Indian Journal of Biochemistry & Biophysics Vol. 51, February 2014, pp. 7-18

Conformational study of N-methylated alanine peptides and design of Aβ inhibitor

Fateh S Nandel* and Radhika R Jaswal Department of Biophysics, Panjab University, Chandigarh 160014, India

Received 01 July 2013; revised 28 October 2013

N-Methylation increases the proteolytic stability of peptides and leads to improved pharmacological and increased nematicidal property against plant pathogens. In this study, the quantum mechanical and molecular dynamic simulation approaches were used to investigate conformational behavior of peptides containing only N-methylated alanine (NMeAla) residues and N-methylated alanine and alanine residues at alternate positions. The amide bond geometry was found to be trans and the poly NMeAla peptides were shown to populate in the helical structure without hydrogen bond with Φ, Ψ values of ~ 0, 90˚ stabilized by carbonyl-carbonyl interactions. Molecular dynamic simulations in water/methanol revealed the formation of β-strand structure, irrespective of the starting geometry due to the interaction of solvent molecules with the carbonyl groups of peptide backbone. Analysis of simulation results as a function of time suggested that the opening of helical structure without hydrogen bond started from C-terminal. Conformational behavior of peptides containing N-MeAla and Ala was used to design Aβ peptide inhibitor and the model tetrapeptide Ac-Ala-NMeAla-Ala-NHMe in the β-strand structure was shown to interact with the hydrophobic stretch of Aβ15-42 peptide.

Keywords: Conformation, N-Methylated alanine peptides, Molecular simulations, Helical structure without hydrogen bond, Carbonyl-carbonyl interactions, Aβ inhibitor design

Peptide drugs due to short half-lives in vivo and lack of oral availability have poor pharmacological profile1. To overcome these limitations, many strategies have been developed to design better drugs molecules, providing new chemical therapies for a range of human diseases2. N-Methylation increases the metabolic stability, intestinal permeability in somatostatin analogs3 and oral availability in cyclosporine (Cs) which is rich in N-methyl leucine4.

Omphalotine is a highly N-methylated compound with potential plant nematicidal properties5. Dictyonamides from marine alga-derived fungus K063 separated from marine rhodophyta Ceratodictyon

spongiosum6 and RHM1 and RHM2 from marine

sponge (Acremonium sp.)7 have shown weak toxicity against murine cancer cell lines and RHM1 also exhibits antibacterial activity. Dragonamides A and E, modified linear lipopeptides rich in N-methylated amino acids from marine cyanobacterium Lyngbya

majuscula also show anti-leishmanial activity with IC50

values of ~6.5 and 5.1 µM, respectively8. Carmabin B and majusculamides A and B8 along with dragonamides, carmabin A and dragomabin from

blue-green alga Lyngbya majuscula Gomont9,10 exhibit anti-malarial activity against chloroquine-resistant protozoan parasite Plasmodium falciparum. Micromide from a species of marine cyanobacterium belonging to the genus Symploca

11, thiocoraline from marine Micromonospora sp.

12 and cyanobacterial peptides13-16 are effective anti-tumor compounds. Pterulamides I-VI from Malaysian fungus (Pterula sp.) composed mainly of non-polar N-Me amino acids are reported to be cytotoxic against P388 cell line17.

N-Methylated peptides of marine origin also exhibit insecticidal and nematicidal properties; discodermin A from sponge Discodermia kiiensis inhibits Bacillus

subtilis, Proteus mirabilis and starfish embryos and polydiscamide from sponge Ircinia sp. shows cytotoxicity against human lung cancer A549 cell line and also inhibits B. subtilis

21. Other marine natural

products containing N-Methyl amino acids are: motuporins (from marine sponge Theonella

swinhoei)18, didemnins (from a tunicate of the genus Trididemnum and family of Didemnidæ)19, cyanobacterial microcystins (from freshwater cyanobacteria belonging to the genera Microcystis, Anabaena, Nostoc, Planktothrix, Anabaenopsis,

Radiocystis and terrestrial cyanobacterium Hapalosiphon) and nodularins (from brackish cyanobacterium Nodularia sp.)20. Thus, N-methylation

__________ *Corresponding author: E-mail: fatehhar@pu.ac.in; fateh_nandel@yahoo.com Tel: 0091-172-253-4125, 0091-9872659689

Fax: 0091-172-254-3101

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is a promising way to improve key pharmacokinetic properties like biological activity, receptor selectivity, enzymatic selectivity, without losing bioactivity1,22-25. N-Methylated peptides have also found application in treatment of diseases, resulting from amyloid-like aggregation, including Prion diseases, type II diabetes mellitus, Huntington’s disease, Parkinson’s disease and Alzheimer’s disease26-29.

Analysis of amino acid sequences of peptides containing N-methylated residues reveals the occurrence of NMe-Val > NMeIle > NMeAla i.e., it is maximum for N-methylated residues having branching at β or γ positions in their side chains. This implies the specific role of N-methylated residues with branched side chains. The N-methylated residues also lack the amide hydrogen, thus precluding the formation of hydrogen bond and it may be the subtle interplay between the directional hydrogen bonds and hydrophobic interactions which imparts these peptides remarkable characteristics.

Due to the wide spectrum utility of N-methylated peptides, much attention has been paid to their synthesis30,31 and the procedure adopted generally includes the chiral centre generation steps32,33. The studies on conformational behavior of poly N-methylated peptides are lacking and majority of the marine peptides containing N-methylated amino acid residues are linear5,21. Here, we report a systematic study of the conformational behavior of poly NMeAla peptides and peptides containing NMeAla and Ala residues at alternate positions with focus on: i) nature of peptide bond geometry and the energy barrier from cis to trans and vice-versa, ii) effect of protecting groups, iii) the type of structure adopted by poly N-methylated peptides, as they lack amide hydrogen, and iv) the interactions stabilizing the adopted structure’s. The elucidation of conformational behavior may aid in the design of potential drugs for specific purposes2.

Computational Methods

Quantum mechanical (QM)

To have the knowledge of global, local and low energy minima, the Φ, Ψ maps and χ potential energy curves for the designed sequences of the NMeAla and Ala were constructed by using standard bond lengths and bond angles34,35. The energy calculations were carried out using the semi-empirical quantum mechanical method PCILO36 (pertubative configuration interaction using localized molecular orbitals) on Sun W, Ultra 5-10; sparc. Energy

minimization was done by the systematic variation of torsion angles, keeping bond lengths and angles constant.

The conformational states for higher poly N-methylated peptides were generated from the knowledge of the global, local and low energy minima in the Φ, Ψ maps and χ curve and their energies were computed. The minimization was further refined by varying Φ, Ψ and χ values in the neighborhood of the minima so obtained in steps of 5 and then in 2 degree steps. In addition, the PCILO result for the peptides containing usual and unusual amino acids were in conformity with ab initio results37,38 and knowledge-based crystallographic data39,40.

Molecular dynamic (MD) simulations

Simulations provide great deal of information with respect to the stability of non-covalent interactions in water. N-Methylated peptides are generally of marine origin, so the MD simulation studies in aqueous systems will throw light on the conformational behavior of these peptides and the structures these peptides adopt will aid in the understanding of their mode of defense action and in designing of potential drug candidates5,21. Simulations were carried out by GROMACS software (version 3.3.1)41 by using force field ffG43a142 which is also suitable for peptides containing unusual amino acids41.

Conformational results by QM calculations were used as the starting geometries for MD simulation studies. The topology files for simulation studies were generated by the Dundee-PRODRG2 server43. Peptide was placed centrally and box was created at a distance of 1 nm from the surface of the protein orthogonally. To perform calculations in an NVT (Number of molecules, Volume and Temperature) ensemble44 with water, maximum box size and minimum water molecules were selected amongst all the conformations of a molecule and afterwards, numbers of water molecules were made to be equivalent with –maxsol

command in all systems. Convergence value (emtol) was set to 1000 kJ mol-1 in energy minimization. The peptide was solvated with water and simple point charge (SPC) water model was used45.

In order to allow equilibration of solvent around the model sequence, position of all residues was restrained for 20 ps at 300 K. MD simulation at 300 K, without any restraints was performed at a constant temperature under NVT conditions. The periodic boundary conditions were applied in all the three dimensions. Time step used was 2 fs46 using Leap

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Frog Algorithm47 and Berendsen scheme48 was used for temperature control using a coupling time constant: τT of 0.1 ps and a reference temperature T0 of 300 K. LINCS algorithm49 was used to restrict all bonds to their equilibrium lengths and the center of mass motion of the system was removed at every step to maintain T0. Pressure was controlled using weak coupling with a time constant of 0.5 ps and a reference pressure of 1 Bar.

For evaluation of coulomb and van der Waals interactions, a cut-off of 0.9 and 1.0 nm, respectively was applied. Long-range forces were updated every 10 fs during generation of the neighbor list. Particle Mesh-Ewald summation method was used to calculate long-range electrostatic interactions50. Initial velocities of all atoms were taken from a Maxwellian distribution at the desired initial temperature. Same force field and other parameters were used in methanol. All atoms of the system were considered explicitly41. All simulations were performed using the NVT ensemble51.

The simulations were carried out in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer52 and parameters for the POPC bilayer systems were taken from lipid.itp, http//moose.bio.ucalgary.ca/

index.php?page=Structures_and_Topologies. The lipid bilayer system consisted of 128 POPC lipids (64 per leaflet) and 2460 water molecules and the peptide Ac-NMeAla6-NHMe in the desired conformation was placed in POPC bilayer system53. The simulations were performed on the pre-equilibrated POPC Protein system for 1 ns and the structural analysis was carried out using VMD software54. Docking of the peptide was performed with the help of Hex 6.12 server55 in shape-cum electrocorrelation type mode. Grid dimension of 0.6 Å was used with ligand and receptor range angle of 180°.

Results and Discussion The results of the conformational behavior of

smaller peptides Ac-NMeAla-NHMe and Ac-NMeAla-NMe2 are summarized in Table 1. It was apparent from the quantum mechanical results that conformational behavior of Ac-NMeAla-NHMe was very labile i.e. it could adopt various conformations with Φ, Ψ values, corresponding to the poly-L-proline type II, collagen type (-23,113˚), left handed (17,70˚) and right handed (-24,-61˚) helices, γ turn (-87,56˚) and helical structures (3,87˚ and 7,-97˚) without

Table 1—Quantum mechanical and simulation results for the model di/tri-peptides

S.N. Φ, Ψ, ω ∆Ea S.N. Φ, Ψ, ω Φ, Ψ, ω ∆Ea

Ac-NMeAla-NHMe Ac-NMeAla2-NHMe

I -23, 113, 180 0.0 I 0, 90, 180 10, 75, 180 0.0 II -24, -61, 180 0.8 II 26, 61, 180 22, 63, 178 2.0 III -87, 56, 180 1.0 III -30, 122, 180 -160, 80,-176 2.1 IV 3, 87, 180 1.3 IV -32, 129, 180 -32, 117,-172 2.3 V 17, 70, 180 1.8 V -170, 80, 178 -165, 77, 180 5.4 VI 7, -97, 180 1.8 VI -157, 71, 180 -95, 110, 180 6.6 VII -160, 85, 180 2.6 VII 50, 76, 24 -14, 107, 176 12.2 VIII -69, 149, 0 4.0 VIII 23, 73, 16 40, 45, 14 26.3 IX -149, 75, 0 5.0 IX 3, 86, -172 33, 75, -17 32.1 X -52, 35, -20 5.0

St gmb simulation St gm simulation

X -108, 124, 7 0.0 IV -119, 138, 156 -103, 149,-167 0.0 I -87, 124, 165 1.3 III -103, 123, 161 -115, 161, 158 1.3 II -89, 119, 167 2.0 VII -128, 93, -171 -121, 112, 163 3.6 III -110, 121,-166 2.8 II 51, 79, 159 -115, 135, 175 3.8 IX -108, 136, -5 5.1 I -121, 102, 168 72, 162, 172 4.8

Ac-NMe-Ala-NMe2 IX -131, 111,-173 63,-178, -8 5.2

I -65, 140, 180 0.0 VIII -93, 151, 172 75, 85, -22 7.1 II -65, 140, 0 3.8

St gm simulation

II -118, 115, 29 0.0 I -131, 127,-170 1.1 akcal/mol, and relative w.r.t most stable state for QM and simulation calculations. bSt gm is starting geometry corresponding to S.N. (Serial Number)

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hydrogen bonding, with amide bond geometry being trans. In other words, the model peptide Ac-NMeAla-NHMe might be realized in different conformations, depending upon environmental conditions and these results were in conformity with the earlier findings56. The states with Φ, Ψ values of -23, 113˚ and -24, -61˚ stabilized by carbonyl-carbonyl interactions are shown in Fig. 1a and b, respectively.

The potential energy curve as a function of ω for the N-methylated amide bond of N-alkyl model peptides Ac-NMeX-NHMe with X = Ala, Val, Ile (Fig. 2) clearly reflected that the N-alkyl amide bond geometry was trans and the barrier to rotation was minimum for NMeAla (~ 4 kcalmol-1) and increased with increase in the bulkiness of the side chain i.e. in going from methyl to iso-butyl side chain. Thus, N-methylation did not support cis conformation57-59 at least in aprotic solvent.

The conformational behavior of model peptide Ac-NMeAla-NMe2 as expected was found to be very

restricted, with Φ, Ψ values corresponding to poly-L-proline type. The simulation results for the peptides Ac-NMeAla-NHMe and Ac-NMeAla-NMe2 under NVT conditions with different starting geometries are summarized in Table 1. Results revealed that due to the interaction of water molecules, the Φ, Ψ values underwent change and this altered the order of stability of the conformers. In all the cases, the molecule adopted structure, with Φ, Ψ values lying in β-sheet region of Ramachandran plot.

Interestingly, simulation studies revealed that in the peptide Ac-NMeAla-NHMe cis amide geometry was favored due to the stronger interaction of water molecules with the carbonyl moiety of the backbone and this also accounted for deviation of ω by ~30˚ (Fig. 3a). It is worth mentioning here that in peptoids where the side chain is shifted from Cα to nitrogen, the amide bond geometry can be cis or trans,

Fig. 1—A graphical view of the peptide Ac-NMeAla-NHMe

Fig. 2—Potential energy curve for ω in N-methylated peptides depicting clearly the trans amide bond geometry

Fig. 3—Molecular view of the peptide Ac-NMeAla-NHMe in the first two most stable states after simulations for 1 ns with Φ,Ψ and ω values of -108, 124, 7˚ and -87, 124, 165˚ with water molecules within 3 Å of peptide surface [Water interactions are more favorable in the trans amide bond geometry, but the cis amide bond geometry is favored due to lesser repulsion between the methyl group of acetyl and tert-nitrogen]

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depending upon the solvent used at the coupling stage between haloacetic acid and amine36. Possibly, nature utilizes this in the cyclization of N-methylated peptides, such as cyclosporins4 and omphalotins5 and both these cyclic peptides contain at least one peptoid residue. Analysis of simulation results as a function of time ruled out the interconversion of peptide bond geometry from cis to trans or trans to cis. It was apparent from the simulation results in Table 1 that for the peptide Ac-NMeAla2-NHMe, trans amide bond geometry was favored over the cis geometry and

for the first three states, the Φ, Ψ values lie in the β region of the Ramachandran map. The state involving conversion of cis to trans, but not from trans to cis lies 3.6 kcalmol-1 higher in energy.

Therefore, conformational behavior of the peptides Ac-NMeAlan-NHMe with n = 4, 6 was investigated by keeping amide bond geometry to be trans and only the results (quantum mechanical) are presented in Table 2. The state with Φ, Ψ values in the neighborhood of 0, 90˚ was populated in all the peptides and was characterized by 3 residues per turn

Table 2—Quantum mechanical results (Φ,Ψ,ω values in degrees) for the model peptides Ac-NMeAlan-NHMe S.N. 1 2 3 4 5 6 7

Ac-NMeAla3-NHMe

I -25,113 180

-7,97 180

-8,96 180

0.0

II -40,135 180

-155,75 180

-26,114 -178

4.4

III 50,70 180

7,82 180

15,70 180

5.7

IV

-37,139 180

-39,135 180

-31,130 180

5.7

V

-161,70 180

-32,125 180

-166,82 180

6.6

VI

-165,80 180

-160,80 180

-162,76 180

8.9

Ac-NMeAla4-NHMe

I -17,104 180

-14,102 180

-11,101 180

-20,111 180

0.0

II -36,129 180

-160,79 176

-24,120 180

-150,67 180

6.5

III -40,135 180

-103,163 -174

-40,140 176

-32,113 -172

7.4

IV

-165,80 180

-32,127 180

-165,80 180

-32,125 180

7.6

Ac-NMeAla5-NHMe

I 16,77 180

0,92 180

-16,104 180

-1,90 180

11,71 180

0.0

II -32,123 180

-155,75 180

-24,117 180

-155,75 178

-32,121 180

7.5

III -165,75 180

-32,126 180

-157,80 180

-39,130 -176

-171,77 180

11.0

IV -57,158 180

-55,160 180

-55,160 180

-60,160 180

-32,120 180

12.8

Ac-NMeAla6-NHMe

I -17,104 180

-22,115 180

-17,103 180

-28,111 180

-7,94 180

13,67 180

0.0

II -26,122 180

-32,128 180

-69,144 -170

-24,115 -178

-32,122 180

-32,114 180

9.4

III -165,80 180

-40,135 180

-165,80 180

-32,125 180

-165,90 180

-24,119 172

12.2

IV

-40,135 180

-156,77 180

-32,122 180

-158,73 180

-32,123 180

-156,70 180

12.5

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and stabilized by carbonyl-carbonyl interactions (dCOi…dOi+1 = 2.23 Å) as depicted in Fig. 4. This kind of structure could be realized in non-polar solvents not capable of hydrogen bonding with the carbonyl-moieties and this state in appearance was similar to the 310 helical structures in peptides and hence was referred as helical structure without hydrogen bond. Earlier, helical structure without hydrogen bond is also reported in α, β dehydropeptides60-62 and in peptoids36. These results were in excellent agreement with the Goodman et al

57, who have shown the existence of the uncharacterized ordered structure for poly N-methylated alanine with trans amide bond geometry in chloroform solution (an aprotic solvent).

The carbonyl-carbonyl interactions can be realized in three motifs — anti-parallel motif, perpendicular motif and sheared parallel motif63-65. In helical state without hydrogen bond, carbonyl-carbonyl interactions correspond to perpendicular motif. It may be mentioned that these interactions have also been reported recently in inorganic systems and others66,67. In silver (I) complexes with 2,6-pyridinediylbis (4-pyridinyl) methanone, all the three carbonyl’s motifs are observed and the strength of carbonyl-carbonyl interactions is comparable to the strength of hydrogen bond68. The collagen-type structure with ~ Φ, Ψ values of -36,124˚ lie 9.4 kcalmol-1 higher in energy and even the states with alternate Φ, Ψ values

of -134, 65˚ and -67,141˚ from crystallographic studies, where the crystal has been grown from methanol/water69 lie high in energy.

Simulations

In order to see the effect of solvent, the simulation studies for the peptides Ac-NMeAlan-NHMe with n = 4, 6 were carried out under NVT conditions with the starting geometries taken from both quantum calculations and the crystallographic data. The results obtained in different solvents (water, methanol and POPC lipid bilayer) summarized in Table 3 revealed the formation of β-strand conformation. Analysis of the results as a function of time showed that opening of the helix started from C-terminal and the driving force for the opening was provided by the interaction of the water molecules with different functional groups. A molecular view of the peptide Ac-NMeAla6-NHMe at different simulation times is shown in Fig. 5a, b respectively, with water molecule within 3 Å of the peptide surface. In methanol, the peptides adopted a perfect β-strand structure, due to strong hydrogen bond (dCO…HOCH3 ~1.6 Å) formation between the methanol hydroxyl group and carbonyl-moieties of the backbone (Fig. 6a), as compared with the interaction with the water molecules.

Simulation studies of the peptide at the lipid/water interface can be of vital importance for drug tailoring properties. The simulation results for the peptide Ac-NMeAla6-NHMe in POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) are given in Table 3. The state with alternate Φ, Ψ values of -67,141˚ and -134, 65˚ was found to be more stable and evolved to adopt a β-strand structure in a short time frame of <20 ps during position-restraint period. The molecular view shown in Fig. 6b clearly depicted that the peptide adopted β-strand structure stabilized by i) carbonyl-carbonyl interactions between the carbonyl groups of peptide backbone and between the peptide backbone carbonyl and the POPC molecule, and ii) hydrogen bond formation between backbone carbonyl at the C-terminal and the water molecules (dO…H

~1.6 Å). Further simulations were carried out for 12 ns period with minor changes in the Φ, Ψ values.

The signature of the CD spectra of the peptide (N-Me-L-Ala)6 was similar to that of the beta sheet structure in α-peptides and the negative band at ~ 220 nm in methanol was red shifted to 228 nm in water by retaining the overall spectrum signature69. This observation might be explained in terms of the interaction of water and methanol molecules with the

Fig. 4—Formation of helical structure without hydrogen bond stabilized by carbonyl-carbonyl interactions between the carbonyl-oxygen of the ith residue and carbonyl-carbon of ith+1 residue for the peptide Ac-NMeAla6-NHMe similar to the 310 helix in peptides

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Fig. 5—Snapshots of the peptide Ac-NMeAla6-NHMe at different simulation times in water reveal the opening of the helical structure without hydrogen bond starting from the C-terminus

Table 3—Simulation results for Ac-NMeAlan-NHMe in different solvents under NVT condition

Solvent St gma 1 2 3 4 5 6 ∆E

Ac-NMeAla4-NHMe

Water I -128,109 178

-106,108 -165

-110,147 175

71,-70 174

0.0

Water II -103,137 -167

-113,127 171

-116,112 -175

-75,141 175

0.2

Water III -130,110 -178

-109,112 -170

-98,137 177

-75,130 175

2.8

Ac-NMeAla6-NHMe

POPC IV -129,133 170

-108,129 178

-104,108 180

-106,143 -171

-114,121 -175

-118,130 -168

0.0

POPC I -73,139 -163

-80,136 171

-112,96 171

-91,122 -176

-92,131 -170

7,91 171

16.8

POPC II -107,126 -156

-99,120 -179

-106,111 -168

-106,103 -178

-87,111 -170

-128,112 174

24.7

POPC III -108,116 175

-110,109 -174

-119,137 167

-117,129 168

-116,83 -178

-118,150 170

42.5

Water I -105,121 -165

-82,127 -175

-108,108 179

-118,132 173

-109,141 175

82,-102 167

0.0

Water III -109,105 -159

-109,105 -179

-99,127 -176

-111,133 177

-100,132 176

-110,94 179

2.5

Water IV -129,104 180

-109,105 173

-126,85 174

-105,143 162

-108,107 -172

-111,101 -177

3.6

Water

II -87,102 -157

-91,136 -174

-81,109 168

-118,129 179

-101,142 -174

8,90 160

6.8

MeOH II -112,115 174

-114,102 173

-120,96 -167

-126,126 -172

-112,117 177

-108,112 172

0.0

MeOH III -126,108 180

-128,112 -179

-108,118 -172

-131,107 166

-111,122 -172

-121,144 -167

3.2

MeOH IV -95,137 179

-141,121 159

-149,97 180

-122,119 -179

-124,101 179

-71,166 170

5.0

MeOH I 117,118 170

-123,126 170

-125,113 172

-119,114 177

-81,135 -175

59,66 178

12.4

aFrom Table 2 POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

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carbonyl groups of the backbone as explained above i.e. methanol was interacting strongly with the backbone than the water molecules. The computational results predicted the formation of a β-strand type conformation which were in good conformity with the CD spectroscopic results69, but were at somewhat variance with the X-ray crystallographic data, where alternate Φ, Ψ values of -134, 65˚ and -67, 141˚ or vice-versa

69 have been reported. The peptide Ac-NMeAla6-NHMe was populated in β-strand conformation in water or in methanol, even with starting geometries having alternate Φ, Ψ values of -134, 65˚ and -67,141˚ or vice-versa. Thus, it was reasonable to think that the X-ray crystallographic results might correspond to intermediate structure realized/arrested in the mixed solvent i.e. methanol-water. In other words, N-methylated alanine peptides having tertiary amide bond were not populated in poly proline or collagen-type structures. Similar kind of conformational behavior was obtained for the peptide H-NMeAla6-COOH without protecting groups in water (Table 4).

It was clear that poly N-methylated peptides adopted β-strand structure in different solvents. These β-strands by interacting with each might lead to the formation of β-sheet which can be explored as biopolymers forming hydrogels, as antibiofoulants70, anticancerous drugs71,72 and as inhibitors of β-sheet type structure formation in neurological diseases like Alzheimer’s26, Parkinson’s and Huntington’s disease, etc27.

Designing of Aβ inhibitor

The N-methylated alanine peptides lacking amide hydrogen are found to adopt amphipathic β-strand structure. These peptides can serve as good candidates for designing of inhibitors for prevention of fibril formation in diseases like Alzheimer’s26 and Parkinson’s, etc27. There are two hydrophobic stretches from residues 16-2273 and from 34-4274,75 in Aβ1-42 protein. Both the stretches prefer structure, with Φ, Ψ values in the second quadrant of the Ramachandran map74. The molecular docking studies of Ac-NMeAla3-NHMe in the β-strand structure with the optimized conformation of Aβ15-42 suggested the absence of interactions between the two molecules. The designed inhibitor must match the conformation in terms of Φ, Ψ values and the overall shape for binding to the target sites. Thus, a peptide composed of N-methylated amino acid residues and a usual amino acid with smaller side chain can be thought of to bind to the amino acid stretch 16-22 of Aβ peptide76. Hence, the conformational behavior of the peptides with alternate sequences of NMeAla and Ala residues was investigated and the results are summarized in Tables 5a and b.

Conformationally, the model tripeptide Ac-NMeAla-Ala-NHMe was found to be very labile as obvious from the results in Tables 5a and b and the peptide Ac-Ala-NMeAla-NHMe was found to adopt a state with Φ, Ψ values of -25,115˚; 15,65˚ for Ala and NMeAla residues, respectively due to the formation of a 10 member hydrogen-bonded ring between the carbonyl-oxygen of the acetyl group and NH of the C terminal amide group. The model heptapeptide Ac-(Ala-NMeAla)3-NHMe with Φ, Ψ values of -25, 115˚ and 15, 65˚ for Ala and NMeAla residues,

Table 4—Conformational results in terms of Φ,Ψ,ω in degrees for the hexapeptide (NMe-L-Ala)6 without protecting groups

S.N. 1 2 3 4 5 6 ∆E I

-,143

0,90 180

-20,110 165

0,90 170

0,95 170

5,75 -170

0.0

II -,143

180,90 180

-170,85 175

180,90 180

180,90 180

-155,65 180

19.2

III -,143

-132,62 -171

-65,143 -177

-136,62 -177

-70,135 -171

-132,62 -171

29.0

St gm

simulation

III -,140

-118,95 174

-106,86 171

-111,115 -171

-116,100 -164

-106,119 167

0.0

II -,142

-108,107 178

-116,121 172

-84,94 -175

-100,129 -160

-128,-159 165

0.3

I -,145

-126,94 178

-83,128 -177

66,67 177

-118,117 -142

72,134 -155

3.0

Fig. 6—Peptide Ac-NMeAla6-NHMe adopts a β-strand structure due to: (a) uniform and strong hydrogen bond formation with methanol molecules, (b) carbonyl-carbonyl interactions between the carbonyl groups of peptide backbone and the POPC molecule.

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respectively was more stable and adopted β-ribbon type structure (Fig. 7) in which alternate 10 member hydrogen-bonded rings were formed between carbonyl oxygen (NMeAla) of the ith residue and HN (Ala) of the ith + 3 residue. This kind of structure could be realized in non-polar solvents and was stabilized by carbonyl-carbonyl interactions i.e. the carbonyl oxygen of the ith residue was interacting with the carbonyl carbon of ith + 1 residue, (dCOi…OCi+1 = 2.2 Å) and the hydrophobic interactions of side chains of ith and ith + 3 residues. In this structure, the carbonyl-carbonyl interactions correspond to perpendicular motif as well as shear parallel motif, depending upon whether the carbonyl-oxygen of NMeAla approached the carbonyl-carbon of Ala or carbonyl-oxygen of Ala approached carbonyl-carbon of NMeAla, respectively.

Simulation results for both the peptides Ac-(Ala-NMeAla)3-NHMe and Ac-(NMeAla-Ala)3-NHMe in Table 5b revealed the formation of folded/bent structure. The folded structure may not be a good candidate for binding to the hydrophobic stretches in Aβ1-42 peptide. The tetrapeptide Ac-(NMeAla-Ala)2-NHMe and pentapeptide Ac-(NMeAla-Ala)2-NMeAla-NHMe were also found

to adopt bent structures after simulations as apparent from the results in Table 6. The Φ, Ψ values of three consecutive residues in peptide Ac-(NMeAla-Ala)3-NHMe were found to lie in β-sheet region of the Ramachandran map and thus, the model tetrapeptide Ac-Ala-NMeAla-Ala-NHMe was found to be the good choice with Φ, Ψ values and adopted collagen type structure after 5 ns simulations in water with different starting geometries (Table 7).

Table 5a—Quantum mechanical and simulation results for peptides containing Ala and NMeAla

S.N. 1 2 ∆E S.N. 1 2 ∆E

Ac-Ala-NMeAla-NHMe

I

-25,115 180

15,65 180

0.0 IV -171,130 178

-167,85 180

5.9

II -22,112 180

-1,87 180

1.6 V -18,-72 -178

-10,-72 -178

7.9

III -172,151 180

175,-80 180

5.4

St Gm MD

III -86,158 171

-110,106 168

0.0 I -137,138 179

85,153 179

4.0

II -119,100 174

-88,106 -159

2.4 V -66,-68 -167

54,-54 -149

10.4

Ac-NMeAla-Ala-NHMe

I -26,112 180

18,62 180

0.0 III -25,-63 -176

-20,-65 180

0.4

II -24,112 180

-23,113 180

0.1 IV -75,60 -178

-75,56 180

0.6

St Gm MD

I -134,84 175

-59,141 172

17.3 II -114,121 -175

-56,119 169

19.1

III -119,109 179

-70,124 -177

18.6 IV -112,120 -166

-140,145 174

19.8

Fig. 7—Formation of a β-ribbon type structure in the peptide Ac-(Ala-NMeAla)3-NHMe with Φ, Ψ values of -25, 115˚ and 15, 65˚ for Ala and NMeAla residues, respectively stabilized by cabonyl-carbonyl interactions and alternate ten member hydrogen bonded ring formation

Table 5bQuantum mechanical and simulation results for peptides containing Ala and NMeAla

S.N. 1 2 3 4 5 6 ∆E

Ac-(Ala-NMeAla)3-NHMe

I -25,115 180

15,65 180

-25,115 180

15,65 180

-25,115 172

15,65 -174

0.0

II -25,115 -176

-1,87 180

-15,105 180

14,67 180

-11,101 178

4,77 -174

0.8

III -30,-60 -170

-17,-73 174

-35,-65 -170

-16,-70 174

-40,-68 -164

-30,-50 -176

19.6

St gm simulation

I -109,140 177

174,68 177

-123,109 -179

67,176 167

-60,137 -178

-120,115 167

0.0

II -83,120 -177

52,-85 -167

-69,137 178

61,22 -176

-53,126 172

65,-111 -171

2.6

III -58,-37 -176

66,-158 180

-126,-48 -178

-112,118 162

-98,145 173

60,97 -169

18.2

Ac-(NMeAla-Ala)3-NHMe

I -20,110 180

-14,102 180

6,80 178

-5,95 174

10,75 -178

-20,110 178

0.0

II 0,90 178

0,90 170

5,70 -174

-15,110 170

22,61 176

20,60 178

3.1

St gm simulation

I -120,123 -177

-61,127 -175

-82,135 -174

-61,111 177

-124,90 25

-130,141 175

0.4

II -152,90 158

-106,124 -160

66,-82 -179

-82,135 172

-104,156 -176

-140,148 179

1.0

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The simulation of Aβ15-42 peptide was carried out for 2 ns in water with starting geometry obtained from spdbv and the Φ, Ψ values for the amino acid residues 16-24 of Aβ15-42 peptide were found to lie in the 2nd quadrant of Ramachandran map; however, Phe20 adopted Φ, Ψ values in the Ist quadrant. The molecular docking studies55,77 of the peptide Ac-Ala-NMeAla-Ala-NHMe in the optimized conformation with the simulated structure of Aβ15-42 peptide suggested that the model tetrapeptide interacted with the Aβ peptide (Fig. 8) through hydrogen bond formation between NH of Val24 and carbonyl-oxygen of Ala3 of the inhibitor (dH…O = 1.72 Å, ∠NHO = 141°) and carbonyl-carbonyl interactions. The carbonyl-carbonyl interactions were between the residues i) Leu17 of Aβ peptide and Ala2 of the inhibitor (dO…C = 3.15 Å), ii) Phe19 of Aβ peptide and Ala2 of the inhibitor (dO…C = 3.09 Å), and iii) Phe19 of Aβ

peptide and Ala1 of the inhibitor (dO…C = 3.14 Å) and the carbonyl…π interactions between acetyl carbonyl group and aromatic ring of Phe20 of Aβ peptide. It may be added that the carbonyl…π interactions have been observed in protein crystals78 and anthraquinones79. The blocking of the hydrophobic stretch may prevent the aggregation of Aβ peptide. The length of the model peptide in the present study was in accordance with the experimental fact that the length of the inhibitor should be less than six residues and with lesser degree of N-methylation80.

Conclusion

Poly N-methylated alanine peptides adopted helical structure without hydrogen bond and were characterized by ~3 residues per turn and stabilized by carbonyl-carbonyl interactions. N-Methylated amide bond geometry was shown to be trans. The simulation studies revealed the opening of the helical structure

Table 6—Quantum mechanical and simulation results for tetrapeptide Ac-(NMeAla-Ala)2-NHMe and pentapeptide

Ac-(NMeAla-Ala)2-NMeAla-NHMe

S.N. 1 2 3 4 5 ∆E Ac-(NMeAla-Ala)2-NHMe

I 0,90 178

0,90 170

5,70 -174

-15,110 170

0.0

II -20,110 180

-14,102 180

6,80 178

-5,95 174

1.1

St gm simulation II -139,117

154 -31,120 165

53,65 164

-145,133 -179

0.0

I -109,138 180

-69,126 -165

55,49 167

-85,144 -177

1.9

Ac-(NMeAla-Ala)2-NMeAla-NHMe I 0,90

178 0,90 170

5,70 -174

-15,110 170

22,61 176

0.0

II -20,110 180

-14,102 180

6,80 178

-5,95 174

10,75 -178

0.9

St gm simulation II -93,104

-174 -61,143 -170

80,165 156

-73,129 -174

-104,144 177

0.0

I 60,172 -161

-61,139 178

-128,120 177

-109,120 178

51,-141 175

2.1

Fig. 8—Molecular view of the designed inhibitor Ac-Ala-NMeAla-Ala-NHMe interacting through various interactions with Aβ15-42 peptide

Table 7—Quantum mechanical and simulation results for the peptide Ac-Ala-NMeAla-Ala-NHMe

QM Simulation

S.N 1 2 3 ∆E St gm 1 2 3 ∆E I

-25,115 180

15,65 180

-25,115 180

0.0

II

-66,130 -171

-122,154 161

-77,138 178

0.0

II -22,112 180

-1,87 180

-22,112 180

0.4 III

-101,137 180

-109,138 178

-80,142 178

0.1

III -172,151 180

175,-80 180

-172,151 180

7.9 I -101,137 180

-103,136 180

80,142 175

0.6

NANDEL & JASWAL: CONFORMATIONAL STUDY OF N-METHYLATED ALANINE PEPTIDES

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without hydrogen bond in water started from the C-terminal due to the interaction of the water molecules with the carbonyl moieties, leading to the formation of the β-strand type conformation. Based on the conformational results, an inhibitor — a model tetrapeptide Ac-Ala-NMe-Ala-Ala-NHMe was designed and was found to interact/block the possible aggregation site of Aβ peptide.

Acknowledgement

RRJ sincerely acknowledges the financial support from Council of Scientific and Industrial Research, Govt. of India, grant 09/135/(0593)/2010/EMR-I.

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