Designing New Hybrid Antibiotics: Proline-Rich Antimicrobial Peptides Conjugated to the Aminoglycoside Tobramycin

Resistance to aminoglycoside antibiotics is a serious problem,typically arising from inactivating enzymes, reduced uptake, or increasedefflux in the important pathogens for which they are used as treatment.Conjugating aminoglycosides to proline-rich antimicrobial peptides(PrAMPs), which also target ribosomes and have a distinct bacterialuptake mechanism, might mutually benefit their individual activities.To this aim we have developed a strategy for noninvasively modifyingtobramycin to link it to a Cys residue and through this covalentlylink it to a Cys-modified PrAMP by formation of a disulfide bond.Reduction of this bridge in the bacterial cytosol should release theindividual antimicrobial moieties. We found that the conjugation oftobramycin to the well-characterized N-terminal PrAMP fragment Bac7(1-35)resulted in a potent antimicrobial capable of inactivating not onlytobramycin-resistant bacterial strains but also those less susceptibleto the PrAMP. To a certain extent, this activity also extends to theshorter and otherwise poorly active fragment Bac7(1-15). Althoughthe mechanism that allows the conjugate to act when its individualcomponents do not is as yet unclear, results are very promising andsuggest this may be a way of resensitizing pathogens that have developedresistance to the antibiotic

An Optimized Workflow for the Discovery of New Antimicrobial Compounds Targeting Bacterial RNA Polymerase Complex Formation

Bacterial resistance represents a major health problem worldwide and there is an urgent need to develop first-in-class compounds directed against new therapeutic targets. We previously developed a drug-discovery platform to identify new antimicrobials able to disrupt the protein-protein interaction between the beta' subunit and the sigma(70) initiation factor of bacterial RNA polymerase, which is essential for transcription. As a follow-up to such work, we have improved the discovery strategy to make it less time-consuming and more cost-effective. This involves three sequential assays, easily scalable to a high-throughput format, and a subsequent in-depth characterization only limited to hits that passed the three tests. This optimized workflow, applied to the screening of 5360 small molecules from three synthetic and natural compound libraries, led to the identification of six compounds interfering with the beta'-sigma(70) interaction, and thus was capable of inhibiting promoter-specific RNA transcription and bacterial growth. Upon supplementation with a permeability adjuvant, the two most potent transcription-inhibiting compounds displayed a strong antibacterial activity against Escherichia coli with minimum inhibitory concentration (MIC) values among the lowest (0.87-1.56 mu M) thus far reported for beta'-sigma PPI inhibitors. The newly identified hit compounds share structural feature similarities with those of a pharmacophore model previously developed from known inhibitors

Peptide inhibitors of bacterial protein synthesis with broad spectrum and SbmA-independent bactericidal activity against clinical pathogens.

Proline-rich antimicrobial peptides (PrAMPs) are promising lead compounds for developing new antimicrobials, however their narrow spectrum of action is limiting. PrAMPs kill bacteria binding to their ribosomes and inhibiting protein synthesis. In this study, 133 derivatives of the PrAMP Bac7(1-16) were synthesized to identify the crucial residues for ribosome inactivation and antimicrobial activity. Then, five new Bac7(1-16) derivatives were conceived and characterized by antibacterial and membrane permeabilization assays, by X-ray crystallography and molecular dynamics simulations. Some derivatives displayed broad spectrum activity, encompassing Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa and Staphylococcus aureus. Two peptides out of five, acquired a weak membrane-perturbing activity, while maintaining the ability to inhibit protein synthesis. These derivatives became independent of the SbmA transporter, commonly used by native PrAMPs, suggesting that they obtained a novel route to enter bacterial cells. PrAMP-derived compounds could become new-generation antimicrobials to combat the antibiotic-resistant pathogens

Il peptide antimicrobico B7-005: biocompatibilità, modalità d'azione e attività antimicrobica contro i patogeni ESKAPE

L'Organizzazione Mondiale della Sanità ha elencato alcuni patogeni resistenti alla maggior parte degli antibiotici e responsabili della maggior parte delle infezioni nosocomiali. Questi patogeni sono indicati con l'acronimo ESKAPE: E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa ed Enterobacter spp. Sono urgentemente necessari nuovi farmaci che superino la resistenza agli antibiotici di questi patogeni. Grandi speranze sono rivolte ai peptidi antimicrobici ricchi di proline (PrAMP). I PrAMP presentano un'attività antimicrobica potente e specifica, penetrano in alcune specie batteriche, si legano ai ribosomi procariotici e inibiscono la sintesi proteica. I PrAMP vengono attualmente ottimizzati per superare uno dei loro più grandi limiti, ovvero lo spettro d’azione limitato solo ad alcune specie di batteri, con il fine ultimo di sviluppare nuovi antibiotici. Nello screening di librerie di PrAMP corti di mammiferi, è stato selezionato il peptide B7-005 perché presenta attività antimicrobica efficace anche a basse concentrazioni, insensibilità alle proteasi del siero e dei dati preliminare hanno suggerito una bassa citotossicità nei confronti delle cellule eucariotiche. I risultati indicano che B7-005 ha una minore propensione a selezionare la resistenza in E. coli rispetto ad altri antibiotici e PrAMP. B7-005 è risultato attivo su tutti i ceppi di riferimento ESKAPE gram-positivi e gram-negativi. La crescita dei preoccupanti patogeni nosocomiali K. pneumoniae e A. baumannii è stata inibita dal B7-005 in un intervallo micromolare basso. Il B7-005 ha inibito la crescita anche di P. aeruginosa, S. aureus ed E. faecium, specie batteriche che spesso non vengono influenzate dai PrAMP nativi, poiché mancano del trasportatore batterico SbmA, sfruttato da questi peptidi per entrare nel citosol batterico. Il B7-005 non solo ha inibito la crescita della maggior parte delle ESKAPE, ma ha anche ucciso attivamente questi patogeni. Tra le specie sono state riscontrate grandi differenze in termini di efficacia, tempo di uccisione e soglia di concentrazione. Per verificare se la morte batterica fosse dovuta a un meccanismo di uccisione non litico, come ci si aspetterebbe per un PrAMP, l'integrità della membrana cellulare e la presenza del peptide intracellulare sono state monitorate mediante citometria a flusso e un derivato fluorescente del B7-005. il B7-005 è stato osservato nel citoplasma delle cellule di E. coli e K. pneumoniae, lasciando allo stesso tempo intatta la membrana batterica. Mentre le cellule delle altre specie di batteri ESKAPE esposte al B7-005 hanno mostrato una destabilizzazione delle membrane, e una ridotta internalizzazione del peptide. Per garantire che l'attività permeabilizzante di membrana di B7-005 osservata in alcune ESKAPE non fosse accompagnata da un aumento della citotossicità, sono stati eseguiti test di biocompatibilità su diverse cellule umane. Le normali cellule endoteliali primarie della vena ombelicale umana (HUVEC) sono risultate tolleranti al peptide anche ad alte concentrazioni e più tolleranti anche dei cheratinociti immortalizzati HaCaT, della linea tumorale polmonare A549 e delle cellule leucemiche MEC-1. I risultati complessivi sono coerenti con i dati della letteratura che indicano che gli AMP spesso colpiscono le cellule tumorali più di quelle non tumorali, principalmente a causa della loro diversa composizione di membrana. Inoltre, sono stati eseguiti test di citotossicità in vivo per valutare la biocompatibilità di B7-005 nel sistema più complesso delle larve di Zebrafish (Danio rerio). B7-005 si è dimostrato sicuro per i pesci fino a concentrazioni millimolari elevate, cioè una dose molto distante sia dalle concentrazionie micromolari definite tossiche per le cellule eucariotiche in vitro, ma ancora di più da quelle battericide.The World Health Organisation has listed a number of pathogens that are resistant to most antibiotics and are responsible for most nosocomial infections. These pathogens are referred to by the acronym ESKAPE: E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter spp. New drugs that overcome the antibiotic resistance of these pathogens are urgently needed. Great hopes rest on antimicrobial proline-rich peptides (PrAMPs). PrAMPs have potent and specific antimicrobial activity, penetrate certain bacterial species, bind to prokaryotic ribosomes, and inhibit protein synthesis. PrAMPs are currently being optimised to overcome one of their major limitations, namely their spectrum of activity limited to certain bacterial species, with the goal of developing new antibiotics. In screening libraries of short mammalian PrAMPs, peptide B7-005 was selected because it exhibits effective antimicrobial activity even at low concentrations, is insensitive to serum proteases, and preliminary data indicate low cytotoxicity to eukaryotic cells. The results suggest that B7-005 has a lower propensity to develop resistance in E. coli than other antibiotics and PrAMP. B7-005 was active in all Gram-positive and Gram-negative ESKAPE reference strains. Growth of the nosocomial pathogens of concern, K. pneumoniae and A. baumannii, was inhibited by B7-005 in the low micromolar range. B7-005 also inhibited P. aeruginosa, S. aureus, and E. faecium, bacterial species that are often unaffected by native PrAMPs because they lack the bacterial SbmA transporter used by these peptides to enter the bacterial cytosol. B7-005 not only inhibited the growth of most ESKAPEs, but also actively killed these pathogens. Large interspecies differences in efficacy, killing time, and concentration threshold were observed. To verify whether bacterial death was due to a non-lytic killing mechanism, as would be expected with a PrAMP, cell membrane integrity and the presence of the intracellular peptide were monitored by flow cytometry, and a fluorescent derivative of B7-005. B7-005 was observed in the cytoplasm of E. coli and K. pneumoniae cells, whereas the bacterial membrane remained intact. In contrast, cells of other ESKAPE bacterial species exposed to B7-005 showed destabilisation of the membrane and decreased internalisation of the peptide. To ensure that the membrane-permeabilizing activity of B7-005 observed in some ESKAPE was not associated with increased cytotoxicity, biocompatibility tests were performed with various human cells. Normal primary human umbilical vein endothelial cells (HUVECs) were tolerant to the peptide even at high concentrations and more tolerant than even immortalised HaCaT keratinocytes, the A549 lung cancer line, and MEC-1 leukaemia cells. The overall results are consistent with literature data indicating that AMPs often affect tumour cells more than non-tumour cells, mainly due to their different membrane composition. In addition, in vivo cytotoxicity tests were performed to evaluate the biocompatibility of B7-005 in the more complex system of Zebrafish larvae (Danio rerio). B7-005 was found to be safe for fish up to very high millimolar concentrations, i.e., a dose far from both micromolar concentrations defined as toxic to eukaryotic cells in vitro, but even more from bactericidal concentrations. The study shows that the peptide B7-005 is an antimicrobial compound with a low propensity to develop bacterial resistance, has a broad spectrum of activity against ESKAPEs, is well tolerated by normal human cells, and is not toxic in an in vivo system such as Zebrafish larvae. B7-005 thus overcomes some of the limitations of AMPs as a therapeutic molecule and is therefore a good candidate for use as a first line of defence against some of the pathogens of concern to human health

Effects of Lipidation on a Proline-Rich Antibacterial Peptide

The emergence of multidrug-resistant bacteria is a worldwide health problem. Antimicrobial peptides have been recognized as potential alternatives to conventional antibiotics, but still require optimization. The proline-rich antimicrobial peptide Bac7(1-16) is active against only a limited number of Gram-negative bacteria. It kills bacteria by inhibiting protein synthesis after its internalization, which is mainly supported by the bacterial transporter SbmA. In this study, we tested two different lipidated forms of Bac7(1-16) with the aim of extending its activity against those bacterial species that lack SbmA. We linked a C12-alkyl chain or an ultrashort cationic lipopeptide Lp-I to the C-terminus of Bac7(1-16). Both the lipidated Bac-C12 and Bac-Lp-I forms acquired activity at low micromolar MIC values against several Gram-positive and Gram-negative bacteria. Moreover, unlike Bac7(1-16), Bac-C12, and Bac-Lp-I did not select resistant mutants in E. coli after 14 times of exposure to sub-MIC concentrations of the respective peptide. We demonstrated that the extended spectrum of activity and absence of de novo resistance are likely related to the acquired capability of the peptides to permeabilize cell membranes. These results indicate that C-terminal lipidation of a short proline-rich peptide profoundly alters its function and mode of action and provides useful insights into the design of novel broad-spectrum antibacterial agents

Structural and biological characterization of shortened derivatives of the cathelicidin PMAP-36

Abstract Cathelicidins, a family of host defence peptides in vertebrates, play an important role in the innate immune response, exhibiting antimicrobial activity against many bacteria, as well as viruses and fungi. This work describes the design and synthesis of shortened analogues of porcine cathelicidin PMAP-36, which contain structural changes to improve the pharmacokinetic properties. In particular, 20-mers based on PMAP-36 (residues 12-31) and 13-mers (residues 12-24) with modification of amino acid residues at critical positions and introduction of lipid moieties of different lengths were studied to identify the physical parameters, including hydrophobicity, charge, and helical structure, required to optimise their antibacterial activity. Extensive conformational analysis, performed by CD and NMR, revealed that the substitution of Pro25-Pro26 with Ala25-Lys26 increased the α-helix content of the 20-mer peptides, resulting in broad-spectrum antibacterial activity against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus epidermidis strains. Interestingly, shortening to just 13 residues resulted in only a slight decrease in antibacterial activity. Furthermore, two sequences, a 13-mer and a 20-mer, did not show cytotoxicity against HaCat cells up to 64 µM, indicating that both derivatives are not only effective but also selective antimicrobial peptides. In the short peptide, the introduction of the helicogenic α-aminoisobutyric acid forced the helix toward a prevailing 310 structure, allowing the antimicrobial activity to be maintained. Preliminary tests of resistance to Ser protease chymotrypsin indicated that this modification resulted in a peptide with an increased in vivo lifespan. Thus, some of the PMAP-36 derivatives studied in this work show a good balance between chain length, antibacterial activity, and selectivity, so they represent a good starting point for the development of even more effective and proteolysis-resistant active peptides

Chemical and Biological Evaluation of Essential Oils from Two Species of Myrtaceae — Eugenia uniflora L. and Plinia trunciflora (O. Berg) Kausel

The chemical composition and antimicrobial activity of essential oils obtained from leaves of two Myrtaceae species–Eugenia uniflora L. and Plinia trunciflora (O. Berg) Kausel–were determined. Analysis by GC/MS as well as determination of Kovatz indexes indicated atractylone (26.78%) and curzerene (17.96%) as major constituents of E. uniflora oil and α-cadinol (19.15%), apiole (11.15%) and cubenol (5.43%) as main components in P. trunciflora oil. Both essential oils were tested for antimicrobial activity against yeasts and bacteria. E. uniflora and P. trunciflora essential oils were active towards two Gram-positive bacteria, Streptococcus equi and Staphylococcus epidermis. In addition, biological activity of both essential oils was detected for pathogenic yeasts of the genus Candida and Cryptococcus. E. uniflora was active towards all yeast tested and exhibited interesting minimal inhibitory concentrations (0.11 to 3.75 mg/mL) across a broad spectrum of activity

Local knowledge of medicinal plants in three artisanal fishing communities (Itapoá, Southern Brazil), according to gender, age, and urbanization Conhecimento local sobre plantas medicinais em três comunidades de pescadores artesanais (Itapoá, sul do Brasil), de acordo com gênero, idade e urbanização

This article analyzes some of the factors affecting the distribution of knowledge about medicinal plants of three artisanal fishing communities in Itapoá Municipality, in the state of Santa Catarina, Brazil. Ninety semi-structured interviews were performed, which resulted in 109 cited plant species. The sample included both men (n=46) and women (n=44), in different age categories: 18-40 years old (n=18), 41-50 (n=21), 51-60 (n=28), and 61+ years old (n=23), and in three different communities: Barra do Saí (n=17), Itapema do Norte (n=31) and Pontal do Norte (n=42). Leaves are the plant parts most used by the communities (62%), and are obtained primarily through cultivation. Cordia curassavica (Jacq.) Roem. & Schult. (Boraginaceae) is the species most cited in the interviews, locally known as 'erva-baleeira' or 'erva-de-baleia'. The elderly have greater knowledge of species, but no gender differences were noticed. Less knowledge of plants was noticed in the more urbanized area when compared to the less urbanized areas. The most used plants are not related to the most frequent ailments. Medicinal plant use is an important resource for the health of these fishing communities, but it is declining mostly due to the lack of interest on the part of the younger members, and to improved access to public health services.<br>Este artigo analisa alguns dos fatores que afetam a distribuição do conhecimento sobre plantas medicinais em três comunidades de pescadores artesanais do Município de Itapoá, Santa Catarina, Brasil. Noventa entrevistas semi-estruturadas foram realizadas, o que resultou em 109 espécies de plantas citadas. A amostra foi composta por homens (n = 46) e mulheres (n = 44), em diferentes categorias de idade: 18-40 anos (n = 18), 41-50 (n = 21), 51-60 (n = 28), e 61 anos ou mais (n = 23), e em três diferentes comunidades: Barra do Saí (n = 17), Itapema do Norte (n = 31) e Pontal do Norte (n = 42). Folhas são as partes da plantas mais utilizadas (62%), sendo obtidas principalmente por cultivo. Cordia curassavica (Jacq.) Roem. & Schult. (Boraginaceae) é a espécie mais citada nas entrevistas, localmente chamada de "erva-baleeira" ou "erva-de-baleia". Os idosos têm um maior conhecimento sobre as espécies, mas não houve diferenças em relação ao sexo dos entrevistados. Um menor conhecimento sobre as plantas foi observado na área mais urbanizada, quando comparada com as áreas menos urbanizadas. As plantas mais utilizadas não estão relacionadas com as doenças mais freqüentes. As plantas medicinais são um recurso importante para a saúde dessas comunidades de pescadores, mas este conhecimento pode estar se deteriorando devido principalmente à falta de interesse dos membros mais jovens e à melhoria do acesso aos serviços públicos de saúde