Design, synthesis, and biological evaluation of stable β6.3-Helices: Discovery of non-hemolytic antibacterial peptides

Abstract

Gramicidin A, a topical antibiotic made from alternating L and D amino acids, is characterized by its wide central pore; upon insertion into membranes, it forms channels that disrupts ion gradients. We present helical peptidomimetics with this characteristic wide central pore that have been designed to mimic gramicidin A channels. Mimetics were designed using molecular modeling focused on oligomers of heterochiral dipeptides of proline analogs, in particular azaproline (AzPro). Molecular Dynamics simulations in water confirmed the stability of the designed helices. A sixteen-residue Formyl-(AzPro-Pro)₈-NHCH₂CH₂OH helix was synthesized as well as a full thirty-two residue Cbz-(AzPro-Pro)₁₆-O^tBu channels. No liposomal lysis activity was observed suggesting lack of channel formation, possibly due to inappropriate hydrogen-bonding interactions in the membrane. These peptidomimetics also did not hemolyze red blood cells, unlike gramicidin A.

Keywords: Antibiotic; Azaproline foldamers; Gramicidin; Liposomes and beta-helices; RBC hemolysis.

Figure 1.

a) Sequence structure of Gramicidin A monomer. R = Trp; B, R = Phe; and C, R = Tyr. b) Conformational states of gramicidin A monomers and dimers in phospholipid membranes [19].

Figure 2.

Na+-ion positions (van der Waals surfaces) of the gD–NaI complex [15].

Figure 3.

Multiple conformations determined by NMR and crystallography [25]. A = Solid-state NMR-derived structure of gA from a lipid bilayer environment: single-stranded, right-handed, and 6.5 residues per turn; PDB = 1MAG [26]. B = X-ray crystallographic structure of crystals prepared from Cs⁺/MeOH solution: double-stranded, right-handed, and 7.2 residues per turn; PDB = 1AV2 [27]. C = A solution NMR structure from an SDS micellar environment: single-stranded, right-handed, and 6.3 residues per turn; PDB = 1GRM [28]. D = An X-ray crystallographic structure of crystals prepared from benzene/methanol solution: double-stranded, left-handed, and 5.6 residues per turn; PDB = 1ALZ [29]

Figure 4:

a) Structures of Gramicidin A (gA) Head-to-Head Dimer Helices (PDB ID: 1NRM [50]); side and top view, b) gA Depicted with pore, c) Helical structure of Pro-AzPro 30-residue helix (30 mer) from molecular modeling; side and top view, d) 30 mer Depicted with pore.

Figure 5:

Flowchart of the procedure for testing candidate dimers for helical quality.

Figure 6.

Torsion distribution plots computed from molecular dynamics simulations of the AzPro-Pro oligomer. a) Torsion angles of all proline residues found in the oligomer. b) Backbone torsions of all azaproline residues found in the oligomer. The Φ angle for azaproline was computed using the α-nitrogen in place of the α-carbon.

Figure 7.

a) Ac-Pro-AzPro-Ac helical structure with ethanolamine embedded demonstrating hydrogen bond bridging between turns. b) Top view demonstrating an unoccluded pore.

Figure 8:

a) Helical structure of Ac-(Pro-AzPro)₈-Ac (16 mer-CaCl₂) helix (trapped with calcium ions from molecular modeling; side view and b) top view.

Figure 9:

CD spectrum comparision of gA and its analogous peptides in methanol (100 μM) at 25 °C. a) gramicidin A (gA) and its analogous peptides (1-3, 8, and 10) hexadecamers and 11 is the full-length 32mer. b) gA, its analog 3 and dimer shortened peptides (39–41). c) gA and Cbz-(AzPro-Pro)_n-O^tBu (n = 16(11), 8 (8), 4(18), 2(29) 1(35)) analogous peptides. d) gA and Boc-(AzPro-Pro)_n-Cbz (n = 8 (10), 4(21), 2(49), 1(46)) analogous peptides.

Figure 10:

Comparison of far-UV CD spectrum of gramicidin A (gA) and its analogous peptides in PBS buffer (100 μM) at 25 °C . a) gramicidin A (gA) and its analogous hexadecamers (1-3) and 11 is the full-length 32mer. b) gA, 2, 3, 11 and dimer shortened peptides (5–7).

Figure 11:

RBC hemolysis comparison: a) gA and its hexadecameric analogous peptides. b) gA and shorter peptide analogs.

Figure 12:

Surface-plasmon resonance sensorgrams of the interaction of liposomes with peptide analogs. Dilution-series binding and dose-response plot of the interaction with POPC liposomes. a), b) Peptide 1; c), d) Peptide 2; e), f) Peptide 8 and g), h) Peptide 10.

Figure 13:

Gramicidin A and its analogous peptide-caused lysis of a), b), c) POPC, and d) DOPC liposomes.

Figure 14:

Inhibition percentage of gA (17 μM) and its analogs against five bacterial pathogens.

Figure 15:

Inhibition percentage (C. albicans = blue bars) of gramicidin A and its analogous peptides against two fungal pathogens. Note that growth of C. neoformans was enhanced (red bars)

Scheme 1.

Synthesis of protected Fmoc-AzPro-Pro-O^tBu dipeptide.

Scheme 2.

Solution phase synthesis of Cbz-(AzPro-Pro)_n-O^tBu oligomeric peptides.

Scheme 3.

Generation of a set of formyl-(AzPro-Pro)-oligomers by limited acid hydrolysis in 4N HCl/dioxane followed by addition of C–terminal ethanolamine to form gA peptidomimetics of different lengths (10, 12, 14 & 16).

Scheme 4.

Synthesis of gramicidin A analogous peptides.

Scheme 5.

Synthesis of Boc-(Pro-Aza)₈-Cbz gramicidin analogous peptide.