Mechanism of action and specificity of antimicrobial peptides designed based on buforin IIb

Buforin IIb-a synthetic analog of buforin II that contains a proline hinge between the two α-helices and a model α-helical sequence at the C-terminus (3× RLLR)-is a potent cell-penetrating antimicrobial peptide. To develop novel antimicrobial peptides with enhanced activities and specificity/therapeutic index, we designed several analogs (Buf III analogs) by substitutions of amino acids in the proline hinge region and two α-helices of buforin IIb, and examined their antimicrobial activity and mechanism of action. The substitution of hydrophobic residues ([F(6)] and [V(8)]) in the proline hinge region with other hydrophobic residues ([W(6)] and [I(8)]) did not affect antimicrobial activity, while the substitution of the first four amino acids RAGL with a model α-helical sequence increased the antimicrobial activity up to 2-fold. Like buforin IIb, Buf III analogs penetrated the bacterial cell membranes without significantly permeabilizing them and were accumulated inside Escherichia coli. Buf III analogs were shown to bind DNA in vitro and the DNA binding affinity of the peptides correlated linearly with their antimicrobial potency. Among the Buf III analogs, the therapeutic index of Buf IIIb and IIIc (RVVRQWPIG[RVVR](3) and KLLKQWPIG[KLLK](3), respectively) were improved 7-fold compared to that of buforin IIb. These results indicate that Buf III analogs appear to be promising candidates for future development as novel antimicrobial agents.     

Shape-selective recognition of DNA abasic sites by metallohelices: inhibition of human AP endonuclease?1

Loss of a base in DNA leading to creation of an abasic (AP) site leaving a deoxyribose residue in the strand, is a frequent lesion that may occur spontaneously or under the action of various physical and chemical agents. Progress in the understanding of the chemistry and enzymology of abasic DNA largely relies upon the study of AP sites in synthetic duplexes. We report here on interactions of diastereomerically pure metallo–helical ‘flexicate’ complexes, bimetallic triple-stranded ferro-helicates [Fe₂(NN-NN)₃]⁴⁺ incorporating the common NN–NN bis(bidentate) helicand, with short DNA duplexes containing AP sites in different sequence contexts. The results show that the flexicates bind to AP sites in DNA duplexes in a shape-selective manner. They preferentially bind to AP sites flanked by purines on both sides and their binding is enhanced when a pyrimidine is placed in opposite orientation to the lesion. Notably, the Λ-enantiomer binds to all tested AP sites with higher affinity than the Δ-enantiomer. In addition, the binding of the flexicates to AP sites inhibits the activity of human AP endonuclease 1, which is as a valid anticancer drug target. Hence, this finding indicates the potential of utilizing well-defined metallo–helical complexes for cancer chemotherapy.     

Recent Advances in Stereoselective Drug Targeting

Reviewed here are some recent examples of medically important protein targets for which stereoselective drugs have been identified. These include heat shock protein 90 (Hsp90) inhibitors as anticancer agents; transient receptor potential vanilloid type 1 antagonists as new analgesics; stereospecific inhibition of human mutT homolog MTH1 for cancer treatment; the stereoselective binding of R‐ and S‐propranolol by the α1–acid glycoprotein transporter; metallohelical complexes that are nonpeptide α‐helical mimetics that enantioselectively target Aβ amyloid for the treatment of Alzheimer's disease; metallohelical assemblies with promising antimicrobial activity that enantioselectively target DNA of resistant bacteria; nonpeptide α‐helical metallohelices that target the DNA of cisplatin‐resistant cancer cells; diastereomeric selectivity of phenanthriplatin‐guanine adducts; and phenazine biosynthetic enzyme active sites that can host both enantiomers of a racemic ligand simultaneously. Chirality 27:589–597, 2015. © 2015 Wiley Periodicals, Inc.     

IQ-motif peptides as novel anti-microbial agents

The IQ-motif is an amphipathic, often positively charged, α-helical, calmodulin binding sequence found in a number of eukaryote signalling, transport and cytoskeletal proteins. They share common biophysical characteristics with established, cationic α-helical antimicrobial peptides, such as the human cathelicidin LL-37. Therefore, we tested eight peptides encoding the sequences of IQ-motifs derived from the human cytoskeletal scaffolding proteins IQGAP2 and IQGAP3. Some of these peptides were able to inhibit the growth of Escherichia coli and Staphylococcus aureus with minimal inhibitory concentrations (MIC) comparable to LL-37. In addition some IQ-motifs had activity against the fungus Candida albicans. This antimicrobial activity is combined with low haemolytic activity (comparable to, or lower than, that of LL-37). Those IQ-motifs with anti-microbial activity tended to be able to bind to lipopolysaccharide. Some of these were also able to permeabilise the cell membranes of both Gram positive and Gram negative bacteria. These results demonstrate that IQ-motifs are viable lead sequences for the identification and optimisation of novel anti-microbial peptides. Thus, further investigation of the anti-microbial properties of this diverse group of sequences is merited.     

Divorcing folding from function: How acylation affects the membrane-perturbing properties of an antimicrobial peptide

Many small cationic peptides, which are unstructured in aqueous solution, have antimicrobial properties. These properties are assumed to be linked to their ability to permeabilize bacterial membranes, accompanied by the transition to an α-helical folding state. Here we show that there is no direct link between folding of the antimicrobial peptide Novicidin (Nc) and its membrane permeabilization. N-terminal acylation with C8–C16 alkyl chains and the inclusion of anionic lipids both increase Nc's ability to form α-helical structure in the presence of vesicles. Nevertheless, both acylation and anionic lipids reduce the extent of permeabilization of these vesicles and lead to slower permeabilization kinetics. Furthermore, acylation significantly decreases antimicrobial activity. Although acyl chains of increasing length also increase the tendency of the peptides to aggregate in solution, this cannot rationalize our results since permeabilization and antimicrobial activities are observed well below concentrations where aggregation occurs. This suggests that significant induction of α-helical structure is not a prerequisite for membrane perturbation in this class of antimicrobial peptides. Our data suggests that for Nc, induction of α-helical structure may inhibit rather than facilitate membrane disruption, and that a more peripheral interaction may be the most efficient permeabilization mechanism. Furthermore, acylation leads to a deeper embedding in the membrane, which could lead to an anti-permeabilizing “plugging” effect.     

Carboxyl-terminal peptides from histone H1 variants: DNA binding characteristics and solution conformation

The carboxyl-terminal domains of the histone H1 proteins bind to DNA and are important in condensation of DNA. Little is known about the details of the interactions between H1 histones and DNA, and in particular, there is little known about differences among variant H1 histones in their interactions with DNA. Questions concerning H1 histone-DNA affinity and H1 conformation were investigated using peptide fragments from the carboxyl terminal domains of four nonallelic histone H1 variant proteins (mouse H1-1, H1-4 and H1^°, and rat H1T). Three of the four peptides showed a slight preference for binding to a GC-rich region of a 214-base-pair DNA fragment, rather than to an AT-rich region. The fourth peptide, H1t, appeared to bind preferentially to the AT-rich region of the 214-base-pair fragment. The results show that these small peptides bind preferentially to a subset of DNA sequences; such sequence preference might be exhibited by the intact H1 histones themselves. CD spectra of the peptides, which are from regions of the proteins that are not compactly folded, showed that the α-helical content of the peptides was minimal if the peptides were in 10 mM phosphate buffer, but increased if the peptides were in 1 M NaClO₄ and 50% trifluoroethanol, conditions that are postulated to approximate certain aspects of binding to DNA. H1-4 peptide, which was predicted to be 70% α-helix, but was not α-helical in 10 mM phosphate buffer, appeared from difference CD spectra to be more α-helical when it was bound to DNA. The regions of the proteins from which these peptides are derived, which are extended in solution, may fold, forming α-helices, upon binding to DNA. © 1996 John Wiley & Sons, Inc.     

Truncated and constrained helical analogs of antimicrobial esculentin-2EM

Esculentin-2EM is a 37-residue, cationic, amphipathic, α-helical antimicrobial peptide isolated from a Korean frog, Glandirama emeljanovi. Many studies revealed that truncation of this peptide results in substantial decreases in its antimicrobial activity. Lee and his colleagues have recently reported that a 23-residue esculentin-2EM analog containing a tryptophanyl substitution at position 16 showed a significant recovery of the antimicrobial activity of the parent peptide. Here we report a new series of 15-residue esculentin-2EM analogs which are constrained into an α-helical conformation via an oct-4-enyl cross-link. The resulting ‘stapled’ derivatives displayed remarkable increases not only in antimicrobial activity but also in helical content and protease resistance compared to Lee’s original 23-residue esculentin-2EM analog. The preliminary data obtained in this work strongly supports the potential of our strategy for the development of a new class of peptide antibiotics.     

Conformation of poly(L-arginine). II. Complexes with polyanions

Conformaitons of poly(L -arginine)/polyanion complexes were studies by CD measurements. The polyanions were the homoplolypeptides poly(L -glutamic acid) and poly(L -aspartic acid); the synthetic polyelectrolytes and polyethylenesulfonate; and the polynucleotides were native DNA, denatured DNA, and poly(U). It was found that poly(L -arginine) forms the α-helical conformation by interacting with the acidic homopolypeptides and the synthetic anionic polyelectrolytes. In each complex, poly(L -glutamic acid) is in the α-helical conformation, whereas poly(L -aspartic acid) is mostly in the random structure. The poly(L -glutamic acid) complex changed into the β-sheet structure at the transition temperature about 65°C in 0.01M cacodylate buffer (pH 7). Even in the presence of 5M urea, this complex remained in the α-helical conformation at room temperature. The existence of the stable complex of α-helical poly(L -arginine) and α-helical poly(L -glutamic acid) was successfully supported by the model building study of the complex. The α-helix of poly(L -arginine) induced by binding with polyacrylate was the most stable of the poly(L -arginine)-polyanion complexes examined as evidenced by thermal and urea effects. The lower helical content of the polyethylenesulfonate-complexed poly(L -aginine) was explained in terms of the higher charge density of the polyanion. On the other hand, native DNA, denatured DNA, and poly(U) were not effective in stabilizing the helical structure of poly(L -arginine). This may be due to the rigidity of polyanions and to the steric hindrance of bases. Furthermore, the distinitive structual behavior of poly(L -arginine) and poly(L -lysine) regarding polyanion interaction has been noticed throughout the study.     

A rigidity-enhanced antimicrobial activity: a case for linear cationic α-helical peptide HP(2-20) and its four analogues

Linear cationic α-helical antimicrobial peptides are referred to as one of the most likely substitutes for common antibiotics, due to their relatively simple structures (≤ 40 residues) and various antimicrobial activities against a wide range of pathogens. Of those, HP(2-20) was isolated from Helicobacter pylori ribosomal protein. To reveal a mechanical determinant that may mediate the antimicrobial activities, we examined the mechanical properties and structural stabilities of HP(2-20) and its four analogues of same chain length by steered molecular dynamics simulation. The results indicated the following: the resistance of H-bonds to the tensile extension mediated the early extensive stage; with the loss of H-bonds, the tensile force was dispensed to prompt the conformational phase transition; and Young's moduli (N/m(2)) of the peptides were about 4 ～ 8 × 10(9). These mechanical features were sensitive to the variation of the residue compositions. Furthermore, we found that the antimicrobial activity is rigidity-enhanced, that is, a harder peptide has stronger antimicrobial activity. It suggests that the molecular spring constant may be used to seek a new structure-activity relationship for different α-helical peptide groups. This exciting result was reasonably explained by a possible mechanical mechanism that regulates both the membrane pore formation and the peptide insertion.     

Identification of an antimicrobial peptide from human methionine sulfoxide reductase B3

Human methionine sulfoxide reductase B3A (hMsrB3A) is an endoplasmic reticulum (ER) reductase that catalyzes the stereospecific reduction of methionine-R-sulfoxide to methionine in proteins. In this work, we identified an antimicrobial peptide from hMsrB3A protein. The N-terminal ER-targeting signal peptide (amino acids 1-31) conferred an antimicrobial effect in Escherichia coli cells. Sequence and structural analyses showed that the overall positively charged ER signal peptide had an Argand Pro-rich region and a potential hydrophobic α-helical segment that contains 4 cysteine residues. The potential α-helical region was essential for the antimicrobial activity within E. coli cells. A synthetic peptide, comprised of 2-26 amino acids of the signal peptide, was effective at killing Gram-negative E. coli, Klebsiella pneumoniae, and Salmonella paratyphi, but had no bactericidal activity against Gram-positive Staphylococcus aureus.     

Pleurocidin-derived antifungal peptides with selective membrane-disruption effect

Pleurocidin (Ple) is a peptide derived from the winter flounder. In our previous study, we reported the antifungal effect of Ple and its mode of action. To develop novel antifungal peptides useful as therapeutic agents, two analogs, with amino acid substitutions, were designed to decrease the net hydrophobicity by Arg (R) or Ser (S)-substitution at the hydrophobic face of Ple without changing the amphipathic structure. By substituting Ser, the hydrophobicity of the peptide (anal-S) was decreased, and by substituting Arg, though the hydrophobicity of the peptide (anal-R) was decreased, the cationicity of this peptide was increased. CD measurements showed the substitution of Arg or Ser decrease the α-helical conformation of analog peptides. Studies with analog peptides have shown decreases in hydrophobicity and α-helicity do not affect antifungal activity but decrease hemolytic activity. These results suggest that highly hydrophobic and α-helical natures are not desirable in the design of antimicrobial peptides.     

Formation of Macromolecule Complex with Bacillus thuringiensis Cry1A Toxins and Chlorophyllide Binding 252-kDa Lipocalin-Like Protein Locating on Bombyx mori Midgut Membrane

P252, a 252-kDa Bombyx mori protein located on the larval midgut membrane, has been shown to bind strongly with Bacillus thuringiensis Cry1A toxins (Hossain et al. Appl Environ Microbiol 70:4604-4612, 2004). P252 was also shown to bind chlorophyllide (Chlide) to form red fluorescence-emitting complex Bm252RFP with significant antimicrobial activity (Pandian et al. Appl Environ Microbiol 74:1324-1331, 2008). In this article, we show that Cry1A toxin bound with Bm252RFP and Bm252RFP-Cry1A macrocomplex, with both antimicrobial and insecticidal activities, was formed. The insecticidal activity of Bm252RFP-Cry1Ab was reduced from an LD?? of 1.62 to 5.05 μg, but Bm252RFP-Cry1Aa and Bm252RFP-Cry1Ac did not show such reduction. On the other hand, the antimicrobial activity of Bm252RFP-Cry1Ab was shown to retain almost the same activity as Bm252RFP, while the other two complexes lost around 30% activity. The intensity of photo absorbance and fluorescence emission of Bm252RFP-Cry1Ab were significantly reduced compared to those of the other two complexes. Circular dichroism showed that the contents of Cry1Ab α-helix was significantly decreased in Bm252RFP-Cry1Ab but not in the other two toxins. These data suggested that the reduction of contents of α-helix in Cry1Ab affected the insecticidal activity of the macrocomplex but did not alter the antimicrobial moiety in the macrocomplex of Bm252RFP-Cry1Ab.     

Role of helicity of α-helical antimicrobial peptides to improve specificity

A major barrier to the use of antimicrobial peptides as antibiotics is the toxicity or ability to lyse eukaryotic cells. In this study, a 26-residue amphipathic α-helical antimicrobial peptide A12L/A20L (Ac-KWKSFLKTFKSLK KTVLHTLLKAISS-amide) was used as the framework to design a series of D- and L-diastereomeric peptides and study the relationships of helicity and biological activities of α-helical antimicrobial peptides. Peptide helicity was measured by circular dichroism spectroscopy and demonstrated to correlate with the hydrophobicity of peptides and the numbers of D-amino acid substitutions. Therapeutic index was used to evaluate the selectivity of peptides against prokaryotic cells. By introducing D-amino acids to replace the original L-amino acids on the non-polar face or the polar face of the helix, the hemolytic activity of peptide analogs have been significantly reduced. Compared to the parent peptide, the therapeutic indices were improved of 44-fold and 22-fold against Gram-negative and Grampositive bacteria, respectively. In addition, D- and L-diastereomeric peptides exhibited lower interaction with zwitterionic eukaryotic membrane and showed the significant membrane damaging effect to bacterial cells. Helicity was proved to play a crucial role on peptide specificity and biological activities. By simply replacing the hydrophobic or the hydrophilic amino acid residues on the non-polar or the polar face of these amphipathic derivatives of the parent peptide with D-amino acids, we demonstrated that this method could have excellent potential for the rational design of antimicrobial peptides with enhanced specificity.     

Viral connectors for DNA encapsulation

Viral connectors are key components of the life cycle of bacteriophages and other viral systems. They participate in procapsid assembly, and they are instrumental in DNA packaging and release. Connector proteins build hollow cylindrical dodecamers that show an overall morphological similarity among different viral systems including a remarkable conserved domain in the central part of the protein. These domains build the wall of the channel forming a 24 α-helices stretch together with an α-β extension. A similar α-helical arrangement is found in other unspecific DNA translocating complexes, suggesting the existence of a common structural signature for channel formation. Preliminary experiments suggest that connectors might be ideal candidates as nanopores for synthetic applications in nanotechnology.     

What can AlphaFold do for antimicrobial amyloids?

Amyloids, protein, and peptide assemblies in various organisms are crucial in physiological and pathological processes. Their intricate structures, however, present significant challenges, limiting our understanding of their functions, regulatory mechanisms, and potential applications in biomedicine and technology. This study evaluated the AlphaFold2 ColabFold method's structure predictions for antimicrobial amyloids, using eight antimicrobial peptides (AMPs), including those with experimentally determined structures and AMPs known for their distinct amyloidogenic morphological features. Additionally, two well-known human amyloids, amyloid-β and islet amyloid polypeptide, were included in the analysis due to their disease relevance, short sequences, and antimicrobial properties. Amyloids typically exhibit tightly mated β-strand sheets forming a cross-β configuration. However, certain amphipathic α-helical subunits can also form amyloid fibrils adopting a cross-α structure. Some AMPs in the study exhibited a combination of cross-α and cross-β amyloid fibrils, adding complexity to structure prediction. The results showed that the AlphaFold2 ColabFold models favored α-helical structures in the tested amyloids, successfully predicting the presence of α-helical mated sheets and a hydrophobic core resembling the cross-α configuration. This implies that the AI-based algorithms prefer assemblies of the monomeric state, which was frequently predicted as helical, or capture an α-helical membrane-active form of toxic peptides, which is triggered upon interaction with lipid membranes.     

Synergistic and antibiofilm activity of the antimicrobial peptide P5 against carbapenem-resistant Pseudomonas aeruginosa

In the search for new antimicrobial molecules, antimicrobial peptides (AMPs) offer a viable alternative to conventional antibiotics, as they physically disrupt the bacterial membranes, leading to membrane disruption and eventually cell death. In particular, the group of linear α-helical cationic peptides has attracted increasing research and clinical interest. The AMP P5 has been previously designed as a cationic linear α-helical sequence, being its antimicrobial and hemolytic properties also evaluated. In this work, we analyzed the feasibility of using P5 against a carbapenem-resistant clinical isolate of Pseudomonas aeruginosa, one of the most common and risky pathogens in clinical practice. After antimicrobial activity confirmation in in vitro studies, synergistic activity of P5 with meropenem was evaluated, showing that P5 displayed significant synergistic activity in a time kill curve assay. The ability of P5 to permeabilize the outer membrane of P. aeruginosa can explain the obtained results. Finally, the antibiofilm activity was investigated by viability analysis (MTT assay), crystal violet and confocal imaging, with P5 displaying mild biofilm inhibition in the range of concentrations tested. Regarding biofilm disruption activity, P5 showed a higher efficacy, interfering with biofilm structure and promoting bacterial cell death. Atomic force microscope images further demonstrated the peptide potential in P. aeruginosa biofilm eradication, confirming the promising application of P5 in multi-resistant infections therapeutics.     

Influence of Proline Substitution on the Bioactivity of Mammalian-Derived Antimicrobial Peptide NK-2

Multidrug-resistant bacteria are emerging as a global threat, making the search for alternative compounds urgent. Antimicrobial peptides (AMPs) became a promising hotspot due to their distinct action mechanism and possibility to be used as an alternative or complement to traditional antibiotics. However, gaining a better understanding about the relationship between antimicrobial peptides structure and its bioactivity is crucial for the development of next generation of antimicrobial agents. NK-2, derived from mammalian protein NK-lysin, has potent antitumor and bactericidal abilities. As proline was considered to be an effective α-helix breaker due to its restricted conformation, to better comprehend the effects of proline in the structure-activity relationship of NK-2, we constructed two NK-2 analogs. We examined the biological activities of NK-2 and its proline substitution analogs and analyzed the resulting conformational changes. Our results showed that introducing proline into the primary sequence of NK-2 significantly decreased the antitumor, antibacterial, and cytotoxic effects, as well as DNA binding activity by changing the α-helix content. However, α-helical content was not the only determining factor, the position of proline inserted was also critical. This study will allow for clearer insight into the role of proline in structure and bioactivity of NK-2 and provide a foundation for future studies.     

N-terminal amphipathic helix as a trigger of hemolytic activity in antimicrobial peptides: A case study in latarcins

In silico structural analyses of sets of α-helical antimicrobial peptides (AMPs) are performed. Differences between hemolytic and non-hemolytic AMPs are revealed in organization of their N-terminal region. A parameter related to hydrophobicity of the N-terminal part is proposed as a measure of the peptide propensity to exhibit hemolytic and other unwanted cytotoxic activities. Based on the information acquired, a rational approach for selective removal of these properties in AMPs is suggested. A proof of concept is gained through engineering specific mutations that resulted in elimination of the hemolytic activity of AMPs (latarcins) while leaving the beneficial antimicrobial effect intact.     

Antimicrobial activity of human α-defensin 5 and its linear analogs: N-terminal fatty acylation results in enhanced antimicrobial activity of the linear analogs

Human α-defensin 5 (HD5) exhibits broad spectrum antimicrobial activity and plays an important role in mucosal immunity of the small intestine. Although there have been several studies, the structural requirements for activity and mechanism of bacterial killing is yet to be established unequivocally. In this study, we have investigated the antimicrobial activity of HD5 and linear analogs. Cysteine deletions attenuated the antibacterial activity considerably. Candidacidal activity was affected to a lesser extent. Fatty acid conjugated linear analogs showed antimicrobial activity comparable activity to HD5. Effective surface charge neutralization of bacteria was observed for HD5 as compared to the non-fatty acylated linear analogs. Our results show that HD5 and non-fatty acylated linear analogs enter the bacterial cytoplasm without causing damage to the bacterial inner membrane. Although fatty acylated peptides exhibited antimicrobial activity comparable to HD5, their mechanism of action involved permeabilization of the Escherichia coli inner membrane. HD5 and analogs had the ability to bind plasmid DNA. HD5 had greater binding affinity to plasmid DNA as compared to the analogs. The three dimensional structure of HD5 favors greater interaction with the bacterial cell surface and also with DNA. Antibacterial activity of HD5 involves entry into bacterial cytoplasm and binding to DNA which would result in shut down of the bacterial metabolism leading to cell death. We show how a moderately active linear peptide derived from the α-defensin HD5 can be engineered to enhance antimicrobial activity almost comparable to the native peptide.     

Copper(II) complexes with sparfloxacin and nitrogen-donor heterocyclic ligands: Structure-activity relationship

Three novel neutral mononuclear copper(II) complexes of the third-generation quinolone antibacterial drug sparfloxacin in the presence of a nitrogen donor heterocyclic ligand 2,2'-bipyridine, 1,10-phenanthroline or 2,2'-dipyridylamine have been prepared and characterized physicochemically and spectroscopically. The resultant complexes are of the type Cu(sparfloxacinato)(N-donor)Cl. Copper(II) is pentacoordinate having a distorted square pyramidal geometry. Molecular modeling calculations have been performed in order to propose the lowest energy model structure of the complexes. The interaction of the complexes with calf-thymus DNA has been investigated with diverse spectroscopic techniques and has shown that the complexes can bind to calf-thymus DNA by the intercalative mode. The antimicrobial activity of the complexes has been tested on three different microorganisms. The Cu(sparfloxacinato)(N-donor)Cl complexes are among the most active ones against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, when compared to the other corresponding copper-quinolone complexes studied by our group and their antimicrobial activity is increased in the order bipyam相似文献