohexanoic moiety must be unable to adopt two
consecu-tive gauche(+) rotamers
The pyrrolidine ring of proline does not hamper the
correct positioning of Leu10 and Met11, because [Pro9]SP is
as potent as SP Ca monomethylation of Gly9 drastically
increases the affinity for the NK-1m binding site of
[Ala9]SP, supporting the formation of a new stabilizing
interaction between this methyl group and a hydrophobic
subsite within the specific NK-1m binding site The CH2b of
the pyrrolidine ring of [Pro9]SP probably fits within this
hydrophobic subsite and thus compensates destabilizing
interactions due to CH2c and CH2d of the pyrrolidine ring
or non optimalF-value However, the introduction of a
second methyl group on the Ca of Gly9 in [Aib9]SP induces
a strong repulsive interaction Although the three b-amino
acid substitutions in position 9 of SP are tolerated by the
NK-1 receptor, the substitution of the flexible Gly by the
even more flexible HGly causes a 30- to 40-fold decrease in
affinity and biological activity Substitution by b3-HAla has
similar effects Only the b2-HAla substitution yields an
analogue that is nearly as potent as SP Thus, a peculiar
orientation of the methyl group in b2-HAla-substituted SP
might be at the origin of a stabilizing interaction
Modelling studies and NMR analysis suggest that the
three b-amino acid-substituted SP analogues [HGly9]SP,
[b2-HAla9]SP and [b3-HAla9]SP may adopt
conforma-tions around residue 9 that are analogous to those
adopted by a-amino acids (Gly, Ala, Sar, Pro) To explain
the slightly higher biological potency of [b2-HAla9]SP
compared to [HGly9]SP and [b3-HAla9]SP, the structures
(backbone and side chain) of the different a and b amino
acids were superimposed Pro, the most constrained
residue, was used as a template for the superimposition
and an extended conformation was chosen, in accordance
with structure–activity relationship studies [21,24]
Low-energy structures of the b-amino acids that best fit are
shown in Fig 6 They all correspond to gauche(+) values
of the h angle The methyl group of b2-HAla occupies a
position close to that of CHb of Pro or the pro S methyl
in Aib or Ala Conversely, the methyl group of b3-HAla occupies a position similar to the pro R methyl of Aib, on the opposite side of the pyrrolidine ring of Pro A parallel can be drawn regarding the differences in biological activities between [Ala9]SP and [Gly9]SP on the one hand, and [b2-HAla9]SP and [HGly9]SP on the other hand In-deed, the higher pharmacological potency of [b2-HAla9]SP compared to [HGly9]SP suggests that the methyl group of
b2-HAla9 may fit within the hydrophobic subsite devoted
to the methyl group of Ala9 The similar biological potencies of [b3-HAla9]SP and [HGly9]SP indicate that even though the backbone of [b3-HAla9]SP may adopt the bioactive conformation, the methyl group of b3-HAla9 may not be orientated towards this hydrophobic stabi-lizing subsite
Finally, as shown herein with the use of ACE that has been reported to cleave SP between residues 8–9 and 9–10 [33], the peptides containing a b-amino acid substitution in position 9 have increased stability compared to the corres-ponding a-amino-acid-containing peptides Therefore it is possible to increase peptide stability at the expense of a minimal decrease in its activity
HGly (named by the authors b-alanine) has been previously introduced in the sequence of the C-terminal heptapeptide of NKA, another peptide of the tachykinin family that binds the NK-1 and NK-2 receptors [HGly8] NKA(4–10) is as potent as NKA and [Ala8]NKA on rabbit pulmonary artery and rat portal vein, two NK-2 receptor bioassays [43] More recently, the same Ala substitution was reported to cause a significant decrease in biological activities of [Ala8]NKA measured in human tissues [44] Indeed, [Ala8]NKA(4–10) was shown to be a weak partial agonist ([HGly8]NKA(4–10) was not tested in this study) These discrepancies have been attributed to the differences
in sequences of rabbit, rat and human NK-2 receptors ( 85% homology)
In conclusion, a b-amino acid could replace an a-amino acid within the sequence of a bioactive peptide provided that the additional methylene group does not cause steric hindrance and does not confine orientations of the side chain to regions of space different from those permitted in the a-amino acid Thus, insertion of a single b-amino acid in
a bioactive peptide could be favourably applied to improve both biological potency and enzymatic stability of the original peptide, as already shown with the empiric design of
a metalloendopeptidase tripeptide inhibitor [45]
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