In silico design of peptides equilibrated in a lipid bilayer with partition free energies indicating probability of antimicrobial activity

Abstract

The present invention provides an evaluating system and the use thereof for the efficacy of antimicrobial peptide, which includes the following steps: (a) constructing a peptide by a first input unit, and load the peptide into an aqueous solution for a first time for equilibration; (b) constructing a lipid bilayer by a second input unit, and load the lipid bilayer into an aqueous solution for a second time for equilibration; (c) Using a first processing unit, simulations are carried out for an aqueous system containing an equilibrated peptide from the first input unit and the equilibrated lipid bilayer from the second input unit; (d) calculating the partition free energy of the peptide by a second processing unit; (e) outputting the prediction by an output unit, wherein the output unit is connected with the first processing unit and the second processing unit.

Claims

1. A method for predicting antimicrobial activity of peptides based on an initial peptide, the initial peptide having a plurality of amino acids, the number of the plurality of amino acids being N1, comprising in silico: (a1) designing a plurality of peptides based on the initial peptide by stepwise circularly reshuffling, wherein the number of the plurality of designed peptides is N11; wherein the number of amino acids of each of the plurality of designed peptides is N1; wherein, if the first one of the amino acids is connected to the last one of the amino acids in each of the peptides, then all the resulting peptides are the same; and wherein the plurality of designed peptides are isomeric peptides; (a2) equilibrating the designed peptides and the initial peptide in an aqueous solution for a first time; (b) designing a lipid bilayer comprising hydrophobic lipids tails and equilibrating the lipid bilayer in the aqueous solution; (c) conducting, for each of the designed peptides and the initial peptide individually, a molecular dynamics (MD) simulation in the aqueous solution in the presence of the equilibrated lipid bilayer; (d) calculating: for each of the designed peptides and the initial peptide, <Ni> as a moving average of the number of heavy atoms in the calculated peptide in contact with the hydrophobic lipid tails during an entire 4 ns window; <No> as a moving average of the number of heavy atoms in the calculated peptide not in contact with the hydrophobic lipid tails during the entire 4 ns window; and a partition free energy based on <N.sub.i> and <No>; wherein the heavy atoms are all non-hydrogen atoms; and (e) outputting a set of designed peptides each having partition free energies lower than the partition free energy of the initial peptide, wherein each such lower partition free energy indicates higher probability of increased antimicrobial activity of the set of designed peptides as compared to the initial peptide.

2. The method of claim 1, wherein step (a1) comprises using PyMol or Discovery Studio Visualizer software; and the MD simulation of step (c) is conducted under physiological conditions by NAMD, VMD, AMBER or GROMACS software with a force field comprising CHARMM, AMBER, GROMOS or OPLS.

3. The method of claim 1, wherein the step (e) comprises: (e1) comparing the partition free energies of the designed peptides with the partition free energy of the initial peptide; and (e2) outputting the partition free energies of any designed peptides which are lower than or equal to the partition free energy of the initial peptide.

4. The method of claim 1, wherein conducting, for each of the designed peptides and the initial peptide individually, the MD simulation in step (c) comprises: making a hydrophobic side of the conducted peptide face the equilibrated lipid bilayer; positioning projections of mass centers of the conducted peptide and the lipid bilayer on an axis perpendicular to the lipid bilayer, wherein the mass centers of the conducted peptide and the lipid bilayer are 35 ngstroms apart from each other; pulling the positioned peptide by a weak force to a level a few Angstroms below an average level of phosphor atoms in an upper leaflet of the lipid bilayer; holding the pulled peptide; releasing the held peptide; and making the released peptide float onto a surface of the lipid bilayer and equilibrate for 10 to 35 nanoseconds.

5. The method of claim 1, wherein, in step (d), the partition free energy is calculated based on G.sub.p=k.sub.BT ln(<N.sub.i>/<N.sub.o>) or G.sub.p=k.sub.BT(<ln N.sub.i>/<ln N.sub.o>), where G.sub.p is the partition free energy, k.sub.B is the Boltzmann constant, T is the absolute temperature, N.sub.i is the number of heavy atoms in the calculated peptide that contact the hydrophobic lipid tails, N.sub.o is the number of heavy atoms in the calculated peptide that do not contact the hydrophobic lipid tails, and the < > symbol denotes a moving averages during the 4 ns window.

6. A method for predicting antimicrobial activity of a peptide, comprising in silico: (a1) designing the peptide; (a2) equilibrating the peptide in an aqueous solution; (b) designing and equilibrating a lipid bilayer in the aqueous solution, wherein the lipid bilayer has a plurality of hydrophobic lipid tails; (c) conducting molecular dynamics (MD) simulation in the aqueous solution with the peptide and the lipid bilayer; (d) calculating: <Ni> as a moving average of the number of heavy atoms in the peptide in contact with hydrophobic lipid tails during an entire 4 ns window; <No> that is moving average of the numbers of heavy atoms in the peptide not in contact with the hydrophobic lipid tails during the entire 4 ns window; and a partition free energy based on <Ni> and <No>; wherein the heavy atoms are all non-hydrogen atoms; and (e) outputting the partition free energy for the peptide, wherein relative low partition free energy correlates with increased antimicrobial activity.

7. The method of claim 6, wherein step (c) includes: making a hydrophobic side of the peptide face the equilibrated lipid bilayer; positioning projections of a mass center of the peptide on an axis perpendicular to the equilibrated lipid bilayer, wherein the mass center of the peptide and the lipid bilayer are 35 ngstroms apart from each other; pulling the peptide by a weak force to a level a few Angstroms below an average level of phosphor atoms in an upper leaflet of the lipid bilayer; holding the pulled peptide; releasing the held peptide; and making the released peptide float onto a surface of the lipid bilayer and equilibrate 10 to 35 nanoseconds.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) The FIG. 1 is the simple figure of the present invention.

(3) The FIG. 2 is the flow chart of the molecular dynamics (MD) simulations system of the present invention.

(4) The FIG. 3 is the figure of the peptide insertion process observed in the molecular dynamics (MD) simulations system of the present invention.

(5) The FIG. 4 is the figure of the hemolytic activities of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

(6) For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections that follow.

(7) Experimental Methods:

(8) The antimicrobial peptide of the present invention is as the following formula: xAP1yBP2zCn(P3vD) (formula I); where the x, y, z, v and n are all positive integers; and x=06, y=0 or 4, z=06, v=04 and n=0 or 1; each of P1, P2 and P3 is -Trp-Leu-Lys-; A, B and C are selected from the group of Trp, Leu and Lys and D is Lys.

(9) In the best embodiment, the result of molecular dynamics (MD) simulations for a series of AMP variants is shown in Table 1.

(10) TABLE-US-00001 TABLE 1 the simulation results for an antimicrobial peptide family (SCR variants) Antimicrobial activity Simulation data (MIC) (g/ml) Peptide G.sub.P Predicted Experimental numbers <Ni> <No> (kcal/mol) values values SEQ ID NO. 1 25.55 107.45 0.88 6.06 3.13 SEQ ID NO. 2 30.03 102.98 0.75 4.39 6.25 SEQ ID NO. 3 29.85 103.15 0.76 4.45 6.25 SEQ ID NO. 4 17.53 115.48 1.16 9.80 12.5 SEQ ID NO. 5 13.93 119.08 1.32 11.98 12.5 SEQ ID NO. 6 28.70 104.30 0.79 4.84 6.25 SEQ ID NO. 7 15.90 117.10 1.22 10.68 12.5 SEQ ID NO. 8 22.78 110.23 0.96 7.21 6.25 SEQ ID NO. 9 16.83 116.63 1.20 10.45 6.25 SEQ ID NO. 10 29.68 103.03 0.76 4.51 6.25 SEQ ID NO. 11 29.98 103.03 0.76 4.40 1.56 SEQ ID NO. 12 37.38 95.63 0.57 1.94 1.56 SEQ ID NO. 13 36.88 96.13 0.59 2.10 1.56 G.sub.P is the partition free energy.

(11) In the best embodiment, the antimicrobial activity of the antimicrobial peptide is shown in Table 2.

(12) MIC: Minimal Inhibitory Concentration of AMPs in MH Medium Against E. coli ATCC 25922

(13) Minimal inhibitory concentration (MIC) represents for the lowest concentration of peptide at which the AMP can still kill 90% of the bacteria. All MIC tests for WLK peptide series SEQ ID NO:1-13 were measured against Escherichia coli strain (ATCC 25922) in triplicate. Peptide concentration was determined by UV spectrophotometer (Ultrospec 1100 pro from Amersham Biosciences) at 280 nm with proper extinction coefficient. (Gill S. C. & Von Hippel P. H., Analytical biochemistry 1989, 182(2), 319-326). All of the samples were prepared from a stock solution containing peptides of concentrations 5, 2.5, 1.25, 0.625, 0.313, 0.156 and 0.078 mg/ml. Bacteria culture in the middle of logarithmic growth at a concentration of 510.sup.5 colony-formation unit (CFU) and protein solution at the aforementioned concentrations were uniformly mixed in a 96-well culture plate (each well contains 1 microliter protein solution and 99 microliter of bacteria culture). After growing the culture at 37 C. for 16 hours, how bacterial growth was inhibited was measured by Thermo Max (Molecular Devices) at the wavelength of 600 nm. The MIC values were determined by triplicates of the above experiments.

(14) Hemolysis Activity Test

(15) Hemolytic activity was determined by measuring hemolysis of human red blood cells. Fresh red blood cells and PBS buffer solution (pH 7.4) was mixed uniformly and centrifugated by relative centrigugal force (RCF) 800 g rpm for 10 minutes, washed and then diluted by the PBS buffer solution to 10% concentration by volume (v/v). All the tested peptides were prepared with different concentrations (800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56 g/ml) and then mixed with PBS buffer in 1:1 ratio (100 L:100 L) by volume. In 1:1 ratio (v/v), the red blood cell solution was mixed with a PBS buffer, with or without 2% Triton X-100, respectively as the positive and negative controls. All samples to be tested were settled at 37 C. environment for 1 hour. Next, the specimen was centrifuged under RCF 800 g for 10 minutes and the absorbance of its supernatant was measured at a wavelength of 450 nm. The hemolytic activity would be measured according to the following formula:
% hemolysis=[A.sub.sampleA.sub.PBS]/[A.sub.TritonX100A.sub.PBS]

(16) TABLE-US-00002 TABLE2 The hemolytic activity, salt resistance, and antimicrobial acitivity of the present invention MIC (g/ml) MIC(g/ml)inLYMmedium inMH NaClconcentration(nM) AMPs Sequence <H> medium control 50 100 200 300 SEQIDNO.1 KKWLKWLKWLKKK 0.647 3.13 1.56 1.56 3.13 6.25 6.25 SEQIDNO.2 KWLKWLKWLKKKK 0.506 6.25 1.56 1.56 6.25 12.5 12.5 SEQIDNO.3 WLKWLKWLKKKKK 0.575 6.25 1.56 3.13 3.13 12.5 25 SEQIDNO.4 LKWLKWLKKKKKW 0.417 12.5 1.56 3.13 3.13 12.5 50 SEQIDNO.5 KWLKWLKKKKKWL 0.663 12.5 3.13 3.13 12.5 25 50 SEQIDNO.6 WLKWLKKKKKWLK 0.700 6.25 1.56 3.13 6.25 12.5 25 SEQIDNO.7 LKWLKKKKKWLKW 0.454 12.5 3.13 3.13 6.25 12.5 50 SEQIDNO.8 KWLKKKKKWLKWL 0.682 6.25 3.13 3.13 3.13 6.25 12.5 SEQIDNO.9 WLKKKKKWLKWLK 0.678 6.25 3.13 3.13 3.13 12.5 50 SEQIDNO.10 LKKKKKWLKWLKW 0.371 6.25 3.13 3.13 3.13 6.25 25 SEQIDNO.11 KKKKKWLKWLKWL 0.555 1.56 1.56 1.56 1.56 1.56 3.13 SEQIDNO.12 KKKKWLKWLKWLK 0.518 1.56 1.56 1.56 3.13 3.13 3.13 SEQIDNO.13 KKKWLKWLKWLKK 0.661 1.56 1.56 3.13 1.56 3.13 12.5

(17) The hemolytic activity, salt resistance, and antimicrobial activity of the novelly designed 12 isomeric peptides (SEQ ID NO: 213) based on the original helical AMP of SEQ ID NO: 1 by the process of stepwise circular reshuffling (SCR) were shown in Table 2. The sink and surface simulation strategy and G.sub.p calculation were used to measure the antimicrobial activity, and the relationship between (G.sub.p) and antimicrobial activity (demonstrated by MIC) of the 13 isomeric peptides (including the original sequence) was analyzed, wherein the correlation coefficient was found greater than 0.8 (MIC=13.50*G.sub.p5.79, R=0.84). Table 2 shows that the 13 peptides in high salt concentration still had high inhibitory effects. Besides, 3 out of 12 peptides were found to have a better antimicrobial activity than the original sequence in the present invention, wherein SEQ ID NO: 12 showed lower hemolytic activity on human erythrocytes. As a result, the current invention provides a new platform whereby a repertoire of more effective antimicrobial peptides can be rationally designed based on known antimicrobial peptides, and their bactericidal activities can be predicted by herein presented computational methods.