Eur J Biochem 270, 939–949 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03456.x Structural 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, Gerard 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-HAlaNHMe 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, Substance 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)-b2HAla or (S)-b3-HAla [(R)-b2-HAla9]SP has pharmacological 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 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 does not affect the overall backbone conformation Altogether these data suggest that [HGly9]SP, [(S)-b3-HAla9]SP and [(R)-b2-HAla9]SP could adopt backbone conformations similar to that of SP, [Ala9]SP and [Pro9]SP In contrast, incorporation of b2-HPhe in position and 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 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 Bioactive a-peptides often present conformational equilibriums 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 conformational 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 conformations, namely six- and eight-membered ring hydrogenbonded structures (C6, C8), extended structures (Ex) and helical structures (He) [11] The C6 structure 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 hydrogenbonded 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 12-, 10-, 12hydrogen-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 commercially 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 C7 structure found with a-amino peptides can be Correspondence to: O Lequin, UMR 7613 Paris 6-CNRS, ´ Laboratoire Structure et Fonction de Molecules Bioactives, ´ Universite Pierre et Marie Curie, Paris, France Fax: + 33 44 27 71 50, Tel.: + 33 44 27 26 78, E-mail: lequin@ccr.jussieu.fr Abbreviations: NK-1, neurokinin-1; SP, substance P (H-Arg-Pro-LysPro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2); NKA, neurokinin A; HGly, homoglycine; b2-HAla, b2-homoalanine; b3-HAla, b3-homoalanine; b2-HPhe, b2-homophenylalanine; Aib (aMeAla), a-aminoisobutyric 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 October 2002, revised 20 December 2002, accepted January 2003) Keywords: b2- and b3-amino acid; secondary structure; molecular mechanics calculations; substance P; neurokinin-1 receptor Ó FEBS 2003 940 S Sagan et al (Eur J Biochem 270) Fig Schematic representation of a-, b2- and b3-amino acids with the torsion angles /, h and w For the b2 and b3-amino acid substituted-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 b2-HAla and (S) for b3-HAla) (CIP, Cahn–Ingold–Prelog.) stabilized 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 somatostatin [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-tetrapeptide 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 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 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-HAlaNHMe have been generated and compared to the canonical structures of the corresponding a-amino acid Ac-Gly-NHMe The corresponding SP analogues substituted in position 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 phospholipase 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 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 the DISCOVER program and AMBER force field [26] The electrostatic potential energy was calculated with a distance-dependent dielectric screening of 4Ỉr and no cut-off was used Minimum-energy conformers of b- and a-amino acids were generated by molecular dynamics at high temperature followed by energy minimization Two thousand structures were generated by molecular dynamics at 1000 K, saving snapshots every ps The time step used was fs and the temperature was controlled by direct velocity scaling Each structure was then submitted to ps of dynamics at 300 K and minimized using steepest descent, conjugate gradient and Newton–Raphson algorithms until the gradient was less than 0.001 kcalỈ ˚ mol)1ỈA)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 mM concentration NMR experiments were recorded at 298 K and 278 K on Bruker Avance spectrometers at a 1H 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 presaturation 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 t2 and 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] Ó FEBS 2003 Effects of b-amino acid insertion in Substance P (Eur J Biochem 270) 941 The 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 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)-b2HAla9]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 37 °C for 60 Degradation was stopped by addition of 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 mLỈmin)1 Retention times were 7.3 for SP, 8.6 for [Ala9]SP, 7.5 for [HGly9]SP, 7.1 for [(R)-b2-HAla9]SP and 7.7 for [(S)-b3-HAla9]SP Enzymatic assays were performed twice and yielded similar results All assays were done in parallel experiments with control at t ¼ for each peptide The percentage of degradation was calculated by comparing the area of the peaks of the intact peptide at t ¼ and t ¼ 60 Binding assays Binding assays were carried out at 22 °C with either [3H][Pro9]SP (0.2–0.5 nM, 65 CiỈmmol)1) for 100 or [3H]propionyl[Met(O2)11]SP(7–11) (2–5 nM, 95 CiỈ mmol)1) for 80 on whole CHO cells expressing the human NK-1 receptor (6 pmolỈmg 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 mM phenylmethanesulfonyl fluoride SP peptide analogues (stored at )20 °C in water at a concentration of 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 determined as described previously [25] Briefly, CHO cells expressing the human NK-1 receptor (6 pmolỈmg protein)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 The accumulation of cAMP was performed in 500 lL Krebs-Ringer phosphate buffer containing mM 3-isobutyl-1-methylxanthine and the peptide to be tested for 10 SP peptide analogues (stored at )20 °C in water at a concentration of mM) were diluted in the assay buffer to the desired concentration just prior to the assay Results Molecular modelling The conformational properties of b-amino acids and b-peptides 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 mechanics 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, C8eq and C8ax), extended structure (Ex) with no hydrogen bonds and helical structures (H12 and H14) The C6 and C8 structures were found to be the most stable conformations Most low-energy structures correspond 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 conformation 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)-b2HPhe7]SP and [(R)-b2-HPhe8]SP have been synthesized 942 S Sagan et al (Eur J Biochem 270) Ó FEBS 2003 Fig (/ – 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), b2-HAla (B) and b3-HAla (C) The contours are drawn within 21 kJỈmol)1 (5 kcalỈmol)1) of the global minimum and are gradually coloured from blue to red (the energy difference between each level is 2.1 kJỈmol)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, antiparallel b-sheet The corresponding (/, w) values of the canonical structures are ()57°, )47°) ()90°, 68°), and ()139°, 135°) by 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] The 1H-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 Stronger upfield shifts of Ha (Fig 3A) and downfield shifts of Ca resonances (Fig 3B) in the 4–8 sequence are observed for [Aib9]SP compared to SP The positive variation observed for CSDCa in 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 underestimation of calculated CSDs These CSDHa and 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, CSDHa and CSDCa indicate that the introduction 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 Ó FEBS 2003 Effects of b-amino acid insertion in Substance P (Eur J Biochem 270) 943 Fig Chemical shift deviations of Ha (A) and Ca (B) for the SP analogues modified in position The CSDCa are not shown for [Pro9]SP Residue X is either Gly (SP), Ala, Pro, Aib, HGly, b2-HAla or b3-HAla residue 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, the 3JNH-Ha coupling constant of Gln5 remains close to 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 confirmed 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, [b3HAla9]SP and [HGly9]SP, indicating more flexible helical structures The C-terminal part of SP undergoes a complex conformational 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 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 Nevertheless, 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 Ó FEBS 2003 944 S Sagan et al (Eur J Biochem 270) Table 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(O2)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 Peptide Ki, NK-1M (nM) EC50, adenylate cyclase Ki, NK-1m (nM) EC50, phospholipase C SP (1)a Propionyl[Met(O2)11]SP(7–11)a [Pro9]SP (2)a [Aib9]SP (3)b [Pro10]SP [Gly9(YCH2CH2)Gly10]SP (4) [Gly9(YCH2CH2)(S)Leu10]SP (5) [Gly9(YCH2CH2)(R)Leu10]SP (6) [Ala9]SP (7) [HGly9]SP (8) [(R)-b2-HAla9]SP (9) [(S)-b3-HAla9]SP (10) [(R)-b2-HPhe8]SP [(R)-b2-HPhe7]SP 1.6 1900 1.1 44 24 190 2.6 73 0.64 64 5.2 53 2400 2500 8±2 >5000 10 ± 125 ± 30c 375 ± 50 1250 ± 50 24 ± 1130 ± 370 4.8 ± 1.2 290 ± 74 40 ± 360 ± 55 >10 000 >10 000 0.13 10 0.13 3.8 3.7 3.0 0.07 4.6 0.0070 3.2 0.54 2.3 240 230 1.0 37 0.7 5.5 3.0 1.2 0.6 2.5 0.69 2.0 2.0 2.9 215 210 a, b ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.4 450 0.1 30 0.5 8.5 0.070 3.5 0.70 300 400 ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.02 0.02 0.4 0.5 0.7 0.01 0.8 0.00065 0.4 0.12 0.3 25 30 ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.2 0.1 1.5 1.0 0.5 0.1 0.7 0.03 0.2 0.4 0.7 25 30 results already published in [24] and [36], respectively c Efficacy 73% that of [Pro9]SP taken as the peptide of reference expressed as Ki for NK-1M (major site) and NK-1m (minor site), and EC50 values 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 (Ki between 0.64 nM and 1.6 nM and EC50 values between 4.8 and nM) [HGly9]SP and [(S)-b3-HAla9]SP are 30 to 45 times less potent than SP (Ki and EC50 values) [(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 (Ki ¼ 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 Ki values for the NK-1m specific binding sites (3.2 and 2.3 nM, respectively), being 20–30 times less potent than SP [(R)-b2HAla9]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 identical (EC50  nM) For comparison, the affinities and potencies of different analogues of SP substituted at position(s) 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 Fig 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 acidcontaining peptide analogues Symbols are the experimental results obtained with data in Table Dotted lines represent theoretical values obtained from equations previously determined with 53 (A) and 22 (B) SP analogues, respectively [39] Ó FEBS 2003 Effects of b-amino acid insertion in Substance P (Eur J Biochem 270) 945 Fig 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(YCH2CH2)Gly10]SP; (5) [Gly9(YCH2CH2)(S)Leu10]SP; (6) [Gly9(YCH2CH2)(R)Leu10]SP; (7) [Ala9]SP; (8) [HGly9]SP; (9) [(R)-b2HAla9]SP; (10) [(S)-b3-HAla9]SP We have previously established that a good correlation exists between EC50 values and the corresponding Ki values, i.e EC50 for cAMP production and Ki for the NK-1M binding site (log(EC50) ¼ 0.8 log(Ki) – 0.6), and EC50 for inositol phosphates production and Ki for 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)-b2HAla9]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 pM to 0.54 nM), the corresponding potency to stimulate PLC is always close to nM (from 0.6 nM to nM), the highest EC50/Ki ratio 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 competitors 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 (v1 and 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 946 S Sagan et al (Eur J Biochem 270) Ó FEBS 2003 A B C b2-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 of SP can accept larger aromatic substituents than position [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 Fig Superposition of selected conformers of b2-HAla (A), b3-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 minimum are 0.88 and 0.86 kcalỈmol)1 for b2-HAla and b3-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 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 are schematically represented in Fig 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 Ó FEBS 2003 Effects of b-amino acid insertion in Substance P (Eur J Biochem 270) 947 relationship 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 competitor at the NK-1 receptor (at that time NK-1M and NK-1m binding sites were not differentiated) The amide bond between residues 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 and 10 Indeed, [Gly9(wCH2CH2)Gly10]SP is a weak competitor 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 aminohexanoic moiety must be unable to adopt two consecutive 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 optimal F-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 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 conformations around residue 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] Lowenergy structures of the b-amino acids that best fit are shown in Fig They all correspond to gauche(+) values of the h angle The methyl group of b2-HAla occupies a position close to that of CH2b 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 Indeed, 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 stabilizing 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 have increased stability compared to the corresponding 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 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Rhaleb, N.-E., Dion, S., Rouissi, N., ´ ´ Tousignant, C., Telemaque, S., Drapeau, G & Regoli, D (1989) Structure–activity studies of neurokinin A Neuropeptides 13, 263– 270 Warner, F.J., Mack, P., Comis, A., Miller, R.C & Burcher, E (2001) Structure–activity relationships of neurokinin A (4–10) at the human tachykinin NK2 receptor: the role of natural residues and their chirality Biochem Pharmacol 61, 55–60 45 Steer, D.L., Lew, R.A., Perlmutter, P., Smith, A.I & Aguilar, M.I (2000) Design and synthesis of inhibitors incorporating b-amino acids of metalloendopeptidase EC 3.4.24.15 J Peptide Sci 6, 470–477 Supplementary material The following material is available from http://www blackwellpublishing.com/products/journals/suppmat/EJB/ EJB3456/EJB3456sm.htm Table S1 Minimum-energy conformers of Ac-HGlyNHMe obtained with DISCOVER and AMBER forcefield Table S2 Minimum-energy conformers of Ac-b2-HAlaNHMe obtained with DISCOVER and AMBER forcefield Table S3 Minimum-energy conformers of Ac-b3-HAlaNHMe obtained with DISCOVER and AMBER forcefield Table S4 NMR Parameters of SP and SP analogues  ... control at t ¼ for each peptide The percentage of degradation was calculated by comparing the area of the peaks of the intact peptide at t ¼ and t ¼ 60 Binding assays Binding assays were carried... [33], the peptides containing a b-amino acid substitution in position have increased stability compared to the corresponding a- amino -acid- containing peptides Therefore it is possible to increase peptide. .. acid incorporation When Phe7 or Phe8 are replaced by b2-HPhe, the corresponding analogues are weak competitors of specific NK-1 binding sites These amino acids are in the helical domain of SP which