Discovery, Synthesis, and Optimization of Peptide-Based Antibiotics.

The rise of multidrug resistant bacteria has significantly compromised our supply of antibiotics and poses an alarming medical and economic threat to society. To combat this problem, it is imperative that new antibiotics and treatment modalities be developed, especially those toward which bacteria are less capable of developing resistance. Peptide natural products stand as promising candidates to meet this need as bacterial resistance is typically slow in response to their unique modes of action. They also have additional benefits including favorable modulation of host immune responses and often possess broad-spectrum activity against notoriously treatment resistant bacterial biofilms. Moreover, nature has provided a wealth of peptide-based natural products from a range of sources, including bacteria and fungi, which can be hijacked in order to combat more dangerous clinically relevant infections.This Account highlights recent advances in the total synthesis and development of a range of peptide-based natural product antibiotics and details the medicinal chemistry approaches used to optimize their activity.In the context of antibiotics with potential to treat Gram-positive bacterial infections, this Account covers the synthesis and optimization of the natural products daptomycin, glycocin F, and alamethicin. In particular, the reported synthesis of daptomycin highlights the utility of on-resin ozonolysis for accessing a key kynurenine residue from the canonical amino acid tryptophan. Furthermore, the investigation into glycocin F analogues uncovered a potent lead compound against Lactobacillus plantarum that bears a non-native thioacetal linkage to a N-acetyl-d-glucosamine (GlcNAc) sugar, which is otherwise O-linked in its native form.For mycobacterial infections, this Account covers the synthesis and optimization of teixobactin, callyaerin A, lassomycin, and trichoderin A. The synthesis of callyaerin A, in particular, highlighted the importance of a (Z)-2,3-diaminoacrylamide motif for antimicrobial activity against Mycobacterium tuberculosis, while the synthesis of trichoderin A highlighted the importance of (R)-stereoconfiguration in a key 2-amino-6-hydroxy-4-methyl-8-oxodecanoic acid (AHMOD) residue.Lastly, this Account covers lipopeptide antibiotics bearing activity toward Gram-negative bacterial infections, namely, battacin and paenipeptin C. In both cases, optimization of the N-terminal lipid tails led to the identification of analogues with potent activity toward Escherichia coli and Pseudomonas aeruginosa.

Incorporation of ß-Silicon-ß3-Amino Acids in the Antimicrobial Peptide Alamethicin Provides a 20-Fold Increase in Membrane Permeabilization.

Incorporation of silicon-containing amino acids in peptides is known to endow the peptide with desirable properties such as improved proteolytic stability and increased lipophilicity. In the presented study, we demonstrate that incorporation of ß-silicon-ß3-amino acids into the antimicrobial peptide alamethicin provides the peptide with improved membrane permeabilizing properties. A robust synthetic procedure for the construction of ß-silicon-ß3-amino acids was developed and the amino acid analogues were incorporated into alamethicin at different positions of the hydrophobic face of the amphipathic helix by using SPPS. The incorporation was shown to provide up to 20-fold increase in calcein release as compared with wild-type alamethicin.

Efficient microwave-assisted one shot synthesis of peptaibols using inexpensive coupling reagents.

A diisopropylcarbodiimide/Oxyma (ethyl 2-cyano-2-(hydroxyimino)acetate) coupling cocktail was successfully incorporated into the automated microwave-assisted synthesis of two peptaibols and one analog, whose previously reported syntheses were complicated by steric hindrance. This method utilizes commercially available reagents and affords alamethicin F50/5 and bergofungin D in high yields and purities along with an appreciable reduction of synthesis time and cost when compared to previously reported methods.

Construction of a Ca(2+)-gated artificial channel by fusing alamethicin with a calmodulin-derived extramembrane segment.

Using native chemical ligation, we constructed a Ca(2+)-gated fusion channel protein consisting of alamethicin and the C-terminal domain of calmodulin. At pH 5.4 and in the absence of Ca(2+), this fusion protein yielded a burst-like channel current with no discrete channel conductance levels. However, Ca(2+) significantly lengthened the specific channel open state and increased the mean channel current, while Mg(2+) produced no significant changes in the channel current. On the basis of 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescent measurement, Ca(2+)-stimulated gating may be related to an increased surface hydrophobicity of the extramembrane segment of the fusion protein.

Total synthesis of Septocylindrin B and C-terminus modified analogues.

The total synthesis is reported of the peptaibol Septocylindrin B which is related to the well documented channel forming peptaibol antibiotic Alamethicin. Several analogues were synthesized with a modified C-terminus, to investigate the SAR of the terminal residue Phaol. All these peptides were tested for their membrane perturbation properties by fluorescent dye leakage assay and for their antibacterial activity.

Metal-assisted channel stabilization: disposition of a single histidine on the N-terminus of alamethicin yields channels with extraordinarily long lifetimes.

Alamethicin, a member of the peptaibol family of antibiotics, is a typical channel-forming peptide with a helical structure. The self-assembly of the peptide in the membranes yields voltage-dependent channels. In this study, three alamethicin analogs possessing a charged residue (His, Lys, or Glu) on their N-termini were designed with the expectation of stabilizing the transmembrane structure. A slight elongation of channel lifetime was observed for the Lys and Glu analogs. On the other hand, extensive stabilization of certain channel open states was observed for the His analog. This stabilization was predominantly observed in the presence of metal ions such as Zn(2+), suggesting that metal coordination with His facilitates the formation of a supramolecular assembly in the membranes. Channel stability was greatly diminished by acetylation of the N-terminal amino group, indicating that the N-terminal amino group also plays an important role in metal coordination.

An automatic solid-phase synthesis of peptaibols.

An automated approach to peptaibols using microwave-assisted solid-phase peptide synthesis is demonstrated with a combination of HBTU and acid fluoride mediated couplings for normal and alpha,alpha-dialkylated amino acids, respectively. The method is utilized for the automated synthesis of several full-length peptaibols, including alamethicin, tylopeptin, ampullosporin, bergofungin, cervinin, trikoningin, trichogin, and peptaibolin, reducing both synthesis time and costs significantly as compared to other approaches. Furthermore, the use of noncommercially available reagents is minimized.

Total syntheses in solution of TOAC-labelled alamethicin F50/5 analogues.

Total syntheses in solution of a set of four selected analogues of the 19-mer component F50/5 of alamethicin, the most extensively studied among the channel-former peptaibol antibiotics, are planned and reported. All analogues bear three Glu(OMe) residues, replacing the Gln residues at positions 7, 18, and 19 of the naturally occurring compound. Three analogues are mono-labelled with the free-radical-containing amino acid residue TOAC at the strategic positions 1, 8, or 16. The fourth analogue is bis-labelled with the same EPR-active residue at both positions 1 and 16. In the native sequence, all of the positions where TOAC replacements have been introduced are characterized by residues of Aib, the prototype of the class of helicogenic C(alpha)-tetrasubstituted alpha-amino acids. All of the TOAC analogues synthesized exhibit significant membrane-modifying properties.

Total synthesis in solution of alamethicin F50/5 by an easily tunable segment condensation approach.

A total synthesis in solution of the 19-mer peptide component F50/5 of alamethicin, the most extensively investigated among the channel-former peptaibol antibiotics, is reported. Three peptide segments (A, B, C) were prepared and assembled, followed by incorporation of the acetylated N-terminal amino acid. The synthetic modules B and C are characterized by three Glu(OMe) residues (at positions 7, 18, and 19) that, after completion of the synthesis, were reacted with ammonia to provide alamethicin F50/5. By use of this general strategy, we also prepared the [Gln7, Glu(OMe)18,19] alamethicin F50/5 analogue. The purity and conformation of the final products were assessed by chromatographic, spectrometric, and spectroscopic techniques. This tunable segment condensation approach will pave the way for an easy synthesis of alamethicin analogues bearing amino acid residues with desired side-chain probes even at the N-terminus and in internal positions of the sequence.

Engineering charge selectivity in model ion channels.

Most ion channel proteins exhibit some degree of charge selectivity, that is, an ability to conduct ions of one charge more efficiently than ions of the opposite charge. The structural origins of charge selectivity remain incompletely understood despite recent advances in the determination of cation-selective and anion-selective channel protein structures. Helix bundle channels formed via self-assembly of the peptide alamethicin provide a tractable model system for exploring the structural basis of charge selectivity. We synthesized covalently-linked alamethicin dimers, with amino acid substitutions at position 18 [lysine (Lys), arginine (Arg), glutamine (Gln), 2,3-diaminopropionic acid (Dpr)] in each helix, to assess the role of this position as a charge-selectivity determinant in alamethicin channels. Of the position 18 substitutions investigated, the Lys derivative exhibited the greatest degree of anion selectivity. Arg-containing channels were slightly less anion-selective than Lys. Interestingly, Dpr channels showed cation selectivity nearly equivalent to that exhibited by the neutral Gln derivative. We suggest that this result is due to a wider pore diameter that permits a greater number of counter-ions leading to enhanced charge screening and a lower effective side-chain positive charge.

Detection of protein-ligand interaction on the membranes using C-terminus biotin-tagged alamethicin.

C-terminal biotin-tagged alamethicin, which has several alpha-aminoisobutyric acid (Aib) residues in its sequence, was synthesized by the preparation of the protected peptide segment using the 2-chlorotrityl resin, followed by conjugation with biotin hydrazide. Suppression of the channel current of the biotin-tagged alamethicin by the addition of streptavidin to the electrolyte was monitorable in real time using the planar lipid-bilayer method. The system was also applicable to the detection of interaction of the biotin-tagged alamethicin with the anti-biotin antibody.

Synthesis and ion conductance behavior of a tetrameric alamethicin ion channel.

[structure: see text] A porphyrin-tethered construct, containing four full-length alamethicin monomers, has been synthesized and characterized. The ion conductance data of the assembly in 1 M HCl display long-lived, albeit noisy, channels that appear to be voltage-independent multiples of only one conductance state. The noise in the data is consistent with the molecular modeling studies, which indicate that the side chain of glutamine 7 of alamethicin does not fit well into the narrow pore of a parallel four-helix bundle.

An anion-selective analogue of the channel-forming peptide alamethicin.

The peptide alamethicin self-assembles to form helix bundle ion channels in membranes. Previous macroscopic measurements have shown that these channels are mildly cation-selective. Models indicate that a source of cation selectivity is a zone of partial negative charge toward the C-terminal end of the peptide. We synthesized an alamethicin derivative with a lysine in this zone (replacing the glutamine at position 18 in the sequence). Microscopic (single-channel) measurements demonstrate that dimeric alamethicin-lysine18 (alm-K18) forms mildly anion-selective channels under conditions where channels formed by the parent peptide are cation-selective. Long-range electrostatic interactions can explain the inversion of ion selectivity and the conductance properties of alamethicin channels.

Structure-function relationships in helix-bundle channels probed via total chemical synthesis of alamethicin dimers: effects of a Gln7 to Asn7 mutation.

Alamethicin channels are prototypical helix bundles that may serve as tractable models for more complex protein ion channels. Solid-phase peptide synthesis of alamethicin analogues using FMOC-amino acid fluorides followed by chemical dimerization of these peptides facilitates structure-function studies of particular channel states in bilayer membranes. State 3 in particular, tentatively assigned to a hexameric helix bundle, is sufficiently long-lived that current-voltage measurements can be made during the lifetime of an individual channel opening. Molecular models of hexameric helix bundles, generated using restrained molecular dynamics with simulated annealing, indicate that a Gln7-->Asn7 (Q7-->N7) mutation will increase channel diameter locally. Experimentally, the conductance of state 3 of the N7-alm channel is found to be larger than that of the Q7-alm channel when ion flow is in the usual direction (cations entering the C-terminal end of the channel). When ion flow is in the opposite direction, no difference in the conductances of state 3 of Q7 and state 3 of N7 channels is observed. These results indicate that the effect of a change in pore diameter at position 7 is dependent on the magnitude of other barriers to permeation and that these barriers are voltage-dependent.

Ferrocenoyl derivatives of alamethicin: redox-sensitive ion channels.

The synthesis and single-channel characterization of two redox-active C-terminal derivatives of alamethicin are herein described. The reduced [Fe(II)] forms of ferrocenoyl-alamethicin (Fc-ALM) and 1'-carboxyferrocenoyl-alamethicin (cFc-ALM) are shown to form voltage-dependent ion channels at cis positive potentials in planar lipid bilayers (PLB) with conductance properties similar to those of alamethicin. In situ oxidation of Fc-ALM [to Fe(III)] in the PLB apparatus causes a time-dependent elimination of channel openings, which can be restored by an increase in the transbilayer potential. In contrast, oxidation of cFc-ALM leads to the formation of shorter-lived channels. Pretreatment of the ferrocenoyl peptides with oxidizing agent alters their single-channel properties in a qualitatively similar manner, establishing that the changes in channel properties in the presence of oxidizing agents are due specifically to ferrocenoyl oxidation. We suggest that the redox sensitivity of these ferrocene-containing ion channels may be governed by a combination of the following factors: (1) changes in hydrophobicity; (2) alteration of peptide molecular dipole; and (3) alterations in tendencies toward self-association. However, oxidation induced changes in peptide conformation cannot be ruled out. Our results provide evidence that it is possible to engineer channel-forming peptides that respond to specific changes in the chemical environment.

Engineering stabilized ion channels: covalent dimers of alamethicin.

The peptide alamethicin forms channels with a variety of conductance states. Selective stabilization of a particular state should simplify the task of understanding conductance in terms of channel structure. We synthesized two different covalent dimers of alamethicin in which peptides were linked at their C-terminal ends by flexible tethers. Both dimeric peptides formed channels with conductances that matched those of alamethicin channels. Particular conductance states were selectively stabilized, however, with lifetimes up to 170-fold longer than the same states observed with monomers. In addition, tethering appeared to limit the size of the structures formed so that, even at higher peptide concentrations, a single predominant conductance state was obtained. We suggest this state corresponds to a channel made from six alamethicin molecules (three dimers).

Ion channel stabilization of synthetic alamethicin analogs by rings of inter-helix H-bonds.

Rings of inter-helix H-bonds due to Gln at position 7, a highly conserved residue in all pore-forming peptaibols, have been suggested to play an important role in the stabilization of alamethicin channels. In an attempt to test this hypothesis, experimental studies have been undertaken on four synthetic alamethicin non-Aib analogs (Alm-dUL) in which the Gln at position 7 (Q7) is substituted by Ala, Asn, or Ser (Q7A, Q7N, or Q7S). Voltage-dependent pore formation by these analogs in planar lipid bilayers is compared at the macroscopic and single-channel conductance levels. As anticipated, the Q7A substitution abolished all channel-forming activity. The voltage dependence of macroscopic current-voltage curves was conserved with the Q7N substitution but reduced in the Q7S analog. Normalized single-channel conductance ratios between substates follow the same pattern, with the Q7S analog yielding the highest unit conductances. Channel lifetimes were the most significantly modulated parameter with markedly faster kinetics when Gln or Asn was replaced by Ser. The effect of the Q7S substitution on channel lifetimes may be explained through a reduced stabilization of bundles by inter-helix H-bonds.

Membrane structure of voltage-gated channel forming peptides by site-directed spin-labeling.

Three spin-labeled derivatives of the voltage-gated peptide alamethicin were prepared with nitroxides at the C-terminal phenyalaninol, and at positions 9 and 15 in the amino acid sequence. In addition, three spin-labeled derivatives of an analog of alamethicin where alpha-methylalanine residues are replaced by leucine were prepared with nitroxide labels at the same positions. Continuous wave power saturation EPR spectroscopy was used to examine the effect of molecular oxygen and water soluble paramagnetic reagents on the saturation behavior of the labeled peptides. Using the gradients of these species which exist through the membrane-solution interface, distances for these nitroxide derivatives from the membrane-solution interface were estimated. The distances show that alamethicin is inserted along the bilayer normal with the C-terminus of the peptide lying in the aqueous solution 3 or 4 A from the membrane interface. In this configuration alamethicin does not completely cross the bilayer, and the N-terminus of alamethicin is within the membrane hydrocarbon approximately 16 A from the phosphate groups on the opposing interface. The analog where leucines replace alpha-methylalanines shows a similar conformation, except that the entire peptide is translated 3-4 A deeper into the membrane than is native alamethicin. The distances that are measured for alamethicin using EPR are consistent with a linear high resolution NMR structure determined in SDS and the X-ray crystal structure. The membrane position and structure of alamethicin found here limit the likely models for voltage-gating of this peptide.

Membrane orientation of the N-terminal segment of alamethicin determined by solid-state 15N NMR.

Alamethicin was synthesized with 15N incorporated into alanine at position 6 in the peptide sequence. In dispersions of hydrated dimyristoylphosphatidylcholine, solid-state 15N NMR yields an axially symmetric powder pattern indicating that the peptide is reorienting with a single axis of symmetry when associated with lamellar lipids. When incorporated into bilayers that are uniformly oriented with the bilayer normal parallel to the B(o) field, the position of the observed 15N chemical shift is 171 ppm. This is coincident with the sigma parallel to edge of the axially symmetric powder pattern for non-oriented hydrated samples. Thus the axis of motional averaging lies along the bilayer normal. Two-dimensional separated local field spectra were obtained that provide a measure of the N-H dipolar coupling in one dimension and the 15N chemical shift in the other. These data yield a dipolar coupling of 17 kHz corresponding to an average angle of 24 degrees for the N-H bond with respect to the B(o) field axis. An analysis of the possible structures and orientations that could produce the observed spectral parameters show that these values are consistent with an alpha-helical conformation inserted along the bilayer normal.

Conformational study of a synthetic analogue of alamethicin. Influence of the conformation on ion-channel lifetimes.

Alamethicin, a 20-residue peptaibol, induces voltage-dependent ion channels in lipid bilayers according to the barrel-stave model. A synthetic analogue (L2) in which all Aib were replaced by Leu shows a conductance behaviour similar to alamethicin, but channel lifetimes are drastically reduced. Among several hypotheses, a different conformation for L2 might be responsible for this phenomenon by increasing the alpha-helical content (alamethicin presents some 3.0(10)-helical parts) and thus decreasing the length of the transmembrane part. A conformational study of L2 was undertaken using FTIR, CD and NMR spectroscopy, and the secondary structure was compared with alamethicin. These techniques showed an enhanced predominant helical structure as compared to alamethicin. Moreover, the NOE pattern showed an exclusively alpha-helical conformation, resulting in a smaller length of the L2 peptide. This shortening somewhat impedes the complete crossing of the membrane, and could then explain the reduction of its ion-channel lifetimes.