Source: https://pubs.rsc.org/en/content/articlehtml/2018/np/c8np00031j
Timestamp: 2019-04-25 14:22:47+00:00

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The increase in the incidence of antibiotic resistant infections is threatening to overwhelm healthcare practices worldwide. Most antibiotics in clinical use are becoming ineffective, so therefore it is imperative to develop new antibiotics and novel therapeutic strategies. Traditionally, the chemical and mechanistic diversity of nonribosomal antibacterial peptides (NRAPs) as lead compounds have meant that their structures are ideal for antibiotic discovery. Here, we summarize the state of our current knowledge about the mechanisms of antibiotic resistance, which can be used to guide the development of new antibiotics. Furthermore, we provide an overview of NRAPs for treating multi-drug resistant bacteria, including innovative approaches for screening NRAPs from new sources and the underlying mechanisms of antibacterial activity. Finally, we discuss the design of NRAP scaffolds for precise medicine and combinatorial NRAP therapies with existing antibiotics to overcome resistance, which will help to control infections in the post-antibiotic era.
Yuan Liu received his PhD from China Agricultural University in June 2018. His PhD study focused on the discovery of novel nonribosomal antibacterial peptides and antibiotic adjuvants to combat multi-drug resistant bacteria. He has been a University Professor at the Institute of Comparative Medicine and College of Veterinary Medicine at Yangzhou University (Jiangsu, China) since July 2018.
Shuangyang Ding is a Professor at the College of Veterinary Medicine at China Agricultural University. She obtained her PhD in 1999 from China Agricultural University. Currently, she is the deputy director of the National Veterinary Drug Residue Reference Laboratory of China. Her research focuses on the development of methods to analyze veterinary drug residues in animal-derived foods and veterinary drug safety evaluation.
Jianzhong Shen is a Professor and the Dean of the College of Veterinary Medicine at China Agricultural University. He has been an academician of the Chinese Academy of Engineering since 2015. His research focuses on animal-derived food safety, antibiotic resistance and the development of novel antimicrobial agents.
Kui Zhu is a Professor at the College of Veterinary Medicine at China Agricultural University. He obtained his Doctor of Veterinary Medicine degree (DVM) from Ludwig Maximilian University of Munich (LMU Munich, Germany). After that, he did postdoc training at Duke University in the USA. His research focuses on the development of novel antibiotics and alternative strategies against multi-drug resistant bacteria.
The rapid emergence and widespread distribution of antibacterial resistance is now recognized as one of the most serious global threats to human health.1,2 Consequently, the Centers for Disease Control and Prevention (CDC) recently revealed that more than two million people suffer from antibiotic-resistant infections and at least 23 000 people die as a result per year in the United States alone.3 Worryingly, the increase in the incidence of multi-drug resistant (MDR) Gram-negative bacteria, such as plasmid-mediated resistance to carbapenems4 and colistin5–7 in Enterobacteriaceae, is threatening to overwhelm healthcare practices worldwide. A similar situation has also been observed for Gram-positive bacteria, such as the notorious methicillin-resistant Staphylococcus aureus (MRSA)8 and vancomycin-resistant enterococci (VRE).9,10 Collectively, it means that no effective antibiotic is available for combating infections caused by either Gram-positive or Gram-negative superbugs. New antibiotics or alternative therapeutics are urgently required for clinical treatments.
To accelerate the process of antibiotic discovery, we need to develop a mechanistic understanding of the diverse ways in which bacteria survive antibiotic treatments. Such an understanding is critical for developing new antibiotics and designing therapeutic approaches to revitalize existing antibiotics. For example, aspergillomarasmine A, a fungal natural product, selectively removes the zinc ion from metallo-β-lactamases such as NDM-1 and VIM-2, to restore their susceptibility to carbapenems in the treatment of Enterobacteriaceae.11 Additionally, given that antibiotic-producers are always equipped with self-resistance to avoid suicide, inspired by the co-evolution between antibiotic-producers and diverse competitors in natural niches,12–14 resistance-based approaches to mining novel antibiotic candidates will be more efficient.
In the golden era of antibiotics, the chemical and mechanistic diversity of antibacterial natural product lead compounds provided interesting and useful scaffolds for antibiotic discovery.15–17 Heretofore, nonribosomal antibacterial peptides (NRAPs), such as penicillin (the first antibiotic introduced in modern medicine) are well-known as antibiotics in the clinical setting.18–20 Additionally, vancomycin21 and colistin22 are recognized as last resort antibiotics against Gram-positive and Gram-negative pathogens, respectively. NRAPs are a subclass of nonribosomal peptides (NRPs) with antibacterial activities, produced by giant nonribosomal peptide synthetases (NRPSs).23 In particular, NRPSs are composed of multiple modular sections, each of which is responsible for the incorporation of one defined amino acid (not limited to the 20 proteinogenic amino acids) into the final peptide-like products.24,25 The flexible biosynthetic mechanism of NRAPs leads to compounds with structural diversity. Nevertheless, the dissemination of multiple resistant genes, such as β-lactamase associated genes and the van and mcr series of genes,5,26 has ultimately paralyzed the use of such antibiotics in the clinical setting. Fortunately, new leads and NRAP scaffolds have been continually reported in the past decade, with potent antibacterial activity against MDR bacteria.
In this review, we will first provide a brief overview of the molecular mechanism of antibiotic resistance to better guide antibiotic discovery. Then, we will describe the recent progress on NRAPs, including new sources, methodologies, structure–activity relationships and modes of action. Finally, further perspectives on developing effective NRAPs and their derivatives will also be discussed.
Antibiotics, used either for treating infections in human beings, as growth promoters in food-animal production, or as the waste of pharmaceutical plants, serve as a driving force to select antibiotic-resistant bacteria from the persistent coexistence of antibiotic-sensitive and antibiotic-resistant bacterial strains in natural environments. Bacteria have evolved multiple strategies to tolerate antibiotic treatments. Here, we will focus on the antibiotic resistance of individual cells, including the inactivation and modification of antibiotics, prevention of access to antibiotics and structural changes of antibiotic targets (Fig. 1A). Antibiotic resistance mediated by a bacterial population, such as quorum sensing and formation of biofilm, and by hosts (in vivo), such as hiding in the cytosols in the form of a “Trojan horse”, will not be discussed here (Fig. 1B).
Fig. 1 Molecular mechanisms of antibiotic resistance in Gram-positive and Gram-negative bacteria. (A) Antibiotic resistance in single bacterial cells, including inactivation and modification of antibiotics by enzymes, reduced permeability due to inactivation or down-regulated expression of porins, increased efflux pumps, and mutation or modification of antibiotic targets. (B) Antibiotic resistance at the levels of individual cells, the population and in vivo. Bacterial population mediated resistance is known for the formation of biofilm, which serves as a barrier or unfavourable environment against antibiotic treatments. In addition, bacteria can invade and survive in host cells, to circumvent the use of antibiotics.
Bacteria have evolved various strategies to impair or tolerate antibiotic assaults, of which bacteria can directly break and/or modify the structures of antibiotics to avoid growth inhibition or being killed. Enzymatic degradation and modification are effective means of antibiotic resistance that has brought about resistance to several major classes of existing antibiotics, including β-lactams and aminoglycosides.
The process of hydrolysis, carried out by a diverse range of hydrolases, has been identified to inactivate multiple antibiotics. The co-evolution of β-lactam antibiotics and β-lactamases is an excellent example to illustrate the arms race between antibiotics and antibiotic resistance. The β-lactamases serve as work horses to degrade β-lactam antibiotics, such as penicillins, cephalosporins and carbapenems, by breaking the core β-lactam ring open, through either serine nucleophilic attack or the metal-based activation of a water molecule. For example, the first β-lactamase discovered was penicillinase,27 which was first isolated from Escherichia coli K-12 even before the introduction of penicillin in the clinical setting.
Unlike β-lactamases, bacterial enzymes can add different chemical groups to vulnerable sites of antibiotics, by preventing modified antibiotics from binding to the corresponding targets. Compared to other antibiotics, aminoglycoside antibiotics tend to be easily modified due to the abundant amide and hydroxyl groups on the surface of aminocyclitol nuclei linked to amino sugars. Nucleotidyl-transferases, phosphotransferases and acetyltransferases are three main classes of aminoglycoside modifying enzymes that can catalyze the modification at different amide or hydroxyl groups of 2-deoxystreptamine nuclei or sugar moieties.32 Worryingly, a novel genomic island that can encode multiple aminoglycoside-modifying enzymes was found in Campylobacter isolates from a food-producing animal origin that conferred high-level resistance to gentamicin and kanamycin.33 Meanwhile, new aminoglycoside-modifying enzymes are still being discovered, even in nonpathogenic susceptible bacterial species. For example, type VIII and type IX aminoglycoside 3′-O-phosphotransferases were reported in Acinetobacter rudis and A. gerneri in 2017,34 respectively.
The accumulation of enough antibiotic in bacteria is a prerequisite for antibacterial activity.35 Decreasing the concentrations of intracellular antibiotics to increase resistance can be achieved through reduced permeability or enhanced efflux.
As well as destroying and modifying antibiotics outside or preventing antibiotic access to intracellular targets, bacteria can form resistance by inducing structural changes of antibiotic targets. Such a strategy is a perfect example of the saying “If you cannot change the world, change yourself”.
Bacteria can circumvent antibiotic therapeutics by altering the original targets, resulting in them being able to survive and cause infections. Similar to the direct modification of antibiotics by various modifying enzymes, protection by modification of antibiotic targets is also widely utilized by different bacterial species. Multiple types of antibiotics can target the ribosome to inhibit or block protein synthesis, such as the classes of phenicols, aminoglycosides and tetracyclines. Correspondingly, methylation of the ribosome by methyltransferases has been characterized in diverse bacterial species to resist their action. For example, wide dissemination of plasmid encoded chloramphenicol–florfenicol resistance (cfr) methyltransferase specifically methylates the adenine of position 2503 in the 23S rRNA,48 which has been observed in the isolates of Gram-positive and Gram-negative pathogens from both human and animal origins.49–52 Such an enzyme thereby confers resistance to a wide range of antibiotics, including phenicols, pleuromutilins, streptogramins, lincosamides, selected 16-membered macrolides and also oxazolidonones (such as linezolid).
To protect critical antibiotic targets for physiological functions, bacteria may evolve different strategies by which to resist antibiotic stresses. Compared to the modification of lipid A with pEtN, recent studies have shown that reduced colistin binding to lipid A can be achieved through the addition of 2-hydroxymyristate, 4-amino-4-deoxy-L-arabinose and palmitate. For example, deletion of mgrB and overexpression of the PhoPQ two-component system increased resistance to polymyxins via different lipid A modifications in K. pneumoniae.64,65 Importantly, these modifications in K. pneumoniae were accompanied by enhanced virulence through lowering the antimicrobial peptide susceptibility and attenuating the activation of early host defense responses. Additionally, despite daptomycin displaying remarkable selectivity against Gram-positive bacteria, the underlying mechanism of daptomycin resistance is not fully understood. The membrane protein multiple peptide resistance factor (MprF) plays a crucial role in the induction of daptomycin resistance, by transferring lysine to modify the membrane lipid phosphatidylglycerol (PG).66 Meanwhile, both LiaF and a GdpD-family protein involved in the cell envelope and cell membrane events, also appear to contribute to daptomycin resistance.67 These results suggest that there is an urgent need to develop new antibiotics to circumvent previously identified targets.
Collectively, elucidation of the resistant mechanisms and better understanding of the diverse ways by which bacteria resist clinically useful antibiotics, will shed light on the design of alternative therapeutic approaches and guide the development of new antibiotics.
NRAPs possess versatile chemical scaffolds, suitable antibacterial activity and unique modes of action,73 making them potent leads for antibiotic discovery. In the past decade, a diverse range of NRAPs have been identified, as shown in Table 1, which indicates that there are still untapped sources for discovering NRAPs with as-yet unknown functions. We will introduce these new compounds in terms of their sources, screening methods and structure–activity relationships (Fig. 2).
a a/b/c, albicidin/tridecaptin A1/telomycin were isolated in 1983/1978/1957 and re-elucidated in 2015/2016/2016, respectively. PBP, penicillin binding protein. LPS, lipopolysaccharide. C55-P, undecaprenyl phosphate.
Fig. 2 Scheme of approaches taken to screen NRAPs for combating multi-drug resistant pathogens. The producers are mainly from soil, marine, insect and human microorganisms. Screening strategies are based on activity, genomics, proteomics and meta-mics.
Fig. 3 Representative NRAPs from different sources of bacteria discovered since 2010. Lysocin E (culturable soil), teixobactin (unculturable soil), ilamycin (marine), sevadicin (insect) and lugdunin (human).
Given the tremendous biodiversity of organisms,80 the marine environment actually represents another prominent source for antibiotic discovery. Sponges, corals and marine animals contain compounds with interesting scaffolds. For example, ilamycins were isolated and identified from deep sea-derived Streptomyces atratus SCSIO ZH16, of which the ilamycins E1/E2 (Fig. 3) were found to show highly potent anti-tuberculosis activity against Mycobacterium tuberculosis.112 Furthermore, gageotetrins A–C, isolated from marine Bacillus subtilis, are a unique class of linear lipopeptides consisting of repeated Leu and Glu units alongside a fatty acid chain.102 Most unexploited marine microorganisms have been isolated for a long time away from many bacterial pathogens that inhabit the earth, which may provide a solution to combat MDR pathogens.
Although we are facing the dilemma that it's difficult to identify novel compound structures due to increased re-discovery of known antibiotics or their analogues from soil microbes such as Actinomycetes, potent antibacterial compounds from other ecological niches may become valuable sources for new types of NRAPs. Taken together, these findings show how attractive NRAPs from new sources are in combating resistant pathogens and give deep insight into the underlying structure–activity relationships.
Screening methods play a critical role in mining potent bioactive compounds. The simplicity and effectiveness of the Waksman platform led to a golden era in antibiotic discovery, contributing to the discovery of most of the existing antibiotics used in the clinical setting. Due to the high re-discovery rates of previous antibiotics and their derivatives, more robust methods are required to exploit antibacterial leads against antibiotic resistant pathogens from new sources.
3.2.1 Activity guided discovery. The traditional activity-guided approach is still full of vitality for screening new NRAPs. The discovery of lugdunin109 and bacaucin111 followed this classic protocol, including the isolation and identification of bacteria, antibacterial testing, separation and purification of active compounds, and structural elucidation. New achievements in biotechnology and chemistry boost such procedures. For example, a multichannel device (iChip) was designed and fabricated to simulate the natural environment of uncultured soil bacteria for antibiotic discovery.76 Teixobactin produced by Eleftheria terrae was successfully identified in this way and was shown to have activity against both MRSA and VRE, and other Gram-positive bacteria. Compared to the small minority of culturable microorganisms in vitro, we expect that more NRAPs or other types of antibiotics will be found by culturing nonculturable species.
3.2.2 Genomics driven screening. To obtain natural products of interest, sophisticated strategies are often involved to optimize the production or activation of the biosynthetic gene clusters (BGCs) of NRAPs in a diverse range of microorganisms. However, such approaches are always limited by the fact that only a small proportion of microorganisms can be cultured under artificial conditions, leaving territory that is yet to be explored. Meanwhile, extensive sequencing of bacterial genomics has revealed that the biosynthetic diversity traditionally accessed represents only a small fraction of what is offered by nature.119 To address these limitations, the high efficiency of genomics driven screening has brought about a new age of NRAP discovery.
Another intriguing challenge is that most microorganisms produce far fewer metabolites of interest under artificial growth conditions than genomes suggest. Many specialized metabolite BGCs are poorly expressed, or not expressed at all, under laboratory growth conditions. Therefore, suitable methods for activating such silent BGCs are a prerequisite to enable the perspective of genomics-driven approaches for the discovery of NRAPs. Summarized strategies to induce the expression of silent BGCs to produce structurally diverse specialized metabolites have been reviewed elsewhere,119 which are generally categorized into two groups including pleiotropic methods and pathway-specific methods. For example, two chlorinated cyclic hexapeptides (ulleungmycins A and B) were discovered from Streptomyces sp. KCB13F003 based on the presence of a cryptic gene cluster encoding NRPS and flavin-dependent halogenase and by manipulating the culture conditions.113 Interestingly, subinhibitory concentrations of ribosome-targeting antibiotics (e.g. chloramphenicol) can enhance the production of NRAPs from Streptomyces through global metabolic perturbation.125 Compared to pleiotropic strategies, pathway-specific approaches provide more genetically stable producers for predictable metabolites through sophisticated operations. Taromycin A, similar to clinically approved daptomycin, was obtained by manipulating pathway-specific regulatory genes in marine Saccharomonospora sp. CNQ490.99 The development of gene editing techniques offers alternative methods to awaken silent BGCs, by engineering the transcription and translation machineries, manipulating regulators, replacing natural promoters and heterologous expression. For instance, a one-step robust CRISPR-Cas9 knock-in strategy was established to active multiple silent BGCs in Streptomycetes species.126 Additionally, malacidins, a class of calcium dependent NRAPs, were discovered very recently to integrate the complete malacidin BGC for heterologous expression in Streptomyces albus.77 These results indicate that genomics driven screening may enable the development of a remarkable range of NRAPs and open avenues for the discovery of new antibacterial compounds for applications in medicine and other fields.
3.2.3 Proteomics and meta-omics based discovery. Despite the great achievements made in genomics driven screening, identifying new NRAPs and their biosynthetic pathways still remains a challenge. Proteomics and meta-omics-based methods used as complementary approaches may accelerate the discovery of antibiotics. An intriguing application of proteomics technology is the proteomic investigation of secondary metabolism (PrISM),127 which has been used to discover a lipoheptapeptide from the Bacillus strain NK2018 with a putative structure highly similar to that of the kurstakins128 and unknown natural products from the hybrid NRPS-PKS zwittermicin A BGC. Meanwhile, an orthogonal active site identification system (OASIS) for the proteomic analysis of PKS/NRPS biosynthetic enzymes has been developed.129 OASIS probes have proven to be successful for Bacillus subtilis, by targeting conserved active sites in NRPS/PKS systems, coupled with analysis through multidimensional protein identification technology (MudPIT) LC-MS/MS analysis. To some extent, PrISM and OASIS approaches represent innovative and complementary methods in NRAP discovery. However, PrISM can be affected by the unanticipated post-lysis proteolytic of NRPSs and its time-consuming and labor-intensive drawbacks limit the synthesis of OASIS probes.
Successes in multi-omics technologies mean that meta-omics is a promising approach for new NRAP discovery. Meta-omics combines the advantages of genomics, proteomics, metabolomics, and transcriptomics analysis tools. The construction of a metagenomics library, direct DNA sequencing and single cell technologies make the sequencing data significantly more available, while bioinformatics facilitates the rapid mining of BGCs.130 Similarly, metaproteomics consistently provides insights into the discovery and analysis of NRPSs, providing the groundwork for NRAP development.
Inspired by versatile heterologous expression systems and achievements in synthetic biology,131,132 meta-omic technologies will enable access to a range broad range of NRAPs that have therapeutic applications in medicine.
The diverse range of new NRAP scaffolds leads to comprehensive structure–activity relationships (SARs) and sheds light on the design of natural NRAP derivatives with improved antibacterial activity. We focus on the SARs of peptide scaffolds, amino acid types and chemical decorations in terms of antibacterial activity, in either newly identified or previously described NRAPs.
3.3.1 Diverse scaffolds. As discussed in Section 2.1, the evolution between β-lactam antibiotics and β-lactamases provides an excellent example to elucidate SARs and to guide the development of new β-lactam antibiotics. To inactivate the activity of various β-lactam antibiotics, MDR bacteria utilize a diverse range of enzymes to specifically catalyze the ring-opening of the core β-lactam ring, rendering the antibiotic useless. In turn, either five-membered or six-membered rings fused to all bicyclic β-lactams combined with the modification of side chains have been developed to resist hydrolysis, as shown in Fig. 4. However, it has been discovered that NDM overwhelms all bicyclic β-lactam antibiotics, while monolactams, such as aztreonam, are still effective. Meanwhile, the N-sulfonated monocyclic β-lactam ring has been well characterized in sulfazecin, due to the unprecedented activity of the thioesterase domain.133 Nevertheless, it turns out that such balance is favorable for bacteria, as shown in a very recent report of an isolate of E. coli CCD1 of food-animal origin in China, which was found to be resistant to all β-lactams tested, including carbapenems and aztreonam.134 Although the underlying mechanism of E. coli CCD1 resistance to aztreonam remains unclear, it indicates that the time where there are no available β-lactam antibiotics to treat CRE in the clinical setting may soon come.
Fig. 4 Co-evolution of β-lactam antibiotics and β-lactamases.
A wide variety of NRAPs produced by microorganisms, that can be structurally categorized into cyclic and linear scaffolds, have been known for decades and show a wealth of activities. Unlike the ring opening of β-lactam antibiotics, which is ineffective, there has been an increasing number of reports that show that linear NRAPs and their analogs demonstrate comparable or even better antibacterial activity against MDR bacteria. Linear NRAPs represent a new class of antibiotic candidates that are structurally distinct from clinically used cyclic NRAPs, such as daptomycin, polymyxins and vancomycin, and may help to circumvent resistance. Linear NRAPs usually contain short peptide chains and different chemical accessories, in particular modified lipid tails, which have been reported for decades in compounds such as cerxins135 and tridecaptins.136 Recently, lots of linear NRAPs have been discovered from a diverse range of sources using different approaches, including lipopeptides such as humimycins, gageotetrins,102 paenipeptins,110 (Fig. 5) and peptides without any further modifications, such as bacaucin-1 and sevadicin. For example, the peptide skeletons of humimycins107 were bioinformatically predicted from primary sequence data of human-associated bacteria and then chemically synthesized by SPPS, and showed moderate antibacterial activity against MRSA and a synergic effect with β-lactam antibiotics (such as carbenicillin and dicloxacillin). Similarly, antibacterial syn-BNP 1 (an N-acylated 13-mer linear peptide) and antifungal syn-BNP 2 (an N-acylated nonapeptide) can be obtained using the same synthetic-bioinformatic natural product (syn-BNP) approach.137 Interestingly, linear heptapeptide bacaucin-1,111 a ring-opened NRAP of bacaucin without lipid modification, has shown specific antibacterial activity against MRSA in both in vivo and in vitro models. It demonstrates an elegant example of improving antibacterial properties by removing the fatty acid tail and opening the ring structure of the parent compound. Bacaucin-1 consists of all L-type amino acids and the cationic guanidino group under physiological conditions plays a crucial role in its selective activity, suggesting that the design of a linear peptide can be used as an alternative for next-generation precise antibiotics.138 Notably, sevadicin (D-Phe-D-Ala-Trp), the shortest natural linear tripeptide, has been found, which shows activity against bacilli.101 Collectively, the absence of a macrocycle within linear NRAPs makes them more easily accessible as they are easier to synthesize.
Fig. 5 Linear NRAPs and derivatives from a diverse range of sources. Humimycin A (human microbiome), paenipeptin C (mushroom), gageotetrins A (marine) and bacaucin-1 (chemically synthetic derivative of bacaucin) (soil).
In addition, nonproteogenic amino acids can also be incorporated into the peptide skeleton, further expanding the diversity of NRAPs and enhancing their antibacterial activities. For instance, teixobactin is able to kill a series of clinically relevant Gram-positive bacteria, including MRSA, VRE, Streptococcus pneumoniae, Mycobacterium tuberculosis, Clostridium difficile and Bacillus anthracis, with values of minimal inhibitory concentration (MIC) ranging from 0.005 to 0.5 μg mL−1. Such ability is mainly due to the introduction of enduracididine, methyl-phenylalanine and four D-type amino acid residues.76 Interestingly, the introduction of rare L-3-nitrotyrosine and L-2-amino-4-hexenoic acid endows ilamycins E1/E2 with highly selective anti-tuberculosis activity, with an MIC value of 9.8 nM.112 Furthermore, lugdunin, the first example of a new class of macrocyclic thiazolidine NRAPs, has been shown to have potent antimicrobial activity against MRSA and VRE, and a wide range of Gram-positive pathogens with MIC values ranging from 1.5 to 12 μg mL−1.109 However, the detailed SARs of these new identified NRAPs remain largely unclear, and further studies are still required.
3.3.2 Modification and optimization. To optimize clinically used NRAPs, a better understanding of the SARs of such antibiotics will improve their therapeutic properties, either by enhancing their antibacterial efficiency or by decreasing their side effects. For example, vancomycin analog 18 was engineered to combat and improve durability against VRE using peripherally and binding pocket modified vancomycin,139 which dramatically enhanced its activity, leading to a decrease in the MIC values from 250 μg mL−1 to 0.005–0.01 μg mL−1 (Fig. 6A). Compared to the approved agent oritavancin, that has the sole introduction of a (4-chlorobiphenyl) methyl (CBP) group to the disaccharide of vancomycin,140 such modifications offer vancomycin analog 18 great advantages against MDR pathogens. Collectively, these modifications not only improve the capability to disrupt the cell membrane integrity, but also inhibit cell wall synthesis. Interestingly, combining C-terminal modification with peripheral CBP addition to the disaccharide in vancomycin achieves three independent and synergistic mechanisms of action, which significantly increases the fitness cost for VRE to obtain new resistance to fight back against such potent antibacterial leads.
Fig. 6 Modification and optimization of NRAP antibiotics. (A) The peripheral modification of vancomycin exhibits enhanced activity against VRE. (B) Structures of polymyxin B and derivatives synthesized through the modifications of N-terminal fatty acyl chains and amino acid substitutions at the third amino acid residue (AA 3). Structures of octapeptin C4 (C) and the derivatives of griselimycin (D).
Polymyxins including polymyxin B and polymyxin E (also known as colistin), are narrow-spectrum antibiotics that are used against MDR Gram-negative pathogens, in particular CRE. Importantly, the side effects of polymyxins involve nephrotoxicity and neurotoxicity in the case of long-term high-dose administration, that accompany their antibacterial activity. To reduce their intrinsic toxicity, systematic SARs have been performed to optimize and design new polymyxin analogs, mainly through the modifications of N-terminal fatty acyl chains and amino acid substitutions. For example, analog CB-182 804 (ref. 141) was obtained by deleting the fatty acid chain moiety and adding 2-chlorophenylisocyanate to the N-terminal free amino group in plymyxin B (Fig. 6B). CB-182 804 shows comparable antibacterial activity to that of plymyxin B/colistin, whereas decreased cytotoxicity in the kidney proximal tubule cells of rats was observed with a half maximal inhibitory concentration (IC50) of more than 1000 μg mL−1.142 On the other hand, reduced cytotoxicity brought about by the replacement of Dab-3 with Gly and using an octanoic acid at the N-terminal was achieved in Analog 38,143 which retained antibacterial activity with low cytotoxicity to HepG2 cells (IC50 > 300 μM). Detailed SARs of polymyxins have been emphasized and recently reviewed in ref. 144. Despite many efforts being made to obtain better analogs of polymyxin, unfortunately, no analog has been approved for clinical use. Therefore, further studies and alternative approaches are still required, especially in the mining of natural compounds. For example, octapeptin C4, a colistin-like NRAP, is active against MDR bacteria, including polymyxin-resistant bacteria (Fig. 6C),114 and also exhibits reduced nephrotoxicity. Compared to polymyxins, octapeptin C4 and its analogs are N-terminally acylated with longer nonanoyl (C9) or decanoyl (C10) β-hydroxy fatty acyl chains. The change in Thr to Leu at position 8 in the heptapeptide ring of octapeptins is the significant difference between polymyxins and octapeptins.
Owing to the urgent need for novel antibiotics and advanced technologies, rich sources of antibacterial lead compounds have been re-discovered to revitalize previously neglected antibiotics. An exciting example of this is the optimization of the cyclic NRAP griselimycin (GM), which is used for tuberculosis therapy.105 GM from Streptomyces with potential antibacterial and antimycobacterial activities has been known for decades,145 but it still has unfavorable pharmacokinetic properties. Given its high activity, new studies have focused on GM and alkylation of the proline ring at position 8 has been shown to improve its pharmacokinetic properties (Fig. 6D). Attractively, the derivative (CGM) with the addition of a cyclohexyl group on the GM was found to be metabolically stable, and can penetrate the thick mycobacterial cell wall due to increased lipophilicity. CGM exhibited enhanced activity with a MIC value as low as 0.06 μg mL−1 and no cross-resistances with current anti-tuberculosis drugs, suggesting that such derivatives possess an unique mechanism of action differing from other mycobactericidal drugs in use.
3.3.3 Calcium-dependent NRAPs. The rich cationic amino acid residues in many NRPAs not only contribute to their antibacterial activity, but also cause unexpected pharmacological effects, such as toxicity, and high affinity to negatively charged serum proteins. The non-cationic scaffolds of NRAPs hold great promise to circumvent such disadvantages. Daptomycin is a cyclic lipopeptide antibiotic encoded by the daptomycin biosynthesis (dpt) gene cluster in Streptomyces roseosporus (Fig. 7A). Daptomycin was initially used for the treatment of severe skin associated infections caused by Gram-positive pathogens, including MRSA and vancomycin-resistant S. aureus (VRSA),146 and was later extended to systemic and life-threatening infections147 owing to the lack of available antibiotics in the clinical setting. However, this application is limited for external conditions, due to toxicity and calcium concentration dependent antibacterial activity. Similarly, taromycin A,99 A54145,85 (Fig. 7B) and friulimicins86 are homologous structures of daptomycin, which also show calcium dependent activity against Gram-positive bacteria. This notion is consistent with the observation that there is a sequence similarity between the dpt gene cluster and the tar/lpt gene clusters, although they contain additional biosynthesis genes in each cluster (Fig. 7A). The conserved peptide motif of Asp-X-Asp-Gly (DXDG)148 has been proposed as a canonical calcium-binding motif to bind to bacterial membrane that is required for sequential oligomerization and pore formation (Fig. 7C1).
Fig. 7 Biosynthetic gene clusters (BGCs) (A), structures (B) and modes of action (C) of four calcium-dependent NRAPs. The whole biosynthetic gene clusters of daptomycin (AY787762), taromycin (KF301601), A54145 (DQ118863) and malacidin (KY654519) were downloaded from GenBank, and the accession numbers are indicated in parentheses. The identities were analyzed against the daptomycin (dpt) gene cluster as a reference, by the Basic Local Alignment Search Tool (BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Intriguingly, a new class of calcium-dependent NRAPs, malacidins,77 are challenging this model. The biosynthesis (mlc) gene clusters carry more accessories, suggesting structural differences between daptomycin, A54145 and taromycin A. Malacidins do not contain the DXDG motif incorporated from rare 3-hydroxyl aspartic acid (HyAsp) and also lack the variable spacer residue (Fig. 7B). Additionally, malacidins can specially inhibit lipid II, which indicates that malacidins may harness a new calcium-binding motif (Fig. 7C2). These observations again prove that natural products are, as always, extremely generous in supplying leads to achieve the same therapeutic purpose. Therefore, non-cationic and calcium-dependent NRAPs will be eagerly expected in the future to achieve robust antibacterial activity with reduced side effects.
Advanced technologies in chemical biology facilitate the understanding of the mode of action (MOA) of many old NRAPs, such as tridecaptin A1 and GM, and recently identified ones, such as teixobactin, ilamycin and humimycin. We will summarize the validated targets (Table 1) for advancing hits in NRAP discovery programs, while the involved cellular pathways leading to bacterial death or growth inhibition are not discussed here. Meanwhile, Brown and co-workers have rigorously reviewed the strategies for target identification up to 2015.149 Heretofore, many unique antibacterial targets have been identified, such as menaquinone, cardiolipin, flippase and translocase I, which open up new avenues to develop leads for combating resistant bacteria. Generally, inhibition of cell wall synthesis, membrane disruption and blocking critical intracellular processes are the major approaches utilized by NRAPs, as shown in Fig. 8.
Fig. 8 Schematic representation of mechanisms of NRAPs against Gram-positive (A) and Gram-negative bacteria (B). MBLs, metallo-β-lactamases. AMA, aspergillomarasmine A.
The cell wall provides bacteria with both structural support for performing sophisticated physiological tasks and protection against harsh environmental stresses and invaders, and is readily accessible in both Gram-positive bacteria and Gram-negative bacteria. The bacterial cell wall is composed of peptidoglycan, which is made of polysaccharide chains constructed from cross-linked peptides, as shown in Fig. 8. Lipid II and lipid III are two membrane-anchored precursors used for the biosynthesis of peptidoglycan and teichoic acid (a type of polysaccharide) in Gram-positive bacteria, respectively. Thus, different types of NRAPs predominantly target the bacterial cell wall biosynthesis machinery. For example, teixobactin inhibits cell-wall biosynthesis in S. aureus by binding to a highly conserved motif of lipid II and lipid III.76 Given that lipid II is only present in bacteria, it serves as a specific antibacterial target in the absence of cytotoxicity to mammalian cells.150 Additionally, no resistant S. aureus was obtained after serial passage at the sub-inhibitory concentrations of teixobactin. Similarly, malacidins bind to lipid II in a calcium-dependent manner,77 which is different to the binding of previously characterized calcium-dependent NRAPs such as daptomycin (Fig. 7C).
In an attempt to reach intracellular components, many antibiotics cannot penetrate the thick cell wall in Gram-positive bacteria or the low permeability of the outer membrane in Gram-negative bacteria. Intriguingly, several analogues of paenipeptin, such as paen 9 (Fig. 8B), have been shown to increase the activity of rifampicin and clarithromycin against carbapenem resistant and polymyxin resistant pathogens.158 This indicates that many hydrophobic antibiotics such as rifampicin, which targets DNA-dependent RNA polymerase, can be revitalized in combination with NRAPs that disrupt the membrane integrity and destroy the thick cell wall.
In fact, NRAPs are often versatile enough to affect several cellular events. An exciting example is lugdunin,13 a new class of macrocyclic thiazolidine NRAP produced by human nasal S. lugdunensis. Lugdunin simultaneously inhibits the biosynthesis of DNA, RNA, protein or cell-wall precursors and leads to the rapid collapse of bacterial energy resources. Such synchronous inhibition of multiple targets dramatically increases the fitness cost for bacteria to evolve resistance.
Although the discovery of NRAPs is blooming, the three crucial obstacles in the development of NRAP candidates for clinical trials are high cost, poor protease stability and nonspecific toxicity in both in vivo and in vitro models. Most NRAPs have unique structures with complicated decorations and tend to be very expensive drugs. Therefore, leads with simple and non-cationic scaffolds are promising candidates. As shown in Fig. 5, increased numbers of linear lipopeptides or their derivatives in the absence of accessories achieve potent activity against bacterial pathogens. The development of linear NRAPs or analogues in lieu of cyclic ones will be economically beneficial, because they can significantly accelerate the synthetic process and thus reduce the cost. Most importantly, a large number of such lead compounds can be produced using the standard protocol of solid-phase peptide synthesis (SPPS). Rationally engineered linear derivatives of paenipeptin A/B/C110 and bacaucin-1 (ref. 111) reveal that the peptide cyclization is sometimes not essential for their antibacterial activity. Surprisingly, bacaucin-1, synthesized by all natural amino acids by SPPS without any fatty acid tail, shows even better antibacterial activity against MRSA than its cyclic parent bacaucin. Collectively, NRAP inspired synthetic linear peptides represent a new paradigm in the discovery of better antibiotics.
To improve the protease resistance of NRAPs, several approaches have been proposed, including the replacement of natural amino acids with mimics including D-type amino acids, non-natural amino-acid analogues and appropriate formulations, to render them protease resistant. For instance, a recent study showed that D(KLAKLAK)2, a membrane active all-D-enantiomer antimicrobial peptidomimetic, is resistant to proteolytic degradation and is a great prototype drug that targets certain Gram-negative pathogens.159 Proteases or other polypeptidases usually recognize specific peptide sequences or certain side chains of amino acid residues to trigger hydrolysis of peptide bonds. For example, trypsin is the work horse in the digestive system of animals and human beings, cleaving peptide chains mainly at the carboxyl sites of lysine or arginine.160 Thus, rearrangement of the peptide sequence based on the elucidated SAR may provide an alternative way of obtaining candidates.
Since the introduction of penicillin for treating bacterial infections, broad-spectrum agents have been advocated due to the lack of sensitive and reliable diagnostic methods and time pressures, which has led to the practice of empirical therapy and overprescription.16 Nowadays, more and more studies are demonstrating that broad-spectrum antibiotics are being challenged in infection control and prevention, because they can result in serious side effects, including the triggering of hyper-inflammatory responses and, most notably, the disruption of the beneficial microbiome.162 Therefore, the treatment of infections should be transformed into an era of precise medicine. Precise antibiotics or narrow-spectrum antibiotics, the cornerstone of precise medicine against infections, are urgently required to perform these tasks.
NRAPs and their derivatives are promising candidates for the next generation of precise antibiotics that can selectively target the bacterial pathogen of choice, without destroying the beneficial microbes in the hosts. For example, ilamycins,112 heptapeptides from marine-derived Streptomyces atratus, show selective activity against two mycobacteria, including M. smegmatis and M. tuberculosis, while they fail to be effective against another six types of bacteria. This indicates that ilamycins are quite prominent as lead compounds for anti-tuberculosis agents. Similarly, bacaucin-1, a heptapeptide derivative of bacaucin, specifically targets MRSA and some Staphylococcus species.111 These findings suggest that NRAPs hold great promise for generating fewer off-target effects on the gut microbiota and decrease the stress that results in the evolution of resistance.
Advanced achievements in the accuracy of point-of-care tests (POCT) and better understanding of the pathogenesis of bacterial pathogens, in particular resistant pathogens, will accelerate the screening, design and development of precise antibiotics. Most notably, the switch to the era of precise antibiotics is not limited to the targeting of specific bacterial components. Alternative strategies for combating bacterial virulent factors and biofilm are promising approaches to promote personalized therapies that exclusively prevent infections.
Studies of mechanisms and resistance evolution of new NRAPs and their derivatives are a mandatory requirement in the screening and development of NRAP-based antibiotics and/or adjuvants. Exposure of hidden targets also contributes to the re-sensitization of resistant bacteria to antibiotic therapy. For example, the activity of penicillin can be reversed to kill MRSA in the presence of statins, cholesterol-lowering drugs, through disassembly of penicillin binding protein (PBP2a) oligomerization.164 In addition, some β-lactam antibiotics such as ceftaroline can specially target PBP2a to destroy the cell wall, which augments the activity of daptomycin against resistant strains in the clinical setting.165 This indicates that knowledge about the underlying mechanisms of resistance and synergy will certainly facilitate the development of new therapeutics to revitalize existing antibiotics and minimize the emergence of resistance. Such a strategy is crucial in the near future, as new antibiotics or solutions are not likely to enter the clinical setting immediately.
The evolution, dissemination and accumulation of multi-resistant pathogens pose a severe threat to human health, and calls for the development of new antibiotics or novel antibacterial strategies. The achievement and development of antibiotics from a diverse range of sources offers promising alternatives to tackle resistance and to treat resistant bacterial pathogen associated infections. Innovative approaches such as high-throughput screening in genome sequencing and bioinformatics tools accelerate the discovery of new NRAPs. Compared to conventional antibiotics, NRAPs are less prone to causing resistance due to their unique mechanisms of action. Furthermore, advances in the understanding of antibiotic resistance at the molecular level will shed light on how to design new scaffolds of NRAPs as precise antibiotics to generate fewer off-target effects on the host microbiome and to minimize the stress that facilitates resistance. Finally, the combination of NRAP based antibiotic adjuvants with existing antibiotics will enhance antibacterial activity and reverse resistance. Such strategy will be well positioned to fill the gap before new antibiotics are introduced into the clinical setting, in particular for infections caused by Gram-negative pathogens, because many NRAPs can disrupt the impermeable outer membrane and increase the accumulation of intracellular antibiotics. Collectively, NRAPs provide highly potent leads for the production of next-generation antibiotics against bacterial pathogens in the resistance era.
This work is supported by the National Key Research and Development Program of China (2017YFC1600305), the National Natural Science Foundation of China (31772796) and the Fund of Modern Agriculture Industry System Innovation in Beijing City Team (BAIC06-2017).
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