Proteomic signatures in antibiotic research

Antibiotics disturb the physiological homeostasis of bacterial cells by interfering with essential cellular functions or structures. The bacterial proteome adjusts quickly in response to antibiotic challenge. This physiological response is specifically tailored to overcome the inflicted damage and, thus, closely linked to the antibiotic target and mechanism of action. In a way, the proteome mirrors the antibiotic insult. This connection can be exploited to guide the development of novel antibiotics. By using structurally different antibiotics, which cause the same physiological disturbance, proteomic signatures diagnostic of the mechanism of action can be defined. These proteomic signatures inform about mechanism-related differential protein expression as well as about protein modifications. This review recapitulates how antibiotic proteomic signatures are established and highlights areas of antibiotic research benefiting most from proteomic signatures. Antibacterial research programs designed to structurally advance existing antibiotic classes profit from rapid in vivo mechanism of action confirmation. What is more, a comprehensive reference compendium of antibiotic proteomic signatures allows rapid mechanism of action identification of those structurally novel compounds that inhibit known targets. Finally, novel proteomic response profiles indicate unprecedented mechanisms. Here, the proteome profile provides evidence on the nature of the antibiotic-caused physiological disturbance leading to testable hypotheses on the mechanism of action.     

Peptide Therapeutics Versus Superbugs: Highlight on Current Research and Advancements

Antibiotics have saved several millions of lives, but its persistent use of antibiotics in the treatment of various infections, whether bacterial, fungal, viral or parasitic has lead to the development of antibiotic resistance. The rapid emergence of antibiotic resistant strains poses a serious challenge to existing antimicrobial therapies. Due to the increase in drug-resistant pathogens and failure of antibiotics the urgent need for the discovery of novel antimicrobials has been continuously emphasized in the global forum. Here we review about antimicrobial peptides (AMPs), their structural insights and recent developments. We had summarized the major classes, mechanism of action and biophysical parameters that modulate therapeutic potency of AMPs. Also, we had briefed the challenges involved in developing therapeutic peptides and the global market potential for peptide therapeutics.     

Impact of ciprofloxacin impurities on bacterial growth,antibiotic resistance development and content assays

In addition to active pharmaceutical ingredient (API), antibiotics may contain small amounts of excipients and impurities and be prone to accumulation of degradation products. There has been limited work characterizing how these substances impact bacterial growth and antibiotic resistance development. We investigated how two ciprofloxacin (CIP) impurities, fluoroquinolonic acid (FQA) and ciprofloxacin ethylenediamine analogue (CEA), impact growth and antibiotic resistance in Escherichia coli. Additionally, we investigated how these impurities impact a frequently used API content assay. Both impurities displayed modest antimicrobial activity compared to the CIP API. The effective antimicrobial activity of a medicine containing increased impurity levels may permit bacterial growth and resistance development. Our results also suggest that increasing exposure concentration and duration to CEA and FQA, independent of CIP, can promote antibiotic resistance development. However, at concentrations of 100% and below the MIC of the API, impurities had limited contributions to resistance development compared to the CIP API. From a methodological standpoint, we found that UV spectrophotometry may be inadequate to account for antibiotic impurities or degradation products. This can lead to incorrect estimations of API content and we propose additional multi-wavelength measures when using UV spectrophotometry to help identify impurities or degradation.     

Microbial environments confound antibiotic efficacy

The increasing prevalence of bacteria that are insensitive to our current antibiotics emphasizes the need for new antimicrobial therapies. Conventional approaches to antibacterial development that are based on the inhibition of essential processes seem to have reached the point of diminishing returns. The discovery that diverse antibiotics stimulate a common oxidative cell-death pathway represents a fundamental shift in our understanding of bactericidal antibiotic modes of action. A number of studies, as discussed above, also provide hints about how intra- and extracellular metabolism can enable antibiotic resistance and tolerance. We have, nonetheless, just begun to understand the repertoire of tactics that bacteria use to evade antibiotics. Biosynthetic pathways for natural antibiotics are ancient, and numerous mechanisms for antibiotic resistance and tolerance are likely to have evolved over the past few million years. Unraveling these mechanisms will require concerted efforts by chemical biologists, microbiologists and clinicians. These efforts will benefit from the use of metabolic models and other network-biology approaches to guide investigation of processes that modulate antibiotic susceptibility. Importantly, by helping to identify common points of vulnerability as well as key differences between pathogens, these models may lead to the development of effective adjuvants, novel antibiotics and new antimicrobial strategies. There is also a crucial need to better understand how bacteria within a population cooperate to overcome antibiotic treatments. Such investigations may benefit from the use of novel chemical probes and experimental techniques to interrogate the physiology and functional dynamics of natural microbial communities. Insights gained from these studies will augment metagenomic models that can be used to identify biomolecules responsible for these cooperative strategies. Leveraging chemical biology methodologies and systems-biology approaches for further studies of microbial environments may reveal a wealth of untapped targets for the development of novel compounds to counter the growing threat of resistant and tolerant bacterial infections.     

Preparative high-performance liquid chromatographic separation and analysis of the Maltacine complex - a family of cyclic peptide antibiotics from Bacillus subtilis

Purification of secondary metabolites from fermentation broths can be a challenging task both due to the complexity of the medium, inherently unstable molecular structures or by the action of enzymes present in the fermentation broth leading to poor isolation yield and loss of antibiotic activity. A combination of different purification techniques has usually been used to arrive at acceptable purities for characterisation of the target molecules. Due to rapid decay of antimicrobial activity a rapid preparative high-performance liquid chromatography (HPLC) method was developed that provided separation and resolution of a family of 18 closely related cyclic peptides within 110 min with minimal loss of activity. Characterisation of the peptides with LC-MS, UV/IR spectroscopy and amino acid analysis disclosed 20 different peptides with cyclic structures (lactones) with molecular weights between 1447.7 and 1519.8 Da. No peptide antibiotics with identical molecular weights have previously been reported in the literature, which lead us to conclude that this peptide complex has not been discovered before. We have named them Maltacines.     

Improved antimicrobial activity of h‐lysozyme (107–115) by rational Ala substitution

The most challenging target in the design of new antimicrobial agents is the development of antibiotic resistance. Antimicrobial peptides are good candidates as lead compounds for the development of novel anti‐infective drugs. Here we propose the sequential substitution of each Ala residue present in a lead peptide with known antimicrobial activity by specific amino acids, rationally chosen, that could enhance the activity of the resultant peptide. Taking the fragment 107–115 of the human lysozyme as lead, two‐round screening by sequentially replacing both Ala residues (108 and 111) by distinct amino acids resulted in a novel peptide with 4‐ and 20‐fold increased antimicrobial activity against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, respectively. These results reinforce the strategy proposed, which, in combination with simple and easy screening tools, will contribute to the rapid development of new therapeutic peptides required by the market. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

ppGpp介导的抗生素胁迫应答机制研究进展

抗生素是由微生物在生长发育后期产生的次级代谢产物,具有杀死或抑制细菌生长的能力,因此被广泛应用于细菌感染的临床治疗。在长期的进化过程中,细菌采取多种方式应对环境中抗生素的威胁。除了广为人知的抗生素耐药性(resistance)之外,细菌还能对抗生素产生耐受性(tolerance)和持留性(persistence),严重影响抗生素的临床疗效。鸟苷四磷酸(guanosine tetraphosphate, ppGpp)和鸟苷五磷酸(guanosine pentaphosphate, pppGpp) (本文统称ppGpp)是细菌应对营养饥饿等不利环境时产生的"报警"信号分子,其能够在全局水平调控基因的表达,使细菌适应不利的环境。越来越多的研究表明,ppGpp与细菌应对抗生素胁迫密切相关。基于此,本文综述了细菌中ppGpp的合成与水解及其作用机制,并重点阐述了ppGpp介导抗生素胁迫应答的分子机制,以期为新型抗生素的开发提供新思路。     

Resistance acquisition via the bacterial SOS response: the inducive role of antibiotics

After the euphoria of the antibiotic discovery and their tremendous action on bacterial infections outcomes, arrives a period of fear with the continuous emergence of bacteria that are resistant to almost all antibiotic treatments. It is becoming essential to better understand antibiotic resistance mechanisms to find new approaches to prevent the worldwide problem of multiresistance. The role of antibiotics on the direct induction of resistance acquisition is known. Recent studies have shown that some antibiotics, by inducing the bacterial SOS response, global repair response after DNA damages, are involved on a broader level in the induction, acquisition and dissemination of resistances in bacteria. We discuss here the role of antibiotics in resistance acquisition via the SOS response through several examples and the interest of identifying the SOS response regulators as the future targets of new families of antimicrobial molecules.     

铁载体-抗生素耦合物:一种新型的抗菌制剂

随着细菌对抗生素耐药性的增强,寻找一种新型抗菌制剂越来越重要。细菌细胞外膜对药物分子的通透性降低是引起致病菌产生耐药性的一个重要因素,克服膜介导耐药性的方法之一是利用铁载体-抗生素耦合物。铁载体是细菌分泌的一种小分子铁离子螯合物,与铁离子螯合后被特定的外膜受体识别并转运至胞浆内供细菌利用。人工合成的铁载体-抗生素耦合物被特定外膜受体识别后主动转运跨过外膜进入胞质内。当铁载体-抗生素耦合物到达细胞质,它们通过释放药物杀死微生物,这可以阻止进一步获取铁离子,并且耦合物自身也可以作为一种抗菌剂。本文综述了铁载体-抗生素耦合物作为一种新型抗菌制剂的研究进展,有助于为进一步研发新型抗菌药物提供理论基础,对治疗耐药性细菌性疾病具有潜在的重要意义。     

A complementary pair of rapid molecular screening assays for RecA activities

The bacterial RecA protein has been implicated in the evolution of antibiotic resistance in pathogens, which is an escalating problem worldwide. The discovery of small molecules that can selectively modulate RecA's activities can be exploited to tease apart its roles in the de novo development and transmission of antibiotic resistance genes. Toward the goal of discovering small-molecule ligands that can prevent either the assembly of an active RecA-DNA filament or its subsequent ATP-dependent motor activities, we report the design and initial validation of a pair of rapid and robust screening assays suitable for the identification of inhibitors of RecA activities. One assay is based on established methods for monitoring ATPase enzyme activity and the second is a novel assay for RecA-DNA filament assembly using fluorescence polarization. Taken together, the assay results reveal complementary sets of agents that can either suppress selectively only the ATP-driven motor activities of the RecA-DNA filament or prevent assembly of active RecA-DNA filaments altogether. The screening assays can be readily configured for use in future automated high-throughput screening projects to discover potent inhibitors that may be developed into novel adjuvants for antibiotic chemotherapy that moderate the development and transmission of antibiotic resistance genes and increase the antibiotic therapeutic index.     

New approaches to antibiotic discovery

New antibiotics are urgently required by human medicine as pathogens emerge with developed resistance to almost all antibiotic classes. Pioneering approaches, methodologies and technologies have facilitated a new era in antimicrobial discovery. Innovative culturing techniques such as iChip and co-culturing methods which use ‘helper’ strains to produce bioactive molecules have had notable success. Exploiting antibiotic resistance to identify antibacterial producers performed in tandem with diagnostic PCR based identification approaches has identified novel candidates. Employing powerful metagenomic mining and metabolomic tools has identified the antibiotic’ome, highlighting new antibiotics from underexplored environments and silent gene clusters enabling researchers to mine for scaffolds with both a novel mechanism of action and also few clinically established resistance determinants. Modern biotechnological approaches are delivering but will require support from government initiatives together with changes in regulation to pave the way for valuable, efficacious, highly targeted, pathogen specific antimicrobial therapies.     

A robust platform for chemical genomics in bacterial systems

While genetic perturbation has been the conventional route to probing bacterial systems, small molecules are showing great promise as probes for cellular complexity. Indeed, systematic investigations of chemical-genetic interactions can provide new insights into cell networks and are often starting points for understanding the mechanism of action of novel chemical probes. We have developed a robust and sensitive platform for chemical-genomic investigations in bacteria. The approach monitors colony volume kinetically using transmissive scanning measurements, enabling acquisition of growth rates and conventional endpoint measurements. We found that chemical-genomic profiles were highly sensitive to concentration, necessitating careful selection of compound concentrations. Roughly 20,000,000 data points were collected for 15 different antibiotics. While 1052 chemical-genetic interactions were identified using the conventional endpoint biomass approach, adding interactions in growth rate resulted in 1564 interactions, a 50–200% increase depending on the drug, with many genes uncharacterized or poorly annotated. The chemical-genetic interaction maps generated from these data reveal common genes likely involved in multidrug resistance. Additionally, the maps identified deletion backgrounds exhibiting class-specific potentiation, revealing conceivable targets for combination approaches to drug discovery. This open platform is highly amenable to kinetic screening of any arrayable strain collection, be it prokaryotic or eukaryotic.     

Facing crises in the 21st century: microfluidics approaches for antibiotic discovery

We urgently need new antibiotics to counteract the rising in the emergence of multidrug-resistant microorganisms. To improve the identification of antimicrobial compounds of microbial origin, numerous multidisciplinary approaches are being implemented. However, the development of innovative microbial cultivation strategies is necessary to exploit the full biosynthetic potential of non-culturable microorganisms. Here, I highlight various articles that employ high-throughput microfluidic-based strategies to identify novel antimicrobial metabolites based on bacterial activities. The rapid development of this technology will likely advance the field of antibiotic discovery.     

The therapeutic applications of antimicrobial peptides (AMPs): a patent review

Antimicrobial peptides (AMPs) are small molecules with a broad spectrum of antibiotic activities against bacteria, yeasts, fungi, and viruses and cytotoxic activity on cancer cells, in addition to anti-inflammatory and immunomodulatory activities. Therefore, AMPs have garnered interest as novel therapeutic agents. Because of the rapid increase in drug-resistant pathogenic microorganisms, AMPs from synthetic and natural sources have been developed using alternative antimicrobial strategies. This article presents a broad analysis of patents referring to the therapeutic applications of AMPs since 2009. The review focuses on the universal trends in the effective design, mechanism, and biological evolution of AMPs.     

Proline-rich antimicrobial peptides: potential therapeutics against antibiotic-resistant bacteria

The increasing resistance of pathogens to antibiotics causes a huge clinical burden that places great demands on academic researchers and the pharmaceutical industry for resolution. Antimicrobial peptides, part of native host defense, have emerged as novel potential antibiotic alternatives. Among the different classes of antimicrobial peptides, proline-rich antimicrobial peptides, predominantly sourced from insects, have been extensively investigated to study their specific modes of action. In this review, we focus on recent developments in these peptides. They show a variety of modes of actions, including mechanism shift at high concentration, non-lytic mechanisms, as well as possessing different intracellular targets and lipopolysaccharide binding activity. Furthermore, proline-rich antimicrobial peptides display the ability to not only modulate the immune system via cytokine activity or angiogenesis but also possess properties of penetrating cell membranes and crossing the blood brain barrier suggesting a role as potential novel carriers. Ongoing studies of these peptides will likely lead to the development of more potent antimicrobial peptides that may serve as important additions to the armoury of agents against bacterial infection and drug delivery.     

Differential Effects of Monovalent and Divalent Ions upon the Mode of Action of the Polyene Antibiotic Candicidin

The polyene antibiotic candicidin is a potent membrane active agent, the action of which can be inhibited by the presence of certain ions. The destruction of the selective permeability of yeast membranes by candicidin allows small molecules to leak into the environment. Loss of intracellular potassium ions inhibits yeast glycolysis. This inhibition may be reversed by extracellular concentrations of potassium or ammonium ions. Monovalent ions did not prevent antibiotic absorption or protect yeast growth from the action of the antibiotic. Divalent ions did not protect yeast glycolysis from the action of candicidin, but were able to reduce antibiotic-induced membrane damage and allowed yeast growth in the presence of antibiotic. It is suggested that divalent ions may interact with membrane sterols creating steric hindrance to subsequent candicidin absorption.     

SAR studies on dihydropyrimidinone antibiotics

There is an urgent need for the development of novel antimicrobial agents that offer effective treatment against MRSA. Using a new class of dipeptide antibiotic TAN-1057A/B as lead, we designed, synthesized and evaluated analogs of TAN-1057A/B. Several novel dihydropyrimidinone antibiotics demonstrating comparable antibiotic efficacy while possessing favorable selectivity were identified.     

In vitro anti-Neisseria gonorrhoeae activity of Terminalia macroptera leaves

In an assessment of antibiotic action on Staphylococcus aureus, we found that distinct changes in intracellular nucleotide pools occur depending on the antibiotic mode of action. In particular, we have quantitated the effect of antibiotics on pools of the nucleotide guanosine 3'-diphosphate, 5'-triphosphate (pppGpp). Intracellular pppGpp levels increased in response to treatment with the isoleucyl tRNA synthetase inhibitor mupirocin, the uncoupler carbonyl cyanide-m-chlorophenylhydrazone, and rifampicin. These compounds were distinguishable by the degree in which they increased the pppGpp pool and by their differential effect on the pools of other nucleotides. This technique has been used to confirm and to refute the expected mode of action of several compounds identified as possible inhibitors of tRNA synthetases. Our results provide the framework for using nucleotide analysis in the assessment of novel antimicrobial compounds with unknown modes of action.